site-directed in vitro mutagenesis of enterovirus ev71

Site-directed in vitro mutagenesis of Enterovirus EV71:

implications for the development of an attenuated

EV71 vaccine

By

Natallia Lazouskaya

This thesis is presented for the degree of Doctor of Philosophy

2013

Environment and Biotechnology Centre

Faculty of Life and Social Sciences

Swinburne University of Technology

Melbourne, Australia

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Abstract

Enterovirus 71 (EV71) is a human pathogen associated both with sporadic

cases and large outbreaks of hand, foot and mouth disease (HFMD) throughout

the world. Young children under 5 are most susceptible to EV71 infection,

although, adult related cases have also been reported. The clinical manifestation

in EV71 patients may vary from mild and self-limited conditions to severe

neurological complications, such as meningitis, brain-stem encephalitis,

meningoencephalitis, poliomyelitis-like paralysis or pulmonary oedema with

lethal outcome. Due to the lack of anti-EV71 specific inhibitors or vaccines

available, treatment of EV71-associated complications relies on symptomatic and

supportive therapies. In the current situation, the major research efforts are being

focused on the development of vaccination strategies against EV71. An

attenuated virus which carries several neutralizing epitopes, both linear and

conformational, and which can induce an immune response similar to natural

infection is the most efficient approach. However, safety issues with respect to

the attenuated vaccine use require comprehensive understanding of the molecular

biology of the virus.

The aim of this study was to explore EV71 molecular determinants, which

can lead to the virus inhibition in vitro. An infectious EV71 cDNA clone was

constructed and subsequently used in site-directed mutagenesis (SDM). The

results demonstrated that among the virus mutants generated within the 5′UTR

domains Vth and VIth only the Sabin3-like EV71 showed slight attenuation in

vitro. However, its phenotype was genetically unstable, and reverted to that of

the wild-type virus during a few passages in cell culture. Stability of the Sabin 3

determinant was enhanced via its compensatory base pairing within the stem of

the domain Vth. Several genetic determinants of the virus temperature sensitivity

were identified by the charged-to-alanine mutagenesis within the VP1 capsid

protein. In particular 162Ala, 164Ala and 213Ala were associated with a

restriction in the virus growth at an elevated temperature. The combination of the

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5′UTR and VP1 mutations resulted in a stronger inhibition of the EV71 mutants

in vitro.

An additional interest of this study was to investigate molecular

determinants of the virus temperature resistance after natural adaptation to an

increased incubation temperature in vitro. A number of mutations were identified

within the VP1, VP2, VP3 and 3D viral genes. SDM within the non-silent

positions demonstrated that the Ser299Thr was mainly responsible for the

temperature resistant phenotype. Modelling of this position on the three-

dimensional 3Dpol structure revealed that the mutation was adjacent to the

catalytic site of the protein. Structure analysis of the conformational changes

within the protein “core” suggested that the Ser299Thr substitution may have a

direct effect on virus replication.

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“Land bridges were everywhere during the extinction, many species were spreading, and there were many diseases”

Robert T. Bakker

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Acknowledgments

I wish to thank A/Prof Poh Chit Laa for giving me the opportunity to undertake this project and for her supervision during the first year of my PhD candidature.

I would like to express my sincere gratitude to my supervisors Dr PA(Tony) Barton and A/Prof Enzo Palombo for their guidance and support throughout the course of this study. Their help in the research related and administrative matters made this project possible throughout all difficulties.

I am grateful to Prof Peter McMinn from the University of Sydney for providing me with the EV71 strain 6F/AUS/6/99 and Vero cell line.

Special thanks to CSL and Prof Ian Harding for the invaluable financial support during research.

I wish to thank Soula Mougos, Chris Key and Ngan Nguyen who provided technical support in the laboratory.

My heartfelt gratitude to my partner Tom for his constant help and encouragements during my PhD course, and for his English proof-reading of the project related materials.

Cheers to all my lab mates – Swarna and Raji Vairavan, Shahanee Mapulage Don, Kristin Kirk, Dhivya Rajasekaran, Hamid Pourianfar, Abdullah Sarrac, and many others who were undertaking their postgraduate studies during the same period. Thank you for your companionship and support in one way or another.

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Declaration

I hereby declare that the work presented in this thesis is an original work

conducted by the author, Natallia Lazouskaya, at the Faculty of Life and Social

Sciences, Swinburne University of Technology. To the best of the candidate’s

knowledge this thesis contains no material accepted for the award of any other degree or

diploma, and has not been previously written or published by another person, except

where due reference is made in the text.

Natallia Lazouskaya

February 2013

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Table of contents

Title page …………………………………………………………………...………...... i

Abstract …………………………………………………..……………………………. ii

Acknowledgments …………………………………………………….……………..… v

Declaration …………………………………………………….…………..………….. vi

Table of contents ………………………………………………………………...…… vii

List of Tables …………………………………………………………………….……. x

List of Figures ………………………………………………………………………… xi

Abbreviations ……………………………………………………………………..…. xiii

1 GENERAL INTRODUCTION AND LITERATURE REVIEW ............................. 2

1.1 Enterovirus 71 .................................................................................................... 2

1.2 Structure and genome organization of enteroviruses ......................................... 3

1.2.1 Genome organization and protein processing of the Picornaviridae.......... 3

1.2.2 Structure of viral particle ............................................................................ 5

1.2.3 5′ untranslated region (5′UTR) of the Picrnaviridae ................................ 10

1.2.4 Viral protein 4 (VP4) of the Picornaviridae ............................................. 16

1.2.5 Viral proteins 2 and 3 (VP2, VP3) of the Picornaviridae......................... 16

1.2.6 Viral protein 1 (VP1) of the Picornaviridae ............................................. 17

1.2.7 Viral 2A protease (2Apro) of the Picornaviridae ....................................... 21

1.2.8 Viral 2B protein (2B) of the Picornaviridae ............................................. 23

1.2.9 Viral 2C protein (2CATPase) of the Picornaviridae .................................... 24

1.2.10 Viral 3A protein of the Picornaviridae ..................................................... 26

1.2.11 Viral 3B protein of the Picornaviridae ..................................................... 28

1.2.12 Viral 3C protease (3Cpro) of the Picornaviridae ....................................... 29

1.2.13 Viral 3D polymerase (3Dpol) of the Picornaviridae .................................. 32

1.2.14 3′ untranslated region (3′UTR) of the Picornaviridae .............................. 39

1.2.15 poly(A) tail of the Picornaviridae............................................................. 40

1.3 Phylogeny of EV71 and its relationships to other human enteroviruses .......... 41

1.4 Pathogenesis of EV71....................................................................................... 46

1.4.1 The neuropathogenic features of EV71 infection ..................................... 46

1.4.2 Tissue tropism and cellular receptors of EV71 ......................................... 48

1.4.3 Molecular basis of EV71 virulence and attenuation ................................. 51

1.5 Development of antiviral treatments and preventive agents against EV71 ...... 59

1.5.1 Agents targeting the EV71 attachment, entry and uncoating .................... 59

1.5.2 RNA interference ...................................................................................... 61

1.5.3 Agents targeting 3C protease .................................................................... 61

1.5.4 Agents targeting 2C protein ...................................................................... 61

1.5.5 Agents targeting 3A protein ...................................................................... 62

1.5.6 Agents targeting 3Dpol ............................................................................... 63

1.6 Vaccine development against EV71 ................................................................. 64

1.6.1 Immunogenic determinants of enteroviruses ............................................ 65

1.6.2 Inactivated vaccine .................................................................................... 67

1.6.3 Attenuated vaccine .................................................................................... 70

viii

1.6.4 Synthetic peptides or epitope peptide vaccine .......................................... 75

1.6.5 Protein sub-unit vaccine ............................................................................ 77

1.6.6 Virus-like particle vaccine ........................................................................ 78

1.6.7 DNA vaccine ............................................................................................. 80

1.7 Project aims ...................................................................................................... 81

2 CONSTRUCTION OF AN INFECTIOUS cDNA CLONE OF EV71 .................. 84

2.1 Introduction ...................................................................................................... 84

2.2 Materials and methods ...................................................................................... 84

2.2.1 Cell culture and virus ................................................................................ 86

2.2.2 Total RNA extraction ................................................................................ 86

2.2.3 Reverse transcription of EV71 RNA......................................................... 87

2.2.4 Design of EV71 gene specific primers...................................................... 87

2.2.5 RT-PCR amplification .............................................................................. 89

2.2.6 Agarose gel electrophoresis of DNA ........................................................ 90

2.2.7 Gel purification of DNA ........................................................................... 91

2.2.8 Cloning of the full-length EV71 genome .................................................. 92

2.2.9 Screening the E.coli transformants ........................................................... 93

2.2.10 Isolation of plasmid DNA ......................................................................... 94

2.2.11 DNA automated cycle sequencing and nucleotide sequence analysis ...... 94

2.2.12 Restriction endonuclease digestion of DNA ............................................. 95

2.2.13 Ethanol precipitation of nucleic acids ....................................................... 96

2.2.14 in vitro transcription of EV71 cDNA clones............................................. 96

2.2.15 RNA electrophoresis ................................................................................. 97

2.2.16 Transfection of Vero cells with in vitro RNA transcripts ......................... 98

2.2.17 Detection and quantitation of EV71 RNA ................................................ 99

2.2.18 Virus titration .......................................................................................... 101

2.3 Results ............................................................................................................ 102

2.3.1 Amplification of the full-length genome of EV71 .................................. 102

2.3.2 Characterization of the EV71 cDNA clones ........................................... 105

2.3.3 Infectivity of the EV71 cDNA clones in cell culture .............................. 109

2.3.4 Role of the poly(A) tail in infectivity of EV71 in vitro RNA transcripts113

2.3.5 Role of the culturing conditions in rescue of EV71 from in vitro RNA transcripts .............................................................................................................. 115

2.4 Discussion....................................................................................................... 121

3 TEMPERATURE RESISTANT MOLECULAR DETERMINANTS OF EV71 UPON NATURAL SELECTION IN CELL CULTURE ............................................. 127

3.1 Introduction .................................................................................................... 127

3.2 Materials and methods .................................................................................... 129

3.2.1 Cell culture .............................................................................................. 129

3.2.2 Viruses and their cDNA clones ............................................................... 129

3.2.3 DNA automated cycle sequencing and nucleotide sequence analysis .... 130

3.2.4 Structure modelling and protein interactions analysis ............................ 130

3.2.5 Site-directed mutagenesis........................................................................ 132

3.2.6 Rescue of the EV71 mutants ................................................................... 136

3.2.7 Temperature sensitivity assay ................................................................. 136

3.2.8 Statistical analysis ................................................................................... 137

3.3 Results ............................................................................................................ 139

3.3.1 Sequence analysis of the ts and TR EV71 viral stocks and their cDNA clones ................................................................................................................. 139

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3.3.2 Structure modelling for EV71 mutants within the structural VP1 and VP3 proteins ................................................................................................................. 142

3.3.3 Structure modelling for EV71 mutant within the 3Dpol .......................... 149

3.3.4 Construction of EV71 mutants ................................................................ 158

3.3.5 Temperature sensitivity of the mutant viruses ........................................ 160

3.4 Discussion....................................................................................................... 163

4 SITE-DIRECTED MUTAGENESIS OF EV71 WITHIN THE 5′UTR ............... 169

4.1 Introduction .................................................................................................... 169


4.2.1 Cell culture .............................................................................................. 172

4.2.2 Design of mutagenic primers .................................................................. 173

4.2.3 Construction and recovery of mutant viruses.......................................... 175

4.2.4 Growth kinetics of the parent and mutant viruses ................................... 177

4.2.5 Stability of the mutant viruses................................................................. 178

4.3 Results ............................................................................................................ 184

4.3.1 In vitro replication of the parental and 5′UTR mutant EV71 .................. 184

4.3.2 Stability of the V stem-loop EV71 mutants in vitro ............................... 189

4.4 Discussion....................................................................................................... 195

5 SCANNING THE VP1 PROTEIN OF EV71 FOR ts-DETERMINANTS BY CHARGED-TO-ALANINE MUTAGENESIS ............................................................ 202

5.1 Introduction .................................................................................................... 202


5.2.1 Site-directed mutagenesis........................................................................ 205

5.2.2 DNA automated cycle sequencing and nucleotide sequence analysis of EV71 mutants ........................................................................................................ 209

5.2.3 Rescue of the EV71 mutants ................................................................... 209

5.2.4 Cell culture assays ................................................................................... 209

5.2.5 Mapping the VP1 mutations on the 3d structure ..................................... 210

5.2.6 Statistical analysis ................................................................................... 211

5.3 Results ............................................................................................................ 212

5.3.1 Effect of the VP1 mutations on virus infectivity .................................... 212

5.3.2 Growth kinetics of the VP1 mutant viruses ............................................ 217

5.3.3 Effect from the combination of the 5′UTR and VP1 mutations on temperature sensitivity of EV71............................................................................ 219

5.3.4 Binding activity of the mutant viruses .................................................... 223

5.4 Discussion....................................................................................................... 227

6 FINAL DISCUSSION AND CONCLUSIONS .................................................... 232

6.1 Contributions of this work .............................................................................. 232

6.2 Final conclusions of the experimental work ................................................... 234

6.2.1 Construction of an infectious cDNA clone of EV71 .............................. 234

6.2.2 Temperature resistant molecular determinants of EV71 due to natural selection in cell culture.......................................................................................... 235

6.2.3 Site-directed mutagenesis of EV71 within the 5′UTR ............................ 235

6.2.4 Scanning the VP1 protein of EV71 for ts-determinants by charged-to-alanine mutagenesis .............................................................................................. 236

6.3 Final comments and future directions ............................................................ 237

7 Bibliography .......................................................................................................... 240

8 WebPages .............................................................................................................. 270

9 Appendices ............................................................................................................ 272

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List of Tables

Table 1-1: Critical amino acid positions of the 3Dpol...................................................... 38

Table 1-2: Molecular classification of Human enteroviruses. ........................................ 42

Table 1-3: Phylogenetic relationships between genome regions of EV71 and other HEV-A viruses. .............................................................................................. 45

Table 1-4: Determinants of temperature sensitivity and attenuation of the OPV strains studied on EV71. ............................................................................................ 54

Table 2-1: Primers used in construction of EV71 cDNA clones, colony PCR and cycle sequencing. ..................................................................................................... 88

Table 2-2: Genetic variation within cDNA clones of EV71. ........................................ 108

Table 2-3: EV71 RNA level after transfection of Vero cell culture with in vitro RNA transcrips. ..................................................................................................... 119

Table 3-1: The MMDB crystal structures used in modelling the EV71 mutations. ..... 131

Table 3-2: List of primers used in SDM of the TR EV71 clone. .................................. 133

Table 3-3: Mutations observed in the conversion of the tss isolate of EV71 to TRs. .... 141

Table 3-4: IBIS predicted interactions within the 3Dpol of EV71. ................................ 154

Table 3-5: Temperature sensitivity of EV71 mutants. .................................................. 162

Table 4-1: Primers used in SDM and PCR/Restriction enzyme cleavage of EV71 5′UTR. .......................................................................................................... 174

Table 4-2: Correlation between substrate recognition sites density and amount of Sau3AI needed to digest 1 µg of the DNA substrate in 1 hour. ................... 182

Table 5-1: List of primers used in SDM within the VP1 encoding region of EV71..... 207

Table 5-2: Infectivity of the EV71 mutants in vitro. ..................................................... 214

Table 5-3: Temperature sensitivity of EV71 carrying mutations within the 5′UTR, VP1 or both genes. ............................................................................................... 222

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List of Figures

Figure 1-1: Genome structure and polyprotein processing of the Picornaviridae............ 4

Figure 1-2: Protein subunits within the picornavirus capsid. ............................................ 6

Figure 1-3: Folding pattern of the structural proteins of PV. ............................................ 8

Figure 1-4: Secondary structure prediction for the 5′UTR of the Picornaviridae. ......... 11

Figure 1-5: Location and secondary structure of PV1 CRE. .......................................... 25

Figure 1-6: VPg is covalently linked to the viral genome. ............................................. 28

Figure 1-7: Three-dimensional model of EV71 3Cpro. .................................................... 31

Figure 1-8: Structure of the PV 3Dpol.............................................................................. 35

Figure 2-1: Construction of EV71 cDNA clones. ........................................................... 85

Figure 2-2: pCR®-XL-TOPO® vector. .......................................................................... 92

Figure 2-3: Agarose gel electrophoresis of the 5′ (A) and 3′-halves (B) of EV71 genome amplified in the 1st round PCR of Strategy 1. ............................................ 102

Figure 2-4: Agarose gel electrophoresis of the full-length genomic cDNA of EV71 obtained in overlapping PCR, Strategy 1. .................................................. 103

Figure 2-5: Agarose gel electrophoresis of the full-length genomic cDNA of EV71 obtained with iProof DNA polymerase. ..................................................... 104

Figure 2-6: Orientation of the EV71 cDNA after cloning into pCR®-XL-TOPO® vector. ......................................................................................................... 106

Figure 2-7: Agarose gel electrophoresis of the EV71 in vitro RNA transcripts. .......... 109

Figure 2-8: CPE in Vero cell culture after transfection with the in vitro transcribed RNA. .......................................................................................................... 110

Figure 2-9: RT-PCR of virus after second round infection from cotransfected RNA showing viable virus from second strategy. ............................................... 112

Figure 2-10: Schematic diagram of the DNA templates used for in vitro RNA synthesis. .................................................................................................................... 114

Figure 2-11: Role of the culturing conditions in rescue of EV71. ................................ 116

Figure 2-12: Comparative quantitation of EV71 RNA isolated from the supernatant and cells after transfection of Vero cell culture with in vitro RNA transcripts. 118

Figure 3-1: Schematic representation of the SDM steps. .............................................. 135

Figure 3-2: Heterogeneity observed within the VP1 gene sequence of EV71 tss. ........ 140

Figure 3-3: BLAST alignment of the VP1 (A) and VP3 (B) proteins of the EV71 TRc against the reference, 3VBF. ...................................................................... 143

Figure 3-4: Mutations within VP1 of EV71 TRc, mapped on 3VBF (Wang et al. 2012) reference structure, (A) top, (B) side and (C) angle views. ....................... 146

xii

Figure 3-5: Mutation within VP3 of EV71 TRc, mapped on 3VBF (Wang et al. 2012) reference structure, side view. .................................................................... 148

Figure 3-6: Sequence alignment and structure modelling of the 3Dpol of the EV71 TRc. .................................................................................................................... 150

Figure 3-7: Multiple BLAST alignment of RDRP of positive-strand RNA viruses. .... 152

Figure 3-8: Interactions formed within the active centre of the 3Dpol during RNA synthesis. .................................................................................................... 156

Figure 3-9: Schematic representation of the EV71 mutant genomes. ........................... 159

Figure 3-10: Temperature dependent reduction in virus titres of EV71 mutants in Vero cells............................................................................................................. 161

Figure 4-1: Predicted secondary structure of EV71 RNA within the 5′UTR. .............. 176

Figure 4-2: Scheme of the PCR/Restriction enzyme cleavage assay used for detection of the wt-sequence revertants within the 473U and 473U/538A mutants. ..... 180

Figure 4-3: In vitro replication of the parental and mutant (473U, 473U/538A) EV71 viruses at 37.0˚C and 39.5˚C in Vero (A) and SK-N-SH (B) cells. ........... 186

Figure 4-4: In vitro replication of the parental and mutant (SL6-CAV8, SL6-CAV16) EV71 viruses at 37.0˚C and 39.5˚C in Vero (A) and SK-N-SH (B) cells. . 188

Figure 4-5: Dynamics of the virus accumulation during serial passaging in cell culture. .................................................................................................................... 190

Figure 4-6: Results of the PCR/Restriction enzyme cleavage assay conducted with the viral RNA of the wt-5′UTR sequence. ....................................................... 192

Figure 4-7: Comparative results of the PCR/Restriction enzyme cleavage assay and sequencing conducted with the EV71 mutant viruses upon serial passaging in Vero cells. .............................................................................................. 194

Figure 5-1: Schematic representation of the SDM steps used to combine the 5′UTR and VP1 mutations within the EV71 genome. .................................................. 208

Figure 5-2: Clusters of charged amino acids within the VP1. ...................................... 212

Figure 5-3: Location of the alanines substituting charged amino acids within the VP1 capsid protein. ............................................................................................ 216

Figure 5-4: In vitro replication of the parental and VP1 mutant viruses at 37.0˚C and 39.5˚C in Vero cells. .................................................................................. 218

Figure 5-5: Temperature dependent reduction in virus titres of EV71 mutants in Vero cells............................................................................................................. 221

Figure 5-6: Location of the 164th and 213th amino acids of VP1 within the viral capsid. .................................................................................................................... 224

Figure 5-7: Effect of substitution of VP1 amino acids at positions 164 and 213 with alanine on binding of EV71 to Vero cells. ................................................. 226

xiii

Abbreviations

3d-structure three-dimensional structure aa amino acid APC antigen-presenting cells BBB blood-brain barrier bp base pairs Br-UTP 5-bromouridine triphosphate BSA bovine serum albumin CAV Coxsackievirus A CBV Coxsackievirus B CHO cells Chinese hamster ovary cells CMV cytomegalovirus CNS central nervous system CPE cytopathic effect CRE cis-acting replication element CTP cytidine triphosphate DC dendritic cells DC-SIGN dendritic cell-specific ICAM-3 grabbing

non-integrin, CD209 DDDP DNA-dependent DNA polymerase DEN Dengue virus dsDNA double-stranded DNA ECV Echovirus eIF4G eukaryotic initiation factor 4G ER endoplasmic reticulum EtBr ethidium bromide EV Enterovirus f mutation frequencies FBS fetal bovine serum FMDV Foot-and-mouth disease virus GTP guanosine triphosphate HAV Hepatitis A virus HFMD hand, foot and mouth disease HHV Human Herpes virus HIV Human Immunodeficiency virus HRV Human Rhinovirus i.c. intracerebral i.m. intramuscular i.p. intraperitoneal ICR mice “imprinting control region” mice IL interleukin IRES internal ribosome entry site IVIG intravenous immunoglobulin KPL1 monoclonal antibody against PSGL-1 receptor MAP multiple antigen peptide MMDB the Molecular Modeling DataBase MMLV Moloney Murine Leukemia virus

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MV measles virus NCBI the National Center for Biotechnology Information NLS nuclear localization signal NPEVs non-polio enteroviruses nt nucleotide o.i. oral inoculation OPF open reading frame OPV oral poliovirus vaccine PABP poly(A)-binding protein PBMCs peripheral blood mononuclear cells PCBP poly(C)-binding protein PCR polymerase chain reaction PDB the Protein Data Bank PFU plaque forming unit Phe (or F) phenylalanine poly(A) polyadenylated tail Pph polyhedron promoter PPi pyrophosphate anion, P2O7

4- PSGL-1 P-selectin glycoprotein ligand-1 PTBP polypyrimidine tract binding protein PV1 Poliovirus type 1 PV2 Poliovirus type 2 PV3 Poliovirus type 3 PVR poliovirus receptor PVR-mouse cells mouse cells expressing the human poliovirus

receptor RDDP RNA-dependent DNA polymerase RDRP RNA-dependent RNA polymerase RNP ribonucleoprotein complex RPC reverse-phase cartridge rRNA ribosomal RNA RT reverse transcription SCARB2 scavenger receptor class B subfamily SDM site-directed mutagenesis SL stem-loop SVDV Swine vesicular disease virus TCID50 50% tissue culture infectious dose Tg mice transgenic mice TM transmembrane TMD transmembrane domain tr temperature-resistant ts temperature-sensitive tshr temperature-sensitive, host range UMP uridine monophosphate VAPP vaccine-associated paralytic poliomyelitis VDPVs vaccine-derived polioviruses VLP virus-like particle VPg viral protein of the genome WSSV White Spot Syndrome virus wt wild type

CHAPTER 1

GENERAL INTRODUCTION AND LITERATURE REVIEW

CHAPTER 1 GENERAL INTRODUCTION AND LITERATURE REVIEW

2

1 GENERAL INTRODUCTION AND LITERATURE REVIEW

1.1 Enterovirus 71

Enterovirus 71 (EV71) was first reported in California, USA in 1969. It

was isolated from the faeces of an infant suffering from encephalitis (Schmidt,

Lennette & Ho 1974). Since that time, many sporadic cases, mid-scale and large

outbreaks have been observed throughout the world. To date, it has been

responsible for large outbreaks of hand foot and mouth disease (HFMD), severe

forms of neurological diseases such as meningitis, brain-stem and/or cerebellar

encephalitis, meningoencephalitis, poliomyelitis-like paralysis, myocarditis,

respiratory infections (herpangina, rash) (Sanders et al. 2006). The most severe

complications which can result in permanent paralysis or death occur in young

children less than 4-5 years old (Chen et al. 2007; Chumakov et al. 1979; Liu et

al. 2000).

It was shown that EV71 has a high prevalence in natural circulation of

enteroviruses (Witso et al. 2006). However, the emergence of highly

neurovirulent EV71 strains and factors which contribute to their spread resulting

in large outbreaks are still puzzling. Comparison of full genome EV71 sequences

from fatal and non-fatal cases in the 1998 Taiwan outbreak showed high genetic

overall homology between strains: 97% and 98% for nucleotide and amino acid

level, respectively, and did not uncover typical amino-acid substitutions which

could contribute to a virulent phenotype (Shih et al. 2000).


3

1.2 Structure and genome organization of enteroviruses

Development of an anti-EV71 vaccine and treatment require detailed

characterization of the structural and genetic organization of the pathogen and its

genes responsible for virulence or induction of immunity. EV71 shares

significant structure similarity with poliovirus (PV) which is considered as a

prototype of the Picornaviridae family.

1.2.1 Genome organization and protein processing of the Picornaviridae

Picornaviruses are small, non-enveloped, icosahedral RNA viruses. They

possess a positive single-stranded RNA genome of approximately 7400

nucleotides (nt) (Brown & Pallansch 1995). The genome comprises a 5′

untranslated region (5′UTR), a long open reading frame (ORF) and a short

3′UTR followed by a polyadenylated (poly(A)) tail. The first uridine

monophosphate (UMP) of the 5′UTR is covalently linked to a small viral protein

VPg (viral protein of the genome) (Figure 1-1) (Lee et al. 1977).


4

“This image is unable to be reproduced online.

Please consult print copy held in the Swinburne Library”

Figure 1-1: Genome structure and polyprotein processing of the Picornaviridae.

Adapted from Fujita, Krishnakumar et al. (2007).

● indicates sites of cleavage by 2Apro; ▲ indicates sites of cleavage by 3CDpro and 3Cpro.


5

The RNA alone is infectious due to the 5′UTR contains the internal

ribosomal entry site (IRES) which allows viral protein translation in a cap-

independent manner (Belsham & Sonenberg 1996). The ORF is translated into a

single large polyprotein of approximately 2100 amino acids (aa) which is divided

into three regions (P1-P3). The polyprotein undergoes a series of processing

events, carried out by virus-encoded proteases, mainly 2A protease (2Apro) and

3CD/3C protease (3CDpro/3Cpro) (Toyoda et al. 1986). The maturation cleavage

of the polyprotein generates structural and non-structural viral proteins. Four

structural proteins, VP1, VP2, VP3 and VP4, encoded by the P1 region,

constitute the virus capsid. Proteins derived from the non-structural P2 (2Apro,

2B, 2BC, 2CATPase) and P3 (3A, 3AB, 3B, 3Cpro, 3CDpro, 3Dpol) regions are most

directly involved in virus replication and those structural and biochemical

changes which are observed within the infected cell (Shen et al. 2008).

1.2.2 Structure of viral particle

The three-dimensional structure of the viral particle has been determined

for a number of picornaviruses many of which are polioviruses. Crystal structure

of EV71 has been reported only recently (Plevka et al. 2012; Wang et al. 2012).

Even though it is very similar to that of other enteroviruses, some structural

details are specific for EV71. According to X-ray crystallography data the viral

capsid of picornaviruses exhibits a pentameric icosahedral structure and consists

of 60 repeating subunits (Hogle, Chow & Filman 1985). Each subunit is

composed of one of four structural proteins, VP1-VP4. Among those, VP4 is

entirely internal, while VP1, VP2 and VP3 are exposed on the surface of the viral

capsid (Figure 1-2, A).


6

A.

B.



Figure 1-2: Protein subunits within the picornavirus capsid.

A. Schematic representation of the picornavirus particle was adapted with some modifications from the Bioinformatics Resource Portal of the Swiss Institute of Bioinformatics (<http://viralzone.expasy.org/all_by_protein/97.html>). B. Diagram of the icosahedral asymmetric unit of EV71 capsid was adapted from Plevka et al. (2012). VP1 is shown in blue, VP2 - in green, VP3 - in red and VP4 - in yellow. Calcium ions and pocket factor are shown in magenta and orange, respectively. Structural features (“canyon”, “puff” and “knob”) of the capsid surface are indicated by dashed lines.


7

Five repeating subunits are arranged into a pentamer with a central vertex

formed by five molecules of VP1. The folding pattern of the VP1, VP2 and VP3

is remarkably similar and consists of a common structural motif, namely β-sheet

or “core”, which is flanked by two helices (Hogle, Chow & Filman 1985). Each

core is composed of 8 antiparallel strands, which form a distorted barrel. When

opened up and laid out flat, the β-sheet exhibits a “jelly-roll” structure, which is

termed Greek key motif (Figure 1-3, A) (Zhang & Kim 2000). The folding

pattern of VP1-VP3 can be summarised to a common structural model shown in

Figure 1-3, B.


8

A. The “jelly-roll” structure of VP1, VP2 and VP3 proteins of the Picornaviridae.

B. Common pattern of the VP1-VP3 structures.

Figure 1-3: Folding pattern of the structural proteins of PV.

Adapted from the web portal of the Department of Biology, University of Hamburg (<http://www.biologie.uni-hamburg.de/lehre/bza/virus/1hxs/1hxsm.htm>).


9

There are extensive interactions between structural proteins in the subunit

(Hogle, Chow & Filman 1985). The C-terminal part of VP1 wraps over VP3

protein on the outer surface of the viral capsid. The outer surface of VP2 is

flanked by VP1amino acid residues 207-237 and 271-295. The inter-subunit

contacts on the inner surface of the capsid are even more extensive and include

interactions between the N-terminus of VP3 and the base of the VP1 barrel, as

well as those formed between the N-terminal part of VP1 and the inner surface of

the VP3 barrel.

The β-sheets are internal to the structure of the protein and relatively rigid

while the loops are on the surface and more “floppy”. The VP1 loops near to the

fivefold axis form peaks protruding from the surface and surrounded by a

depression called a “canyon” (Reisdorph et al. 2003). The amino acid residues

within the canyon are invariant and interact with cell receptors providing the

attachment of the virus to the host cell (Colonno et al. 1988; He et al. 2000). The

amino acid residues on the rim of the canyon are variable and only these residues

can interact with antibody. Additionally, neutralizing antigenic sites were

suggested to contribute to the docking process of virions to cell receptors (Harber

et al. 1995). This hypothesis is supported by the fact that PV exists only in three

serotypes in spite of high pressure from the immune system and a genetically

unstable viral genome (Wimmer, Hellen & Cao 1993).

The VP1 loops of EV71 are smaller than in other enteroviruses, and, as a

result, the canyon is shallower (Plevka et al. 2012). It was suggested, that due to

this fact, the EV71 canyon cannot provide binding for cell receptors with

immunoglobulin-like fold. However, protrusions on the virus surface formed by

prominent loops of VP2 and VP3 and named “puff” and “knob”, serve as binding

sites for non-immunoglobulin-like receptors (Figure 1-2, B).

Another recent study suggested that the GH loop of EV71 VP1 might act

as an adaptor-sensor during virus attachment to the cell (Wang et al. 2012).

Interactions between cellular receptors and mature virus trigger rearrangement of

the adaptor-sensor region with the following conformational changes in viral

capsid and formation of the expanded uncoating particle. Structural studies of the


10

expanded particle revealed formation of the capsid channels opening at the base

of the canyon and at the 2-fold axis, which might allow viral RNA release.

Similar to other picornaviruses, conversion of EV71 to the expanded

particle is accompanied by the exit of the pocket-factor from the hydrophobic

pocket buried bellow the canyon floor (Figure 1-2, B) (Plevka et al. 2012; Wang

et al. 2012). Unlike other enteroviruses, the EV71 pocket-factor is partially

exposed to the solvent and interacts with polar residues on the canyon floor. It

was suggested that anti-EV71 compounds which bind into the pocket and inhibit

virus uncoating might require a hydrophilic head group in contrast to the

inhibitors designed for other enteroviruses (Plevka et al. 2012).

Since the virus capsid contains the binding sites for host cell receptors it

contributes significantly to the pathogenicity of the virus and is considered as one

of the main targets in antiviral drug development.

1.2.3 5′ untranslated region (5′UTR) of the Picrnaviridae

The 5′UTR is approximately 746 nucleotides in length. Within this region

there are seven to eight AUG codons upstream of the initiator AUG. Most of

these are poorly conserved between strains and do not appear to be recognized by

the translational machinery. Point mutations introduced at the first nucleotide of

the AUG codons of PV2 (Lansing strain) RNA resulted in viruses with a wt

plaque morphology except a mutant in AUG7 (Pelletier et al. 1988a). Mutation in

that codon produced a virus with a small plaque phenotype that suggested the

AUG7 may play a positive role in poliovirus RNA translation.

The 5′UTR of enteroviruses is predicted to contain extensive secondary

structure (Fig. 1-4) which is highly conserved even in the absence of high

sequence conservation (Belsham & Sonenberg 1996).


11



Figure 1-4: Secondary structure prediction for the 5′UTR of the Picornaviridae.

Adapted from Wimmer (1993).

The boundaries of the IRES are indicated by a dotted line, the polypyrimidine tract and AUG elements are represented by shaded and black rectangles, respectively.


12

Functionally, the 5′UTR of enteroviruses can be divided into two regions:

the 5′–terminal cloverleaf (nt 1 to 89) and the internal ribosome entry site (IRES,

nt 123 to 602). The cloverleaf structure was found to be important both for

translation and RNA replication (Gamarnik & Andino 1998; Parsley et al. 1997).

It carries overlapping cis-acting signals that control the switch between these two

processes via interaction with the cellular poly(rC)-binding proteins 1 and 2

(PCBP) or viral polymerase precursor, 3CD. The binding of the cellular PCBP to

stem-loop B of the cloverleaf facilitates viral translation up to 10-fold. In

contrast, interaction of the viral protein 3CD with the cloverleaf stem-loop D

represses translation and enhances negative-strand RNA synthesis (Andino et al.

1993; Gamarnik & Andino 1997; Vogt & Andino 2010). Substitutions within the

3CD binding site of the 5′UTR cloverleaf in the positive strand, but not negative

strand, disrupted the ribonucleoprotein complex, inhibited virus growth and

affected positive-strand RNA accumulation (Andino, Rieckhof & Baltimore

1990). The defect on positive-strand synthesis could be fully or partly a

consequence of a primary reduction in negative-strand synthesis (Gamarnik &

Andino 1998).

A large fragment (about 450 nucleotides) within the 5′UTR, which is

required for the internal ribosome entry, is IRES (Figure 1-4). This sequence

directs initiation of viral RNA translation in a cap-independent manner. Viral

RNA translation causes dramatic inhibition of host protein synthesis via

proteolytic degradation of the cap-binding protein complex and inhibition of

cellular mRNA translation (Belsham & Sonenberg 1996; Gan & Rhoads 1996;

Roberts, Seamons & Belsham 1998). Members of the Picornaviridae family

have 2 types of IRES: type I IRES (entero- and rhinoviruses) and type II IRES

(cardio-, aphthoviruses and hepatovirus). The core region of type I IRES consists

of the nucleotides between positions 134 and 585 (numbering according to PV2,

Lansing strain) (Nicholson et al. 1991). A 5′-end deletion extending to nucleotide

134 reduced translation four-fold, whereas further deletion to position 155, which

removed most of the stem-loop II, was deleterious for translation. Removal of the

stem-loop structure III, encompassing nucleotides 189 to 223, had only a small


13

effect on virus growth. In contrast, deletions within stem-loops IV and V

abolished viral RNA translation (Nicholson et al. 1991).

Two short motifs within the stem-loop IV structure, termed stem-loop A

and B, were first identified by Jackson et al. (1994). It is believed that those

motifs are involved in long-range tertiary interactions with helical regions (Pley,

Flaherty & McKay 1994). Modification of the GNRA consensus (where N

designates any nucleotide and R stands for purine) in stem-loop A of EMCV or

PV greatly reduced internal initiation of translation (Jackson et al. 1994). The

loop B motif is C rich, but its functional significance is unknown. A short single-

stranded sequence (443UCCUCC448) linking domains IV and V is essential for PV

IRES function. Mutations within nt 425-449 decreased in vitro RNA translation

up to 10 fold (Belov et al. 1995). All viable viruses with restored activity of the

RNA template demonstrated a reversion at position 444. Attempts to insert

restriction sites within UCCUCC sequence of PV yielded non-viable viruses

(Gromeier et al. 1999).

At the 3′ end of IRES element, all picornaviruses have a polypyrimidine

tract, first noted by Kuhn et al. (1990). A 3′ deletion in the poliovirus 5′UTR up

to the stem-loop VI had no effect on translation (Meerovitch, Nicholson &

Sonenberg 1991; Pelletier et al. 1988b). A further 3′ deletion including the stem-

loop VI reduced translation efficiency fivefold, whereas deletion extending on

the polypyrimidine tract abolished translation (Meerovitch, Nicholson &

Sonenberg 1991). Thus, the polypyrimidine tract was found to play a critical role

in viral proteins synthesis. The most important part of the polypyrimidine tract is

its 5′ half including nucleotides UUUC which are conserved amongst all

picornaviruses. Uridine residues in the middle and 3′ half do not play a crucial

role but nevertheless have a stimulatory effect in RNA translation (Nicholson et

al. 1991). There is a functional difference in the sequence requirement for the

polypyrimidine tract between different picornaviruses. For instance, modification

of the polypyrimidine tract sequence by partial substitutions with purines

(UUUCC to UAAAC) reduced the translational efficiency of PV1 (Mahoney

strain) to about 2% of wild type (Belsham & Sonenberg 1996). Point mutations


14

in the polypyrimidine tract (UUUCCUUU to UGUGGUUG) of foot-and-mouth

disease virus (FMDV) decreased the efficiency of RNA translation up to 5%

(Kuhn, Luz & Beck 1990). In contrast, a remarkable insensitivity of the

polypyrimidine tract to mutations has been demonstrated for cardioviruses

(Kaminski, Belsham & Jackson 1994). Substitution of all pyrimidines by purines

(mainly by A residues) reduced initiation efficiency by not more than 30-35%.

When two of the pyrimidines were left unchanged, the translation initiation was

decreased by only 10-15%.

Approximately 20 nt downstream from the polypyrimidine tract, there is

an AUG codon which is highly conserved in picornaviruses. In aphthoviruses

and cardioviruses it is the translation initiation codon. In rinoviruses and

enteroviruses the initiator AUG is located approximately 30 or 150 nucleotides

downstream, respectively. Although the upstream AUG codon (AUG7) of PVs is

not functional as an initiator, mutations in this codon were shown to result in

inhibition of translational efficiency (Meerovitch, Nicholson & Sonenberg 1991;

Pelletier et al. 1988a; Pilipenko et al. 1992a). Nucleotide substitutions on the

opposite side of the stem had no effect. That allowed concluding that the primary

nt sequence encompassing the AUG7, and not the secondary structure in this

region, is important for poliovirus replication (Meerovitch, Nicholson &

Sonenberg 1991).

Importance of the 20 nt fragment between the polypyrimidine tract and the

AUG7 was shown in PV2 (Lansing strain). A 13 nucleotides deletion reduced

translation significantly (Nicholson et al. 1991). The result was consistent with a

proposed mechanism of initiation of picornavirus RNA translation whereby

ribosome binds directly to the polypyrimidine tract and then may “scan” the

RNA to reach the authentic AUG initiator (Jackson, Howell & Kaminski 1990).

The 18S rRNA has at least 3 regions which are complementary to the 3′ end of

the polypyrimidine stretch and evolutionarily conserved among prokaryotes and

eukaryotes. Two of those regions are not base-paired and might interact with the

picornavirus mRNA (Nicholson et al. 1991). The mechanism by which the

ribosome misses the AUG7 and translocates to the start codon is unknown.


15

The 3′ terminus of the 5′UTR of enteroviruses, up to 150 bases upstream

from the initiation codon, is known as the spacer region. The role of this highly

variable region is not fully understood. It can be greatly modified and causes

little effect on the virus. An insertion of 72 nucleotides at position 702 in 5′UTR

of PV1 (Sabin strain) resulted in small-plaque phenotype mutants which easily

reverted to the parent phenotype (Kuge, Kawamura & Nomoto 1989). The

revertants showed lack of genomic sequences including all or a part of the

insertion or had point mutations. All substitutions occurred in the AUG triplet

existing at positions 56 to 58 of the insert. An insertion of the 72-nucleotide

sequence containing C at position 57 (AUG→ACG) resulted in viruses with the

parent phenotype. The data showed that the insertion of AUG codon was

harmful for virus replication whereas extending the spacer region up to 72

nucleotides did not affect virus growth in a tissue culture system. Another study

showed that deletion of the spacer region in PV1 chimera did not affect growth

phenotype of the virus (Gromeier et al. 1999).

The interaction between the IRES and host receptor proteins is an

important determinant of host specificity for virus replication. Artificially

induced mutations between nt 128 to 134 blocked virus replication at IRES-

dependent translation initiation in transgenic mice but have no effect on

replication in primate cells (Shiroki et al. 1997). Other studies showed that

mutations located within stem-loop V are responsible for viruses that failed to

replicate in neural tissue (Agol et al. 1989; La Monica & Racaniello 1989;

Malnou et al. 2002). Genetic determinants which correlated with the attenuating

phenotype of chimera PV1 in human neuroblastoma SK-N-MC cells, CD155

transgenic mice and Cynomolgus monkeys were mapped to the upper loop

sequences of domains V and VI (Gromeier et al. 1999). Replacing one or both of

those regions with the human rhinovirus type 2 (HRV2) IRES was sufficient

alone, without a contribution of the Sabin capsid attenuating determinants, to

create chimera viruses with a high degree of attenuation of neurovirulence.


16

1.2.4 Viral protein 4 (VP4) of the Picornaviridae

Viral protein 4 (VP4) of EV71 is composed of 208 amino acid residues. In

contrast to the compact structures of VP1-VP3, VP4 has a more extended

conformation. The only compact structure in VP4 is a two-stranded antiparallel

β-sheet near the NH2-terminus of the protein. The VP4 is only exposed to the

inner surface of the picornavirus particle and together with NH2-terminal parts of

VP1, VP2 and VP3 forms the interior surface of the viral capsid (Hogle, Chow &

Filman 1985). The most significant interaction of the inner surface is the

interaction between VP4 and NH2-terminus of VP3 at the fivefold axis. The NH2-

terminal parts of five VP3-subunits intertwine and form a five-stranded tube

which is flanked on its lower surface by β-sheets from five subunits of VP4. This

structure may direct pentamer formation and contributes significantly to its

stability once the pentamer has formed.

1.2.5 Viral proteins 2 and 3 (VP2, VP3) of the Picornaviridae

Viral protein 2 (VP2) and viral protein 3 (VP3) form a broad plateau at the

threefold axes of the picornavirus particle (Hogle, Chow & Filman 1985). The

plateau is broadened by two outward projections. The smaller projection is

composed of amino acid residues 53-69 of the VP3. The larger projection is

formed by residues 127 to 185 of the VP2 and is supported at its base by the

COOH-terminal part of the VP2 (residues 261-272), and by residues 207-237 and

271-295 of the VP1.

Amino acid residues 72 to 75 and 240 to 244 in VP2, and 75 to 81 and

196 to 206 in VP3 form loops which are exposed at the surface of the particle.

The VP2 protein of CBV3 was shown to contribute to the induction of cell

apoptosis (Henke et al. 2000).

Adaptive mutations at VP2-K149 of EV71 were identified to be important

for efficient virus replication in rodent cells (Arita et al. 2008; Chua et al. 2008;


17

Miyamura et al. 2010; Wang et al. 2004). However, it is unknown if those

mutations have a direct effect on viral interaction with host cell receptors, or if

they act in a receptor-independent manner.

1.2.6 Viral protein 1 (VP1) of the Picornaviridae

Viral protein 1 (VP1) of EV71 is composed of 297 amino acids. The

structure and folding pattern of VP1 have been extensively studied on

polioviruses. The loops of the five VP1 proteins have been shown to form peaks

at the fivefold axis of the viral capsid subunit (Hogle, Chow & Filman 1985).

Loops which are composed of amino acid residues 96-104 (BC loop), 142-152

(DE loop), and 245-251 (HI loop) are exposed at the summit of the peak. The

loop 96-104 is the most protruding from the surface and contains the trypsin-

sensitive site in the Sabin 1, Sabin 3 and Leon strains of PV.

The structure of the BC loop is defined by several interactions (Wien et al.

1997). The base of the loop is stabilized by a hydrogen bond between residues

Asp93, Leu104 and water. The sides of the loop are stabilized by a β-turn from

residues Ala96-Thr99 and a hydrogen bond between Thr99 and Lys101. In

addition, the BC loop makes non-polar interactions with His248 and Lys252

from the HI loop. Structural arrangement of the BC loop is to a large extent

provided by Pro in position 95. Its side chain makes a large number of van der

Waals contacts in the centre of a local hydrophobic core. Those interactions

involve Thr at position 99 at the top of the loop and Lys103 at the far side of the

loop. The substitution of Pro95 with Ser breaks the interaction between the BC

and HI loops and exposes surfaces of the viral capsid which are normally buried.

Those structural changes result in instability of the virion. Other residues which

tend to be tightly packed in a hydrophobic region of VP1 include Met158,

Val160, Val166, Ile239 and Ala241. Substitution at any of these positions very

likely interferes with tight hydrophobic packing and correlates with

compensating substitution(s) at other positions within this region. In addition,


18

this area is found immediately adjacent to a highly conserved sequence motif

amongst picornaviruses, Pro-X-Gly-Z-Pro (161-165 residues), with X and Z

being Pro and Ala in polioviruses and rhinoviruses. This sequence is located in

an interface between symmetry-related protomers and therefore probably plays a

role in virus assembly or disassembly. Mutations which introduce structural

changes and result in instability of the viral capsid are suggested to play a role in

virus adaptation and its ability to overcome defects in host cell receptors (Wien et

al. 1997).

The GH loop of the VP1 of polioviruses contains a highly conserved

amino acid triplet (GDS, position 225-227) which is located on the canyon rim

and participates in viral binding to host cells (Harber et al. 1995). D226A

mutation resulted in defective binding of PV1 to mouse cell lines expressing

poliovirus receptor (PVR). Residue D226 was suggested to form a salt bridge to

residue R172 of VP2 (Hogle, Chow & Filman 1985).

A consensus structural model for the EV71 VP1 was first developed by

Ranganathan et al. based primarily on the homologous structure of the bovine

enterovirus (Ranganathan et al. 2002). The amino acids positioned within the

hydrophobic pocket were determined to be identical or conservatively substituted

compared to the template. Conserved positions comprised I111, D112, M129,

F131, F135, F137, V192, M195, Y201 and N228 whereas I113, T114, F155,

V190, W203, M230 and F233 differed from the template but were conserved

among the eight EV71 strains taken for the analysis. The main structural feature

that distinguished VP1 of EV71 from the template structure was only the 5

additional residues EGTTN (positions 98-102) in the ‘BC loop’ between β-strand

B and β-strand C. This region is exposed in the pentameric capsid and has been

shown to affect neurovirulence in PV1 (Bouchard, Lam & Racaniello 1995). The

BC loop is completely conserved among EV71 strains except for position 98 (E

or K). The combination of these completely conserved and unique (compare to

other enterovirus sequences) amino acid positions within the segment 92-107 is

considered as an important immunogenic site in EV71 VP1 (Foo et al. 2008a;

Ranganathan et al. 2002).


19

Lal et al. (2006) reported that full-length VP1 protein of EV71 is capable

of self-association. The major domain involved in dimerization of the VP1 was

determined to consist of amino acids 66-132 of the protein. Additionally, the C-

terminal region between amino acids 132 and 297 was shown to play an

important role in increasing the strength of the VP1-VP1 interaction. The self-

association activity of the truncated protein spaning amino acids 66-297 was

equivalent to that of the full-length VP1. A deletion of residues 66-132 or 220-

297 caused a reduction in the self-association capability of VP1.

Ke et al. (Ke, Chen & Lin 2006) used the homology modelling technique

in order to construct the three-dimensional structure (3d-structure) of EV71 VP1

based on the structures of the template proteins 1EAH (VP1 of PV2 complexed

with antiviral agent Sch48973), 1PIV (VP1 of PV3 complexed with disoxaril)

and 1D4M (VP1 of CAV9 complexed with disoxaril). Although the amino acid

sequence identity between EV71 VP1 and the templates was 36-38% it was

possible to identify nine major β-strands composed of amino acid residues: 88-

95, 107-114, 125-129, 132-140, 149-156, 178-182, 189-195, 230-238 and 247-

262. Predicted structural model of VP1 revealed that the attachment site or

“binding pocket” for EV71 inhibitors, such as ligand WIN51711 or lig20, is

apparently formed by several β-strands similar to that observed in the template

1PIV. Most of these β-strands were identified approximately at the same genome

regions in EV71 and PV3. The binding pocket was found to be rather

hydrophobic and consisted of amino acid positions I111, I113, A133, F155,

P177, V192, M195 and M230 (Ke, Chen & Lin 2006; Ke & Lin 2006). The

binding pocket in the constructed protein model was conserved among VP1

proteins of bovine enterovirus, poliovirus, rhinovirus and EV71.

The structure of the attachment site in VP1 is important and is regarded as

the prime target for antiviral drug development that may prevent the attachment

of virus to the host cell. It was shown that even a single mutation of the VP1

sequence could result in virus resistance to antiviral compounds with capsid-

binding mechanism of activity. Shih et al. have demonstrated that an alteration of


20

the amino acid at position 192 (V192M) confers resistance to the inhibitory

effect of the imidazolidinone BPR0Z-194 (Shih et al. 2004b).

Interactions between capsid proteins of EV71, mainly VP1, and host cell

proteins contribute to pathogenicity of the virus. Therefore, the identification of

the host proteins involved in this type of interaction leads to a better

understanding of the potential pathogenic consequences of EV71 infection. By

screening of brain and bone marrow cDNA libraries, Yeo et al. (2007)

discovered three human proteins with strong binding affinity to VP1 of EV71.

They were ornithine decarboxylase (ODC1) and KIAA0697 from the brain

cDNA library, and gene trap repeat (GTAR) protein from the bone marrow

cDNA library.

ODC1 is an enzyme in polyamine biosynthesis. During EV71 infection,

interaction between VP1 and ODC1 diminishes transcription and translation of

ODC1 that adversely affects polyamine biosynthesis and macrophage cytotoxic

activity (Yeo & Chow 2007). ODC1–VP1 complexes displayed a cytoplasmic,

mainly perinuclear localization.

KIAA0697 gene was originally identified as a cDNA clone derived from

the human brain (Ishikawa et al. 1998). However, RT-PCR analysis showed that

KIAA0697 gene is expressed at similar levels in all 10 different tissues taken into

analysis: heart, brain, lung, liver, skeletal muscle, kidney, pancreas, spleen, testis

and ovary. The polypeptide KIAA0697 used by Yeo and Chow (2007) in

experiments on binding affinity to EV71 VP1 contained only a partial sequence

(571bp) of KIAA0697 gene including only a half of the ankyrin motif. The

polypeptide interacted with VP1 predominantly in cytoplasm. In contrast,

GTAR–VP1 interaction displayed nuclear localization, including around the

nucleolus. GTAR protein is known as a product of the KIAA0697 gene. The

interacting GTAR polypeptide contained six and a half repeated ankyrin motifs.

Therefore, the authors assumed that multiple ankyrin repeats may be necessary

for the complex to enter the nucleus (Yeo & Chow 2007). Ankyrin repeats are

the most common protein-protein interactions motifs in known proteins. They

were found to interact with the L1 vertebrate cell adhesion molecule and


21

disruption of the binding resulted in the CRASH neurological syndrome (corpus

callosum hypoplasia, retardation, adducted thumbs, spasticity and

hydrocephalus) (Needham, Thelen & Maness 2001). Disease manifestation in

some patients with EV71-related acute flaccid paralysis during the HFMD

outbreak in Taiwan (1998) was very similar to CRASH symptoms (Chen et al.

2001; Yeo & Chow 2007).

Additionally, the VP1 sequence contains two potential monopartite

nuclear localization signals (NLS): RRK (amino acids 120-122) and KHVR

(amino acids 256-259). NLS were found in a number of viral proteins including

VP1 of the simian virus 40 (KRK and KKPK motifs), and VP3 (or VP2) of avian

polyomavirus (Ishii et al. 1996; Johne & Muller 2004). The NLS were shown to

allow transport of the polyomavirus capsid protein complexes from cytoplasm to

nucleus that resulted in formation of virus-like particles (Johne & Muller 2004).

The mechanism by which EV71 VP1 enters the nucleus is unclear. It has

been suggested that its translocation from the cytoplasm or its translation in the

nucleus are possible (Yeo & Chow 2007).

Interactions between EV71 VP1 protein and host proteins mediating the

virus entry into the cell are being discussed in section 1.4.2.

1.2.7 Viral 2A protease (2Apro) of the Picornaviridae

Viral 2A protease (2Apro) of Picornaviridae is required during processing

of the viral polyprotein. It cleaves at two sites: first, at the C-terminus of the

capsid protein precursor P1 and, second, within the 3CD fragment to generate the

3C protease and the 3D polymerase (Yu & Lloyd 1991).

The 2Apro plays a significant role in the initiation of viral RNA translation.

The 2Apro of enteroviruses cleaves the eukaryotic initiation factor 4G (eIF4G).

That in turn severely restricts cap-dependent synthesis of host cell proteins, but

permits cap-independent initiation of translation to proceed from the IRES in


22

viral RNAs (Belsham & Sonenberg 1996; Goldstaub et al. 2000; Thompson &

Sarnow 2003).

The 2Apro of EV71 was shown to induce apoptotic cell death by cleavage

of the cap-binding complex (eIF4G), cellular DNA and by degradation of

poly(ADP-ribose) polymerase, which is essential for DNA repairing (Kuo et al.

2002). Induction of apoptosis has been demonstrated to be an important

component of virus-induced cell damage in the CNS of infected mice (Girard et

al. 1999; Oberhaus et al. 1997).

Molecular-genetic and biochemical studies of PV 2Apro showed its

similarity to the trypsin-like serine protease family (Yu & Lloyd 1991). A

catalytic triad identified in the 2Apro displays identity among a number of the

picornaviruses studied (PV, EV71, CBV1, CBV3, CBV4, HRV2, HRV14) (Kuo

et al. 2002). The catalytic triad comprises amino acids residues H20, D38 and

C109 (Yu & Lloyd 1991). Site-directed mutagenesis revealed that D38 could be

replaced with glutamic acid with no effect on the autocatalytic function of the

enzyme. Amino acid residues Y88, Y89 and T124 have been suggested to lie in

loops involved in substrate binding.

In addition to proteolytic activity, the 2Apro of poliovirus was shown to be

involved in viral RNA replication (Li et al. 2001). The stimulation of RNA

replication is provided by a negatively charged cluster of amino acids E/D

E/DEAME at the C-terminus of 2Apro. The motif was found to be conserved

among all enteroviruses. Deletion of the EEAME stretch from poliovirus 2Apro

was lethal but did not affect the proteolytic activity of the enzyme. In HRV2, the

C-terminus of 2Apro is composed of EEQ. Addition of the EAME sequence to the

2Apro of HRV2 resulting in an EEEAMEQ C-terminus, stimulated RNA

replication of a mutant HRV2 by 100 fold. Since silent mutations in C-terminal

sequence of 2Apro coding region did not affect virus proliferation, it was

concluded that the C-terminal motif is essential in viral replication at the protein

level.


23

1.2.8 Viral 2B protein (2B) of the Picornaviridae

The 2B protein of enteroviruses consists of approximately 100 amino

acids. It is classified as a viroporin and functionally involved in the alteration of

the plasma membrane permeability of the infected cell (Agirre et al. 2002;

Madan et al. 2007). The 2B is produced intracellularly, localizes at the

endoplasmic reticulum (ER) and the Golgi apparatus, and acts as a pore-forming

protein during late viral infection (Martínez-Gil et al. 2011).

The 2B protein has two highly hydrophobic regions. The first stretch

includes amino acids 32-55 (in PV) and is known as the amphipathic domain

(Agirre et al. 2002). The second region constitutes amino acids 61-81 (in PV) and

represents the transmembrane domain (TMD). Both domains combine in a

monomer via the reverting loop and form a hairpin-like structure. According to a

proposed model, each pore formed by the picornavirus 2B protein consists of

four self-aggregated hairpin monomers with their amphipathic domains

constituting the hydrophilic pore and their TMDs spanning the bilayer and acting

as a transmembrane anchor (Nieva et al. 2003). The pore lining motif within the

amphipathic domain consists of three positively charged lysines followed by a

polar residue serine (Patargias et al. 2009). The side chains of those residues are

oriented toward the aqueous lumen of the pore, while hydrophobic residues face

the lipid bilayer of the membrane. An estimated diameter of the pore is 6Å which

allows transport of solutes with molecular weight under 1000 Da. As an example,

it has been shown that expression of 2B protein of CBV3 causes the influx of

extracellular Ca2+ and releases Ca2+ from the endoplasmic reticulum, which

results in disruption of the membrane and release of viral particles (van

Kuppeveld et al. 1997a).

Additionally, there is evidence that 2B protein of PV can trigger cell

apoptosis via the mitochondrial pathway (Madan et al. 2010). The amphipathic

domain of 2B is used by the pore-forming peptide P3 to translocate through the

plasma membrane and target mitochondria.


24

Mutations altering either the amphipathic domain or TMD resulted in a

defect in membrane permeability and virus replication (van Kuppeveld et al.

1997a; van Kuppeveld et al. 1997b).

1.2.9 Viral 2C protein (2CATPase) of the Picornaviridae

The 2C non-structural protein of EV71 comprises 329 amino acid

residues. In Picornaviridae it has an ATP-ase activity essential for the synthesis

of the negative-strand RNA (Barton & Flanegan 1997).

It was proposed that 2CATPase may coordinate the formation of

membranous ribonucleoprotein complexes (RNP) from the appropriate proteins,

CRE (2C), 5′UTR cloverleaf and 3′UTR of the viral RNA (Steil & Barton 2009).

In Enteroviruses the 2C gene contains a cis-acting replication element

(CRE) (Brown et al. 2005; Goodfellow et al. 2000; Paul et al. 2000b) (Figure 1-

5).


25



Figure 1-5: Location and secondary structure of PV1 CRE.

Adapted from Steil and Barton (2009).


26

The CRE is predicted to fold into a stem with a 14 base loop where the 1st,

5th, 6th, 7th and 14th residues are purines. The 5th and 6th adenines of the conserved

5′-A5A6A7C8A9-3′ motif serve as template nucleotides during uridylylation of

VPg by RNA-dependent RNA polymerase, 3Dpol (Paul et al. 2003b). Silent

mutations that destroyed the CRE structure within polioviral RNA prevented

positive-strand RNA replication but allowed accumulating negative-strand RNA

at a wild-type level. This indicated that uridylylated form of VPg (VPgpUpUOH)

was absolutely required for the positive-strand RNA synthesis of poliovirus

(Morasco et al. 2003; Murray & Barton 2003). Similar results have been received

for the coxsackievirus B3 (CVB3) (van Ooij et al. 2006b). Additionally, a

substitution of the templating A5 residue with a guanine abolished both the

uridylylation reaction and negative-strand RNA synthesis of CVB3 in vitro.

1.2.10 Viral 3A protein of the Picornaviridae

The 3A protein is a cleavage product of the 3AB precursor by viral 3C and

3CD proteases. The 3A protein itself, and when it is within the 3AB precursor,

exhibits different function during viral replication. The 3AB precursor was

shown to be necessary for interaction of the 3CDpro with the 5′ cloverleaf and 3′

terminus of the picornaviral RNA (Harris et al. 1994; Xiang et al. 1995).

Additionally, the 3AB accelerates autoprocessing of the 3CDpro which yields

3Cpro and 3Dpol (Molla et al. 1994). This cleavage activates 3Dpol activity and

stimulates RNA synthesis on a poly(A) template nearly 100-fold (Lama et al.

1994; Plotch & Palant 1995). The interaction between the 3AB and 3Dpol happen

through the sequences located in the 3B protein while the 3A allows anchoring to

the membrane through its hydrophobic C-terminus (amino acids 1-13) (Hope,

Diamond & Kirkegaard 1997; Towner, Ho & Semler 1996; Xiang et al. 1998).

This interaction results in formation of non-transmembrane (non-TM)

complexes. In this non-TM configuration the 3AB itself serves as a template in

proteolysis by the 3CDpro (Lama et al. 1994). After release of the 3B protein


27

(known as VPg) off the 3AB precursor, the 3A protein can exist both in non-TM

and TM forms (Fujita et al. 2007).

The 3A protein consists of approximately 90 amino acids, which form two

main domains: a soluble cytosolic (N-terminus, aa 1-58) and hydrophobic (C-

terminus, aa 59-81) (Fujita et al. 2007). It has been shown that 3A inhibits

secretion of the cellular cytokines (interleukin-6 (IL-6), IL-8, beta interferon)

which results in reduction of the native immune response and inflammation

caused by the virus (Dodd, Giddings & Kirkegaard 2001). Additionally, 3A

inhibits membrane trafficking between the ER and Golgi complex by binding a

cellular regulatory protein, the guanine nucleotide exchange factor GBF1 (Dodd,

Giddings & Kirkegaard 2001; Teterina et al. 2011). Interestingly, this function

was observed for PV and CVB3 3A proteins, but not for HRV,

encephalomyocarditis virus, FMDV or hepatitis A virus (HAV) (Wessels et al.

2006). The inhibitory function of 3A was first related to its cytosolic domain, as

mutagenesis within the first 16 N-terminal amino acids could abolish the

function, while 3A protein amounts remained at the wild-type level (Doedens,

Giddings & Kirkegaard 1997). Later studies showed that proline residues within

the N-terminus of 3A were of prime importance in inhibition of ER-to-Golgi

transport (Wessels et al. 2005). Replacement of the N-terminus of HRV 3A with

the related sequence of CVB3 3A allowed gaining the GBF1-binding ability by

the chimeric 3A protein (Wessels et al. 2006).

Except for those functions, there is evidence that 3A protein interacts with

2B viroporin and 2CATPase during viral replication. Mutations introduced within

the N-terminal amphipathic helix of 2CATPase resulted in compensatory changes

within either 3A or 2B (Teterina et al. 2006).


28

1.2.11 Viral 3B protein of the Picornaviridae

Viral protein 3B is 22 amino acids long. It is also known as the Viral

Protein of the genome (VPg). It is covalently linked by its tyrosine hydroxyl to

the 5′ terminal uridine of viral RNA via a phosphodiester (Ambros & Baltimore

1978; Lee et al. 1977) (Figure 1-6).



Figure 1-6: VPg is covalently linked to the viral genome.

Adapted from Steil and Barton (2009).


29

The VPg and its uridylylated form (VPgpUpUOH) function as primers for

negative and positive-strand RNA synthesis (Morasco et al. 2003; Paul et al.

1998; Paul 2002). It has been proposed, that CRE(2C)-dependent VPg

uridylylation catalysed by the 3Dpol initiates only plus strand RNA synthesis

(Murray & Barton 2003; Paul et al. 2003a). After the uridylylation, the

VPgpUpUOH probably remains associated with the 3Dpol molecule and, via

unknown mechanisms, is translocated from the CRE(2C) RNA template to the 3′

terminus of negative-strand RNA. Priming of positive-strand RNA replication

occurs between the uridine residues in VPgpUpUOH and two adenosine residues

at the 3′ end of negative-strand RNA (Sharma, O'Donnell & Flanegan 2005).

To initiate minus strand RNA synthesis, the poly(A) tail of the viral RNA

is required for VPg uridylylation. The VPgpUpUOH cannot prime the positive-

strand RNA if the adenosine residues are internal from the 3′ end of negative-

strand RNA (Herold & Andino 2000).

1.2.12 Viral 3C protease (3Cpro) of the Picornaviridae

Viral 3C protease (3Cpro) possesses multiple activities. One important

function is that it accomplishes cleavage of Gln-Gly pairs in P2-P3 regions

during proteolytic maturation of the viral polyprotein (Kitamura et al. 1981;

Pallansch et al. 1984). The catalytic motif of picornavirus 3Cpro was reported to

comprise amino acids H40, E71 and C147 (Hammerle, Hellen & Wimmer 1991).

Changing two of them (H40G and C147G) in the EV71 3Cpro sequence resulted

in the loss of proteolytic activity by the enzyme and inhibition of apoptosis (Li et

al. 2002). Another highly conserved position between different picornaviruses,

namely Asp85, was suggested not to form part of the active site of 3Cpro, but to

be important for the specific recognition of cleavage sites within P2 (Kean et al.

1991). Site-directed mutagenesis at this position (D85E) led to ts phenotype of

PV1.


30

Similar to 3Cpro of PV and HRV, the 3C protein of EV71 was

demonstrated to interact specifically with the 5′UTR of the viral RNA (Shih et al.

2004a). Two regions were found to be essential for RNA binding, namely

KFRDI (amino acid positions 82-86) and VGK (positions 154-156). The amino

acid substitution R84Q completely abrogated RNA-binding ability of EV71

3Cpro. Other mutations within the KFRDI motif (K82Q, D85N, and I86A)

resulted in a significant reduction of the RNA binding by the protein.

Surprisingly, the R84K mutant showed only a slight defect when compared to

wild-type 3Cpro. Single amino acid substitutions in the VGK binding site (V154T,

V154S, G155A and K156Q) also strongly reduced RNA-binding activity.

In spite of the catalytic and RNA-binding domains of the 3Cpro being

structurally distinct, certain mutations within the RNA-binding sequences are

shown to interfere with the proteolytic activity of the enzyme. The cleavage

activity of 3Cpro was completely eliminated by the deletion of the KFRDI site or

substitution at position 86 (I86A). The G155A and K156Q mutants displayed

lower proteolytic activity when compared to the wild-type protease. In contrast,

K82Q, R84Q, R84K, D85N, V154T and V154S mutants possessed cleavage

activities equal to that of the wild-type enzyme (Shih et al. 2004a).

On the other hand, mutations within the catalytic site of 3Cpro did not

influence the RNA-binding ability of the protein. The generated mutant 3Cpro,

H40D or C147S, resulted in loss of the proteolytic activity both in vitro and in

vivo. However, both mutant proteins retained the ability of viral RNA-binding

(Shih et al. 2004a).

The model structure of EV71 3Cpro was built by homology modelling

using the X-ray structure of HRV2 3C as a template (Shih et al. 2004a). Structure

analysis showed that the catalytic triad was located at the interface of KFRDI and

VGK domains of the enzyme (Figure 1-7).


31



Figure 1-7: Three-dimensional model of EV71 3Cpro.

Adapted from Shih, Chiang et al. (2004a)

The catalytic motif (H40, E71, and C147) is located between the KFRDI and VGK structural domains of the enzyme. The side chains of the catalytic triad atoms are in ball-and-stick representation.


32

The 3Cpro plays an important role in pathogenesis of the EV71 infection.

Degradation of cellular DNA in nucleosomes and generation of apoptotic bodies

were observed in the human glioblastoma SF268 cell line as result of the

transient expression of the wild-type 3Cpro of EV71 (Li et al. 2002). The 3Cpro

triggered an activation of caspases, which are proteases in the ICE (interleukin-1-

β-converting enzymes) family. Caspases, in turn, cleaved (ADP-ribose)

polymerase and caused apoptotic cell death. The cleavage took place within 24 h

after 3C-transfection and could be blocked by the caspase inhibitors.

The 3Cpro sequence of enteroviruses is distinct from that of mammalian

proteases. Therefore, 3Cpro is considered to be a good molecular target for

developing anti-EV71 agents. Compounds which target both the catalytic domain

and RNA-binding regions of the enzyme, potentially, may offer higher efficacy

(Shih et al. 2004a).

1.2.13 Viral 3D polymerase (3Dpol) of the Picornaviridae

The 3D region of the EV71 genome encodes the 3D viral polymerase

(3Dpol). The enzyme consists of 462 amino acids and exhibits RNA-dependent

RNA polymerase (RDRP) activity. The 3Dpol is responsible for the synthesis of

both plus- and minus-strand viral RNA. The 3Dpol activities include RNA

binding, NTP binding, RNA strand displacement, template-dependant elongation

and terminal transferase (TNTase) activity (Neufeld et al. 1994).

Genetic studies indicate that despite wide variations among viruses in

sequences of their structural proteins, the polymerase sequences have regions

with a high level of conservation. Kamer and Argos (1984) first reported YGDD

span, which was found common both in RDRP and RNA-dependent DNA

polymerase (RDDP) sequences. Four years later this similarity was expended to

the DNA-dependent DNA polymerases (DDDP) (Argos 1988). Later Poch O. et

al. (1989) demonstrated that there are at least 4 conserved motifs, including


33

YGDD span, which are common in the sequences of all RDRP encoded by plus-,

minus- and double-strand RNA viruses.

The YGDD span (positions 327-330 in PV1) was found to be flanked by

several hydrophobic amino acids on each side. Consistent homology in the 14-

residue sequence motif containing this span as a core region was found among

plant, animal and bacterial viruses. It suggested that the YGDD span represents

an active site or recognition region for the polymerases (Kamer & Argos 1984).

The aspartates were proposed to be important in polymerase function by acting

directly in catalysis and/or by binding magnesium while tyrosine may possibly

act in phosphate binding (Argos 1988). A substitution of the aspartates with

alanine abolished RNA synthesis and TNTase activity of RDRP (Ranjith-Kumar

et al. 2001).

Four highly conserved motifs (A, B, C and D) were found in RDR/RDD

polymerases encoded by retroviruses, viral and non-viral retroposons, plus-,

minus- and double-strand RNA viruses (Poch et al. 1989). The motifs form a

large domain of 120 amino acids with 4 strictly and 18 conservatively maintained

residues. The high level of conservation within these motifs may indicate their

crucial importance as functional units after proper folding of the protein. For

instance, invariant Asp residue (position 233 in PV1 3Dpol) of motif A and the

first invariant Asp residue (position 329 in PV1 3Dpol) of motif C were found to

be critical for polymerase activity of the reverse transcriptase of HIV1 (Larder et

al. 1987). Site-directed mutagenesis on these positions totally destroyed the

polymerase activity. Within the other mutated amino acids falling in the

conserved motifs, drastic loss of activity was observed when Tyr residue

(position 327 in PV1 3Dpol) of motif C was mutated. These site-directed

mutagenesis experiments did not involve invariant Gly (position 290 in PV1

3Dpol) of motifs B and Lys (position 360 in PV1 3Dpol) residue of motif D.

However, another study showed that insertion of amino acids at position 3 or 10

of motif B induced the loss of reverse transcriptase activity (Hizi, Barber &

Hughes 1989; Hizi, McGill & Hughes 1988). The key role of the Gly (position

328 in PV1 3Dpol) of motif C was shown in experiments with Qβ coliphage


34

(Inokuchi & Hirashima 1987). Substitutions of the Gly by Ala, Ser, Pro, Met or

Val residues totally suppressed phage RNA synthesis.

Another comparison of 46 amino acid sequences of RDRP of 30 positive-

strand RNA viruses revealed 8 distinct blocks of amino acid residues which

could be considered conserved motifs (Koonin 1991). Motifs IV, V and VI

corresponded to motifs A, B and C which had been described previously (Poch et

al. 1989). Of 380 positions aligned only 5 amino acids were invariant in all

sequences and 1 of them was newly identified: Lys (position 159 in PV1 3Dpol).

Several residues were highly conserved, being substituted in only a few

sequences and usually by functionally similar residues. These newly identified

positions were: Ser/Thr/Met289, Ser/Thr299, Phe/Ser/Tyr/Ile/Val374.

Additionally several amino acids residues were specifically conserved in the

group which included PV and FMDV. These residues were Glu/Ala161,

Lys/Arg167, Arg174, Gly211, Ser/Thr235, Gly/Val/Ile/Ser286 and Lys/Ser376.

The crystal structure of RDRP determined from a number of

picornaviruses, such as PV, HRV and FMDV, displays a similar overall fold. The

first crystal structure of PV 3Dpol determined by Hansen et al. was incomplete

due to missing N-terminus (Hansen, Long & Schultz 1997). Several years later

Thompson and Peersen (2004) described a complete crystal structure of the

enzyme (Figure 1-8). Kok, CC and McMinn, P (2008) summarised both data

sets.


35

A.



B.



Figure 1-8: Structure of the PV 3Dpol.

Adapted from Thomson and Peersen (2004).

A. Top view of the 3Dpol. B. Bar representation of the 3Dpol sequence with structural elements highlighted according to picture A. The “right hand” analogy is used to describe a structure of nucleic acid polymerases. Based on this model PV 3Dpol consists of several sub-domains: the “palm”, “thumb” and “fingers” (Hansen, Long & Schultz 1997).


36

The “palm” sub-domain includes 4 amino acids sequence motifs (from A

to D) that are highly conserved among polymerases. These motifs form the core

region of the “palm”. The active site, namely GDD span, which is found in all

RDRP, locates in motif C. The fifth conserved motif (E) lies between the “palm”

and “thumb” and is a unique feature of RNA-dependent polymerases. During the

polymerisation process amino acids of motif E interact with the nascent strand.

The “thumb” sub-domain is encoded by the 3'terminus of the 3Dpol gene.

Its structure mostly consists of α-helices with a β-strand at the N-terminal end

(Hansen, Long & Schultz 1997).

The “fingers” sub-domain (“index”, “pinky”, “ring” and “middle”)

consists of amino-acid sequences which are tightly intertwined. Interactions

between the “thumb” and “finger” sub-domains form structures which encircle

the active GDD site and create an NTP entry tunnel at the rear of the polymerase

(Thompson & Peersen 2004).

It was demonstrated that purified 3Dpol of PV can prime the RNA

synthesis in vitro. When inside the cell, the polymerase molecules oligomerize

into lattice structures or replication complexes bound to the membrane by the

viral protein 3AB (Lyle et al. 2002). Interaction between 3Dpol molecules itself

forms Interface I which involves residues from the front of the thumb subdomain

(Leu446, Arg455, Arg456) of one molecule, and residues from the back of the

palm subdomain (Asp339, Ser341, Asp349) of the next molecule (Hansen, Long

& Schultz 1997; Pathak et al. 2002). The membrane-bound replication

complexes provide more efficient RNA synthesis and allow recombination

events during viral replication due to proximity of 3Dpol molecules (Egger &

Bienz 2002).

Replication of the picornaviruses occurs in the cytoplasm of the host cell;

however, it inhibits all three host cell transcription systems, which are distinctly

nuclear functions. The inhibition of the host cell RNA polymerases I, II and III is

associated with a cleavage of a number of cellular factors by viral 3Cpro

(Weidman et al. 2003). The 3Cpro does not contain NLS and enters the nuclei of

infected cells in the form of the 3CD precursor in 2-4 h post infection (Amineva


37

et al. 2004; Sharma, Raychaudhuri & Dasgupta 2004). Several potential NLS

have been described within the 3Dpol of picornaviruses. KKKRD motif (amino

acid positions 125-129) was mapped within the PV 3D protein. This sequence is

highly conserved among enteroviruses. Alanine substitutions of the NLS within

PV 3Dpol were found to be lethal for the virus (Diamond & Kirkegaard 1994).

Another potential NLS which is similar to the NLS of yeast ribosomal proteins

was mapped near the N terminus of 3Dpol of the rhinovirus: PnKTKLnPS

(Amineva et al. 2004). That region is conserved among rhino-, polio- and

enteroviruses. In addition, the amino acids 167-174 (KIKKGKSR motif) were

identified as a potential NLS within the 3Dpol of EV71 (Yeo & Chow 2007).

The most critical amino acid positions within the 3Dpol are summarised in

Table 1-1.


38

Position* Function Reference G1, G64, G239, G241

The terminal amino group of G1 forms hydrogen bonds with the carbonyl oxygens of G64, G239 and G241. The interaction buries the essential D238 into the active site of the 3Dpol. Deletion of G1 inactivates the polymerase. The G64S mutation increases fidelity of the polymerisation process and results in a ribavirin resistant virus.

(Pfeiffer & Kirkegaard 2003; Thompson & Peersen 2004)

D233 Coordinates two catalytically necessary Mg2+ ions towards the catalytic center. Interacts with a highly conserved D328. Mutations on this position totally inactivate the polymerase.

(Gong & Peersen 2010)

D238 Conserved in RDRP. In RDDP (retroviruses) there is Y at this position. During the polymerisation process, it selects a nucleotide with a correct sugar and correct base. The backbone of the residue interacts with the 3′-OH of the ribose. Substitutions at this position abolish the enzymatic activity.

(Gohara, Arnold & Cameron 2004; Gohara et al. 2000; Thompson & Peersen 2004)

S288, T293 Form hydrogen bonds with the side chain of the D238 and, hence, stabilize its position in the nucleotide-binding pocket.

(Gohara, Arnold & Cameron 2004)

N297 Conserved in RDRP. In RDDP (retroviruses) there is F at this position. It selects for the 2′-OH group of the incoming ribo- (in RDRP) or desoxyribonucleotides (in RDDP) during the polymerization process. 3Dpol mutants on this position exhibit reduced catalytic activity.

(Gohara, Arnold & Cameron 2004; Gohara et al. 2000)

D328, D329

Both aspartates are highly conserved in RDRPs. They are positioned in the core of the 3Dpol and interact with D233. Mutation on D328 results in an inactive polymerase. Mutation on D329 alters metal specificity.

(Jablonski, Luo & Morrow 1991)

Table 1-1: Critical amino acid positions of the 3Dpol.

* amino acid numbering is in accordance with their positions in 3Dpol of PV1


39

1.2.14 3′ untranslated region (3′UTR) of the Picornaviridae

The 3′UTR of picornaviruses has been proposed to play a significant role

in minus strand RNA replication. There have been no studies conducted on EV71

3′UTR. In polioviral genome, the 3′UTR was shown to form a pseudoknot

structure with 3Dpol (Jacobson, Konings & Sarnow 1993). Disruption of this

structure by an 8-nucleotide insertion resulted in ts mutant virus (Sarnow,

Bernstein & Baltimore 1986). However, mutants with 2- or 10-nucleotide

insertions at the same site exhibited wild-type growth phenotypes.

According to the proposed structure by Pilipenko et al. (1992b; 1996), the

3′UTR consists of X and Y stem-loops with a tertiary “kissing” interaction

between them. The destabilization of this interaction due to site-directed

mutagenesis resulted in a severe suppression of the viral RNA synthesis, but the

mutant transcripts were infectious. The X-Y structure represents the oriR

replicative cis-element, which controls the length of the poly(A) tail during

positive strand RNA synthesis (van Ooij et al. 2006a).

Complete deletion of the 3′UTR from a full-length PV cDNA construct

was shown to have a negative effect on viral replication (Todd et al. 1997).

Transfection of HeLa cells with RNA transcripts missing the 3′UTR showed a

long delay before the detection of cytopathic effects (CPE) and viral RNA

accumulation (Brown et al. 2005). However, subsequent infections with the

recovered mutant virus resulted only in a moderate defect in RNA synthesis.

Further analysis of the recovered mutant virus suggested that the original severe

defect in viral replication was due to non-viral sequences in RNA transcripts

rather than the 3′UTR deletion. Additionally, the RNA replication defect was

more prominent in the SK-N-SH cells, which are limited in the cellular factors

required by the virus to replicate or to restore the original 3′-end (Brown et al.

2004).


40

1.2.15 poly(A) tail of the Picornaviridae

The genomic RNA of Picornaviruses is tailed with a 3′-poly(A) tract of

approximately 80-90 nucleotides in length (van Ooij et al. 2006a). The poly(A)

tail is genetically coded by a 5′-poly(U) tract of the negative strand RNA (Larsen

et al. 1980). The 5′-poly(U) consists of approximately 20 nucleotides and,

according to the proposed reiterative hypothesis, serves as a template in snap-

back synthesis of a longer poly(A) tail by the viral RDRP. The process is

controlled by the oriR, a cis-element located in the 3′UTR of the positive strand

RNA, immediately upstream of the poly(A) (van Ooij et al. 2006a). Another

possible mechanism of the poly(A) tail elongation is a non-template

polyadenylation catalysed by a cellular poly(A) polymerase or a terminal

adenylyl transferase (TATase). A TATase activity was demonstrated for RDRP

of some viruses including poliovirus (Neufeld et al. 1994; Ranjith-Kumar et al.

2001).

The poly(A) tail is essential for efficient viral replication. It interacts with

the 5′ cloverleaf RNA structure via the poly(A) binding protein (PABP). The

interaction results in formation of a circular RNP complex which is required for

initiation of negative strand RNA synthesis (Herold & Andino 2001). Viral RNA

with the poly(A) tail of less than 20 nucleotides in length is replicated less

efficiently due to dissociation from the PABP (Silvestri et al. 2006).

Experiments in a cell-free system demonstrated that the poly(A) tail

stimulates translation of picornaviral RNA (Bergamini, Preiss & Hentze 2000).

Increase (from 3 to 10 folds) in translation varies between three types of IRESs

probably due to different mechanisms of their interaction with host cell proteins,

such as eIF4GI, eI4GII and PABP.


41

1.3 Phylogeny of EV71 and its relationships to other human

enteroviruses

Genome analysis of EV71 showed that it belongs to the genus Enterovirus

of the family Picornaviridae (Brown & Pallansch 1995; Schmidt, Lennette & Ho

1974). Traditionally, human enteroviruses were grouped according to their

serotypic identity in neutralization assay with serotype-specific antisera. By the

end of the 1990's, sixty six immunologically distinct serotypes of human

enteroviruses had been identified (Muir et al. 1998). Introduction of molecular

approaches such as PCR and sequencing have facilitated the diagnosis of

enterovirus associated infections and become the most predominant techniques

used for typing of enteroviruses (Oberste et al. 1999a; Poyry et al. 1996; Santti,

Vainionpaa & Hyypia 1999). Hence, the classification of human enteroviruses

was revised and now consists of four species named Human Enterovirus species

A, B, C and D (Table 1-2) (<http://www.ictvonline.org).


42

Species Members Human Enterovirus A (HEV-A)

● CAV: 2-8, 10, 12, 14, 16; ● EV: 71, 76, 89-91

Human Enterovirus B (HEV-B)

● CAV: 9 ● CBV: 1-6 ● ECV: 1-7, 9, 11-21, 24-27, 29-33 ● EV: 69, 73-75, 77-88, 100-101

Human Enterovirus C (HEV-C)

● PV: 1-3 ● CAV: 1, 11, 13, 17, 19-22, 24

Human Enterovirus D (HEV-D)

● EV: 68, 70

Table 1-2: Molecular classification of Human enteroviruses.

Adapted with modifications from Khetsuriani, Lamonte-Fowlkes et al. (2006).


43

Molecular studies of the P1 capsid region of EV71 strains revealed that

they have only 46% amino acid identity with the poliovirus strains (Brown &

Pallansch 1995). The EV71 has a closest genetic relationship to CAV16, and

together with other non-polio enteroviruses (NPEVs) such as CAV7 and CAV14

forms a distinct genetic sub-genogroup within cluster Human Enterovirus species

A (Oberste et al. 1999b). The prototype strains of CAV16 (G10: GenBank

accession # U05876) and EV71 (BrCr: GenBank accession # U22521) share

significant capsid sequence similarity and antigenic cross-reactivity (Oberste et

al. 2004).

The capsid protein VP1 is most often used in phylogenetic analysis due to

its high degree of diversity and lack of involvement in recombination (Bible et al.

2007; Oberste et al. 1999b; Yoke-Fun & AbuBakar 2006). Based on the VP1

sequences of EV71 isolates, three distinct genogroups: A, B and C, have been

identified (Brown et al. 1999). Genogroup A is solely represented by the original

prototype virus from California (BrCr strain). Genogroups B and C can be sub-

divided into B1-B5 and C1-C5 (Chan, Sam & AbuBakar 2010). Based on the

original definition established by Brown et al. (1999), EV71 strains within the

same genogroup should not differ from each other by more than 12%. However,

sequence variation among strains belonging to two different genogroups may

reach 16-19%. Sub-genogroups within B and C clusters may vary by 6-11% and

6-10%, respectively.

Since 1995, when the first complete genome sequence of EV71 was

obtained, almost 190 EV71 strains have been sequenced in full (the Nucleotide

database <http://www.ncbi.nlm.nih.gov/nuccore>, search results on 14 March,

2012). Recently, a comprehensive comparative analysis of the complete genome

sequences of EV71 and other HEV-A viruses was performed by Yoke-Fun Chan

and AbuBakar Sazaly (2006). The total length of the genomes ranged from 7395

to 7434 nucleotides due to size variations of the 5′UTR, structural protein genes

and 3′UTR. The P2 and P3 genomic regions did not contain deletions or

insertions suggesting that all HEV-A viruses have constant length within these

genes.


44

Phylogenetic analysis conducted on separate genes showed that clustering

EV71 isolates with other HEV-A viruses varies (Table 1-3) (Yoke-Fun &

AbuBakar 2006). Only in the VP1 genome region were EV71 isolates distinct

from other viruses and showed further segregation on sub-genotypes with a

bootstrap value of 100%. The average sequence divergence within VP1 region of

EV71 isolates was 17-22% at nucleotide level and 3-8% at amino acid level. The

average nucleotide and amino acid sequence divergences between the VP1 of

EV71 and other HEV-A viruses were 22-29% and 10-20%, respectively.


45

Genome region

Description of phylogenetic position of EV71 isolates

5′UTR ● EV71 BrCr prototype strain segregates together with CAV2 and CAV3 and away from other EV71 genotypes. ● EV71 subgenotype C2 is closer to CAV8. ● EV71 genotype B and C4 form one cluster.

VP1 ● EV71 isolates cluster distinctly from all other existing HEV-A viruses and subdivide into the three genotypes: A, B and C.

P1 ● EV71 isolates cluster together and closer to CAV16. ● No significant segregation observed within EV71 isolates (low bootstrap support).

P2 ● EV71 BrCr prototype strain clusters with CAV2, CAV3, CAV6, CAV10, CAV12 and CAV16/Tainan5079. ● EV71 subgenotype C2 is closer to CAV8. ● EV71 subgenotype B2 and C4 cluster together with CAV4, CAV5, CAV14 and CAV16/G10.

P3 ● EV71 BrCr prototype strain clusters with CAV2, CAV3, CAV6, CAV10, CAV12 and CAV16/Tainan5079. ● EV71 subgenotype C2 is closer to CAV8. ● EV71 subgenotype B2 and B4 cluster together and close to CAV5. ● EV71 subgenotype B3 and C4 cluster with CAV4, CAV14 and CAV16.

3′UTR ● EV71 subgenotype B3 and C4 cluster with CAV4, CAV14 and CAV16. ● Other EV71 isolates show no significant segregation (<30% bootstrap support).

Table 1-3: Phylogenetic relationships between genome regions of EV71 and other

HEV-A viruses.


46

1.4 Pathogenesis of EV71

A significant variation in the spectrum of clinical symptoms associated

with EV71 infection is evident. Since its discovery, EV71 has been responsible

for sporadic cases and outbreaks of HFMD and herpangina with mild

manifestation, as well as severe CNS complications such as aseptic meningitis,

brain stem encephalitis or poliomyelitis-like acute flaccid paralysis. HFMD

caused by EV71 is generally associated with more severe forms of the disease

compared to CAV16 cases, where patients normally recover without requiring

any special attention. This observation suggests that despite the significant capsid

sequence similarity and antigenic cross-reactivity between EV71 and CAV16,

the virulence determinants and host cell receptors used by both viruses might

differ.

The knowledge of the mechanisms behind EV71 neuropathogenesis and

host cell receptors determining tissue tropism of the virus still remain very

limited. Furthermore, there is no clear understanding of the factors that contribute

to the different clinical manifestations in patients infected with genetically

similar or identical EV71 strains (Shih et al. 2000). For instance, it has been

suggested that hypersensitivity to EV71 might occur in a patient during active

co-infection with another enterovirus, such as CAV16 (Ho et al. 1999).

1.4.1 The neuropathogenic features of EV71 infection

EV71infections with neuropathogenic manifestation were first reported on

a large scale during an outbreak in Bulgaria in 1975. Poliomyelitis-like paralysis

was diagnosed in 21.1% of patients and the fatality rate within that group reached

29.5% (Shindarov et al. 1979). Since that time the tropism of EV71 for spinal

cord and brain-stem grey matter has been well established (Chumakov et al.

1979; Lum et al. 1998). It was shown that the neuropathogenicity of EV71 is


47

similar to that of wild poliovirus, which causes poliomyelitis in 0.1 to 0.5% of

infected individuals (Fine & Ritchie 2006). Similar to that, for instance, the case

severity rate of the EV71 outbreak in Taiwan (1998) was estimated at about 0.3%

(Ho et al. 1999).

However, a clear difference was demonstrated between the histological

changes in the CNS after infection with EV71 or PV1 in a monkey model.

Intravenous inoculation with EV71 BrCr-tr strain resulted in diffuse

panencephalitis with involvement of both the pyramidal and extrapyramidal

systems. CNS damage induced by PV1 was mainly restricted to the motor

neurons of the pyramidal tract (Nagata 2004).

Ho M. et al (1999) reported that eighty three percent of the patients who

died during the outbreak in Taiwan in 1998 had pulmonary oedema or pulmonary

hemorrhage. The pathogenesis of these diseases in enteroviral infections is not

well understood. According to Chang et al. (1998), who described the clinical

symptoms in an 8 year old girl who died from neurogenic pulmonary oedema,

the disease was caused by direct invasion of spinal cord and medulla by EV71

that resulted in death. Further necropsy showed that EV71 presented in brain,

medulla, cervical, thoracic, and lumber spinal cords as well as in rectal and throat

swabs. No virus was isolated from blood, lung, liver, heart, or pancreas.

However, another study from the same outbreak reported EV71-cases of acute

encephalomyelitis with mild symptoms of myocarditis and pancreatitis (Yan et

al. 2000). EV71 was detected in all organs with inflammatory reaction. However,

most prominent lesions were observed in the brain stem and spinal cord.

The mechanisms by which EV71 can enter the CNS are currently

unknown. The retrograde axonal transport theory suggests the peripheral nerves

to be a major route for EV71to spread from the skeletal muscles to the CNS

(Chen 2007). The theory is based on the observed pattern of the virus

dissemination in a mouse model after i.m. inoculation into hind or forelimb.

Additionally, in the same study, treatment with colchicine, an axonal transport

inhibitor, resulted in reduction of EV71 titres in CNS in a dose-dependent

manner.


48

An alternative theory is a hematogenous transmission of EV71 through the

blood-brain barrier (BBB). According to this theory, systemic viremia during

EV71 infection increases permeability of the BBB by triggering production of

tumour necrosis factor in the brain tissue (Chen 2007).

Immunopathogenesis associated with EV71 infection probably plays a

significant role in the development of severe neurological symptoms. Elevated

levels of white blood cells both in blood and cerebrospinal fluid were observed in

patients with a fatal outcome (Chang et al. 2004; Hsia et al. 2005). Additionally,

high levels of cytokines (IL-6, TNF-α, IFN-γ) triggered by EV71 infection were

suggested to be responsible for inflammatory damage of the CNS (Lin et al.

2003; Wang et al. 2003; Wang et al. 2007b).

1.4.2 Tissue tropism and cellular receptors of EV71

Identification of the specific cellular receptors is crucial for understanding

the mechanism of virus-host interactions and pathogenesis.

It has been shown that the difference in neurovirulence of CAV16 and

EV71 in mice is most likely determined by their structural genes and interaction

with host cell receptors. Though both EV71 and CAV16 viruses are closely

related, it was suggested that different receptors are utilized for the virus entry

into the different tissues, which resulted in different disease manifestation and

outcome in murine model (Chan & AbuBakar 2005).

Cellular tropism of EV71 isolates from mild and severe cases in humans

also differs. Kung et al. (2007) showed that the encephalitis isolate

TW98NTU2078 was tropic to peripheral blood mononuclear cells (PBMCs) and

replicated in human astrocytoma cells (HTB-14) 100-times faster than the

herpangina isolate TW98NTU1186. The authors assumed that PBMC-tropism

may benefit transport of the encephalitis isolate to the BBB and subsequent

productive infection of astrocytes.


49

First data regarding EV71-specific cellular receptors only became

available very recently. In 2009, three types of receptors which are partially

involved in EV71 entry into human cells were described. One of those, SCARB2,

is a scavenger receptor class B subfamily (Yamayoshi et al. 2009). It is a double-

transmembrane protein, located mostly in lysosomes and endosomes and

participating in membrane transport. It was shown that human cell lines, such as

RD, HeLA, Hep-2, 293T and HepG2, have a different level of surface expression

of SCARB2, which is well correlated with progression of EV71 infection in

those cell lines. In contrast, mouse L929 cells, which normally did not express

SCARB2, were highly resistant to EV71. However, when L929 cells were

transformed with RD cells genomic DNA they became EV71 susceptible. Anti-

SCARB2 antibody could block EV71 infection in RD cells in a dose-dependent

manner. Amino acids 142 to 204 within the human SCARB2 were identified to

be involved in binding of the EV71 virions (Yamayoshi & Koike 2011). Another

research revealed that residues 144-151 in human SCARB2 were critical for

interaction with EV71 VP1, and could convert an inefficient murine SCARB2 to

an efficient EV71 receptor (Chen et al. 2012). Additionally, in the VP1 protein,

amino acid residue Gln-172 and the surrounding canyon were identified as a

binding site. SCARB2-mediated EV71 entry into host cells was described as

clathrin-mediated endocytosis which was dependent on acidic pH and availability

of intact membrane cholesterol (Lin et al. 2012).

The second EV71 receptor in humans is P-selectin glycoprotein ligand-1

(PSGL-1) (Nishimura & Shimizu 2012; Nishimura et al. 2009). It is a sialomucin

membrane protein which is expressed on all circulating leukocytes. The N-

terminal amino acids (from 42 to 61) of PSGL-1 were shown to interact with

EV71. Infection was inhibited in PSGL-1 expressing cells by KPL1 (a

monoclonal antibody against PSGL-1) or pretreatment of EV71 with a soluble

form of PSGL-1. EV71 replication was not blocked by KPL1 in non-leukocyte

cell lines, such as RD, Hep-2c, SK-N-MC and Vero, none of which expressed

PSGL-1. It was suggested that an alternative mechanism of host cell EV71

interaction exists in cells of non-leukocyte origin. Interestingly, when tested in


50

co-immunoprecipitation assay, PSGL-1 exhibited different binding efficacy with

different EV71 strains. Out of eight strains tested, only five bound soluble PSGL-

1. Additionally, two strains with distinct differences in the ability to interact with

PSGL-1 belonged to the same genotype C1 and differed in their capsid proteins

only by four amino acids. It suggested that as little as a few positions in capsid

proteins of EV71 can determine the receptor-specific phenotype and possibly

pathogenesis of the strain.

A post-translational modification of the PSGL-1, such as tyrosine

sulfation of its N-terminal region (Y46, Y48 and Y51), was reported to be critical

for the PSGL-1-EV71 interaction and virus replication in leukocytes (Nishimura,

Wakita & Shimizu 2010).

The third receptor described is the dendritic cell-specific ICAM-3

grabbing non-integrin (DC-SIGN) (Geijtenbeek et al. 2000). It is a type II

membrane protein, which is expressed in immature dendritic cells (DC). It plays

an essential role in the primary immune response through initiation of resting T

cells to proliferate (Hart 1997). However, it has been reported that the DC-SIGN

mediates transport and cellular entry of some viruses to susceptible cells,

including members of the Retroviridae (HIV-1, HIV-2, SIV), Filoviridae (Ebola

virus), Herpesviridae (CMV, HHV-8) and Paramyxoviridae (MV) (Alvarez et al.

2002; de Witte et al. 2006; Halary et al. 2002; Pohlmann et al. 2001a; Pohlmann

et al. 2001b; Rappocciolo et al. 2006). In the EV71 infection of DC, the DC-

SIGN plays only a partial role as a pre-treatment of the DC with anti-DC-SIGN

antibody reduced viral tire by about 50% (Lin et al. 2009).

In 2011, Yang et al. identified an EV71 VP1-binding protein from RD

cells. That protein, annexin II (Anx2), was previously known to interact with

phospholipid membranes and cellular factors, and to be involved in the

regulation of endo-/exocytosis, and generation of plasmin (Yang et al. 2011).


51

1.4.3 Molecular basis of EV71 virulence and attenuation

It was shown that EV71 can be considered as one of the most common

enteroviruses detected both from the samples of clinical cases and healthy

children (Khetsuriani et al. 2006; Witso et al. 2006). But knowledge of the

molecular basis for observed differences on EV71 virulence is still limited.

Analysis of EV71 strains at the molecular level in some cases can help to explain

the varied clinical patterns of EV71 infection and answer the question why some

viral strains are associated with mild HFMD outbreaks while others cause severe

complications in CNS (Kung et al. 2007; Kung et al. 2010).

1.4.3.1 Attenuating determinants of the oral poliovirus vaccine (OPV)

strains studied on EV71

Due to the significant similarity in the genome structure of EV71 and PVs,

and common mechanism of the replication/translation, it has been suggested that

mutations described for PV strains might have an importance in understanding

the molecular basis of EV71 pathogenesis (Arita et al. 2005). To date, a number

of critical determinants of virulence and attenuation have been found in the

5′UTR, VP1, VP2, VP3, VP4, 2Apro, 2B and 2C regions of PVs (Evans et al.

1985). Some of those determinants caused attenuation when introduced into the

EV71 genome (Arita et al. 2008) (Table 1-4).


52

Region Mutation site, nt (aa)* Strain**

Mutation-associated phenotype in PVs Mutation-associated phenotype in EV71

I. Single mutations

5′UTR C472U P3/Leon/37 to Sabin 3

A strong attenuation determinant of Sabin 3 OPV strain (Evans et al. 1985; Westrop et al. 1989). The mutation is associated with the reduction of viral RNA translation in human neuroblastoma cells (La Monica & Racaniello 1989). The mutation is located in the IRES.

The position corresponds to C474 in EV71 (BrCr). The EV71 mutant (C472U) derived from the BrCr-TR strain showed small-sized plaques in Vero cells, similar to BrCr-ts variant, and slight ts phenotype (∆36/39ºC =2.25 log)*** (Arita et al. 2005).

A480G Mahoney to Sabin 1

A major attenuation determinant of Sabin 1 OPV strain (Nomoto et al. 1982; Ren, Moss & Racaniello 1991). However, the mutation has very little effect on viral temperature sensitivity (Kawamura et al. 1989). The mutation is responsible for limited growth in neuroblastoma cells (Agol et al. 1989).The mutation is located in the IRES.

The position corresponds to A485 in EV71 (BrCr). The EV71 mutant derived from the BrCr-TR strain showed a slight ts phenotype (∆36/39ºC=2.0 log) and medium-sized plaques in Vero cells (Arita et al. 2005).

G481A Lansing to Sabin 2

An attenuation determinant of Sabin 2 OPV strain (Ren, Moss & Racaniello 1991). It attenuates neurovirulence in transgenic mice expressing human poliovirus receptors. However, another study showed that the mutation alone contributes very little to the attenuated phenotype (Rezapkin et al. 1999). Revert substitution to wild-type sequence did not increase neurovirulence in monkeys. The mutation is located in the IRES.

The position corresponds to G486 in EV71 (BrCr). The EV71 mutant derived from the BrCr-TR strain showed a slight ts phenotype (∆36/39ºC was 2.25 log) and medium-sized plaques in Vero cells (Arita et al. 2005).


53

3D U6203C (Y73H) Mahoney to Sabin 1

The mutation contributes to the ts phenotype of poliovirus Sabin 1 (Georgescu et al. 1995). The mutant is defective in VPgpUp(U) and VPg-poly(U) synthesis at 39.5°C (Paul et al. 2000a). Also, it was shown to be involved in the attenuation of Sabin 1 in mice (Tardy-Panit et al. 1993).

The position corresponds to Y73 in EV71 (BrCr). The mutation has been studied in combination with C363 (in 3Dpol) and A7409 (in 3′UTR) (see “triple mutations” bellow).

II. Combined mutations

3D

and

3′UTR

U6203C (Y73H), C7071U (T362I) and A7441G Mahoney to Sabin 1

The ts phenotype of poliovirus Sabin 1 results from the combination of these genetic determinants (Georgescu et al. 1995). Mutants carrying the single mutations (U6203C or C7071U) within 3Dpol did not have a growth defect at 40°C. Temperature sensitivity was enhanced by introducing the 3′UTR-mutation. Contribution of these mutations to attenuated phenotype of Sabin 1 is not clear and based on contrasting results, which might be due to different animal model used and different approaches applied to quantify the neurovirulence (Bouchard, Lam & Racaniello 1995).

The “triple mutations” analogous to Sabin 1. The positions correspond to Y73 and C363 within 3Dpol, and A7409 within 3′UTR of EV71 (BrCr). The mutant virus, EV71(3′), showed a strong ts phenotype (∆36/39ºC >3.5 log versus 1.25 log for BrCr-TR) but still medium- plaque phenotype. Its growth kinetics in Vero cells at 36ºC was similar to that of the parental strain. Monkeys intravenously inoculated with 107 CCID50 of the EV71(3′)-mutant showed mild neurological manifestation and histological changes. Virus was detected in the spinal cord (50% of the animals) and brain stem (100%), but not in the cerebellum and cerebrum as was BrCr-TR. Viruses recovered from the CNS retained a strong ts phenotype, similar to the original EV71(3′) (Arita et al. 2005).

5′UTR

and

3D

A480G and U6203C (Y73H),

as above The “triple mutations” combined with the attenuating determinant within 5′UTR of Sabin 1 (A480G). The mutant virus, EV71(S1-3′), showed phenotype and growth kinetics similar to those of


54

and

3′UTR

C7071U (T362I) and A7441G Mahoney to Sabin 1

EV71(3′). Neurological manifestation in monkeys was similar to that of EV71(3′). Virus was detected in the spinal cord (50% of the animals) but not in the brain stem, as was EV71(3'). Viruses recovered from the CNS retained a strong ts phenotype, similar to the original EV71(S1-3′) (Arita et al. 2005). Cooperative effect of these genetic determinants on attenuation was further observed in a NOD/SCID mouse model (Arita et al. 2008).

Table 1-4: Determinants of temperature sensitivity and attenuation of the OPV strains studied on EV71.

* numbering of nt and aa mutations are in accordance with their positions within the PV genome or specific genes, respectively. ** GenBank accession # of the strains as follows: Mahoney (J02281), Sabin 1 (V01150), Lansing (M12197), Sabin 2 (X00595), P3/Leon/37 (K01392), Sabin 3 (P3/Leon/12a1b, X00596). *** The logarithmic difference of TCID50 at 36 and 39ºC (∆36/39ºC or ∆TCID50). Temperature sensitivity from 2.0 to 2.75 logarithmic differences was defined as a slight ts phenotype, when more than 2.75 - as a strong ts phenotype.


55

The determinants within the 5′UTR affect the translational efficiency of

the viral RNA, and hence a single nucleotide substitution may be sufficient to

change growth and neurovirulence phenotypes in isolates (Evans et al. 1985;

Macadam et al. 1994). The crucial attenuating mutations in the Sabin vaccine

strains are located between residues 472 and 481 within the IRES, domain V of

the 5′UTR (Ren, Moss & Racaniello 1991; Svitkin et al. 1988; Westrop et al.

1989). It was demonstrated, that those mutations inhibit initiation of the

poliovirus RNA translation in a cell dependent manner (Guest et al. 2004; Haller,

Stewart & Semler 1996; Svitkin et al. 1990). Attenuated viruses failed to

replicate efficiently in neural tissue or in neuroblastoma cells while their

replication in gut and in HeLa cells was not affected. In contrast wild-type

viruses replicated efficiently in all cell types (Agol et al. 1989; La Monica &

Racaniello 1989). The substitutions in the IRES are major attenuating

determinants of the Sabin 1 and Sabin 3 vaccine strains. The precise contribution

of G481A mutation to attenuated phenotype in type 2 OPV is unclear (Rezapkin

et al. 1999). The reverse substitution (A to G) introduced into the Sabin 2 strain

did not increase neurovirulence in monkeys. Additionally, the nucleotide

sequence of an alternative strain (Chung 2) used in the OPV production in China

showed that it contained wild-type 481G, but possessed an extremely low level

of neurovirulence.

Several mutations have been found to restore the original stem-loop

structure in the IRES of vaccine-derived polioviruses (VDPVs) isolated both

from healthy OPV recipients and patients with vaccine-associated paralytic

poliomyelitis (VAPP) (Minor & Dunn 1988; Muzychenko et al. 1991).

Reversions (U472C, G480A and A481G) were shown to occur within 1 week of

vaccination and resulted in prolonged periods of virus excretion as compared to

other vaccines where reversions did not occur (Minor & Dunn 1988).

Additionally, suppressing mutations were found at the base pairing positions in

all 3 types of VDPVs: U525C complements to G480 in VDPVs of Sabin 1,

U398C complements to G481 in VDPVs of Sabin 2 and G537A complements to

U472 in VDPVs of Sabin 3 (Muzychenko et al. 1991).


56

The main attenuation determinants of Sabin OPV strains within the 5′UTR

(G480, A481 and U472) introduced into EV71 (BrCr) genome resulted in slight

ts phenotypes in Vero cells (Arita et al. 2005). When the attenuation

determinants within the 5′UTR were combined with the “triple mutations” within

the 3Dpol and 3′UTR, the mutant EV71 showed reduced neurovirulence in

monkeys. That cooperative affect was further confirmed in the NOD/SCID

mouse infection model with an EV71 mutant of the mouse-adapted Nagoya strain

(Arita et al. 2008).

Other attenuating determinants of Sabin OPV strains were mapped within

VP4 (Sabin 1: nt position 935), VP3 (Sabin 1: nt position 2438, Sabin 3: nt

position 2034) and VP1 (Sabin 1: nt positions 2795 and 2879, Sabin 2: nt

position 2908, Sabin 3: nt position 2493) (Bouchard, Lam & Racaniello 1995;

Ren, Moss & Racaniello 1991; Westrop et al. 1989). However, mutations

corresponding to those attenuating determinants of OPV strains have never been

studied in EV71.

1.4.3.2 Attenuating determinants described in EV71 clinical isolates

Mutation from U to C at position 575 or 576 of the polyadenylation site of

the 5′UTR was found in EV71 herpangina isolates from the Taiwan outbreak in

1998 (Kung et al. 2007). A clinical isolate carrying the mutation showed a 100-

fold lower replication capacity in human astrocytoma cell culture (HTB-14 cells)

when compared to the encephalitis isolate. Additionally, the herpangina isolate

could not replicate in human PBMC-adherent cells. It was suggested, that

U575/576C mutation could have a negative effect on viral replication and

pathogenesis. However, experimental conformation has thus far not been

attempted.

The EV71 isolates harbouring the A170V mutation within VP1 capsid

protein were isolated during the Western Australia epidemic (Perth, 1999) and


57

linked to severe neurological disease (McMinn et al. 2001a). However, a precise

role of this residue in EV71 neurovirulence has not been determined.

In another study, sequence analysis of EV71 strains isolated from patients

with different clinical symptoms identified an amino acid change within the 3Dpol

(V1994I, numbering according to BrCr polyprotein corresponds to V263I in

3Dpol) (Chang et al. 2010). Strains carrying the mutation failed to cause disease in

mice irrespective of the route of inoculation (i.c. and i.p). The inoculated mice

had a weight loss during the first week post inoculation, but recovered and

survived throughout the period of observation.

Interestingly, a single amino acid change within the same domain of 3Dpol

(T251I) reported in EV71 isolates from Taiwan outbreaks, 1998 and 2000, was

suggested to be associated with an increase in viral virulence of the circulating

strains (Kung et al. 2010). The reverse mutation from T to I in the EV71

infectious clone resulted in a strong temperature sensitive phenotype in SK-N-SH

cells and delayed the death of the infected mice.

Another interesting study was performed with two unadapted clinical

isolates of EV71, 237 and 4643, obtained in Taiwan in 1986 and 1998,

respectively (Yeh et al. 2011). Virus 237 was isolated from a case of HFMD

whereas virus 4643 was associated with a fatal case of encephalitis. Virulence

determinant of those clinical isolates was mapped to the 158th nucleotide position

(C in 237 or U in 4643 isolate) of the stem-loop II within the 5′UTR.

Detailed studies are important to understand if differences observed

between EV71 clinical isolates at the genome level are associated with disease

severity in naturally occurring human EV71 infections.

1.4.3.3 Attenuating determinants of EV71 selected in vitro

The ts variant of the BrCr strain was selected in CMK cells and was

shown to have a less neurovirulent phenotype in monkeys (Hagiwara, Yoneyama

& Hashimoto 1983). Among nine nucleotide differences observed between the


58

TR and ts variants three were non-synonymous and resulted in amino acid

changes: A253T in VP2, Y116H in VP1 and T305I in 2C protein. The amino

acid change within VP1 protein was found to be responsible for both ts and

small-plaque phenotype of the BrCr-ts. The BrCr-TR strain carrying Y116H

mutation in VP1 showed small-sized plaques in Vero cells and strong ts

phenotype (∆36/39ºC >2.75 log versus 1.25 log for BrCr-TR). The corresponding

substitution introduced into BrCr-ts strain (TR2784 mutant) resulted in a slight ts

phenotype (∆36/39ºC =2.0 log versus >3.00 log for BrCr-ts) and a large-plaque

size. Monkeys intravenously inoculated with 107 CCID50 of the TR2784 showed

mild neurological manifestation and histological changes. Virus was detected in

the spinal cord (100% of the animals), the brain stem (100%) and cerebrum

(50%), but not in the cerebellum as BrCr-TR (Arita et al. 2005).


59

1.5 Development of antiviral treatments and preventive agents

against EV71

The high risk of mortality in patients with CNS involvement due to EV71

infection requires early aggressive antiviral treatment in order to increase the

chance of a positive outcome (Wang et al. 1999). However, currently there is no

effective antiviral therapy available for EV71-associated neurological disease.

Treatment used in patients with acute EV71 infection with neurological

manifestation basically aim to reduce symptoms of the disease (Jan et al. 2010;

Wang et al. 2006). Additionally, the intravenous immunoglobulin (IVIG)

administered to children with neurological complications did not improve the

clinical outcomes (McMinn et al. 2001b). To date, there have been several potent

antiviral inhibitors tested in vitro against EV71. However, none has been

evaluated against EV71 in clinical trials. Compounds which showed anti-EV71

activity can be classified according to their antiviral mechanism of action.

1.5.1 Agents targeting the EV71 attachment, entry and uncoating

This group of EV71 inhibitors includes antibodies, which can block host-

cell receptors used by EV71 for attachment. For example, anti-SCARB2 and

KPL1 could inhibit EV71 infection in cells expressing SCARB2 and PSGL-1

receptors, respectively, in a dose-dependent manner (Nishimura et al. 2009;

Yamayoshi et al. 2009). However, experiments demonstrated that the inhibition

was not complete, suggesting the possible involvement of additional receptors in

EV71 attachment. Furthermore, cellular receptors utilised by EV71 to interact

with neural cells have not been discovered to date, which prevents using this

therapeutic approach in treatment of severe CNS cases.

Another group of inhibitors includes capsid-binding compounds, such as

the WIN series (e.g. Pleconaril), the R77975-related compounds (e.g. Pirodavir,


60

BTA39), and the SCH series (Andries et al. 1992; Buontempo et al. 1997; Pevear

et al. 1999). These molecules are a result of computer modelling and designed to

inhibit EV71 uncoating by occupying the hydrophobic pocket within the VP1

protein and stabilizing the viral capsid (Shia et al. 2002). These type of inhibitors

have been evaluated in a cell culture or animal model and some of them

produced promising results (e.g. WIN-derivatives: BPROZ-compounds and a

Pirodavir-derivative: BTA-compounds) (Barnard et al. 2004; Chen et al. 2008b).

It suggests that there is a potential to use those compounds in the treatment of

EV71 infection upon completion of the clinical trials.

As a result of screening the LOPAC1280 drug library, several compounds

were identified to have anti-EV71 activity (Arita, Wakita & Shimizu 2008).

Among those, NF449 was shown to inhibit EV71 infection in RD cells via

targeting VP1 protein. After 5 passages in cell culture, NF449-resistant mutations

were observed in the BC loop of VP1 near the fivefold axis of viral capsid (aa

positions 98 and 244). It was suggested, that NF449 might inhibit EV71 binding

via an ionic interaction with the site near the fivefold axis.

Among natural agents which possess antiviral activity, lactoferrin (Lf) is

known to prevent virus attachment and absorption by intestinal cells for a

number of viruses (e.g rotavirus, PV, adenovirus) (Seganti et al. 2004). The anti-

viral mechanism of action of Lf is mainly due to its binding both to viral particles

and cell receptors. The anti-viral activity of Lf against EV71 was observed in

vitro (RD and SK-N-SH cells) and in vivo (mice). Additionally, Lf reduced

production of IL-6, which is associated with inflammatory reaction caused by

EV71 infection (Weng et al. 2005).


61

1.5.2 RNA interference

RNA interference (RNAi) is a promising therapeutic approach allowing

viral inhibition through the silencing of viral genes by introduction of specific

dsRNA molecules (Stram & Kuzntzova 2006). Those molecules are small

interfering RNA (siRNA) and short hairpin RNA (shRNA) which are designed to

target highly conserved sequences within the viral genome. In EV71, various

genome regions (3′UTR, 2C, 3C and 3D) have been tested with chemically

synthesized siRNA and the inhibitory effect was observed in a dosage-dependent

manner (Lu et al. 2004; Sim et al. 2005; Tan et al. 2007; Wu et al. 2009).

1.5.3 Agents targeting 3C protease

Compounds targeting the 3Cpro of the EV71 are represented by derivatives

of rupintivir (AG-7088), an inhibitor of the rhinovirus 3Cpro (Kuo et al. 2008).

The AG-7088 was shown to exhibit antiviral activity against broad spectrum of

HRV and HEV isolates due to its covalent irreversible interaction with highly

conserved amino acid residues within the 3Cpro (Binford et al. 2005; Tsai et al.

2009). The 10b compound (an improved formula of rupintivir) showed great

inhibitory activity with no cytotoxicity when tested against EV71 in RD cells.

However, this potent inhibitor still needs to be evaluated for its efficacy in vivo.

1.5.4 Agents targeting 2C protein

Metrifudil and its derivative, N6-benzyladenosine, have been identified as

strong anti-EV71 compounds with no cytotoxicity at the concentrations sufficient

to inhibit EV71 infection of RD cells (Arita, Wakita & Shimizu 2008). Mutations

associated with the emergence of the resistant phenotypes of EV71 to those

compounds were found after 3 passages and were located within the C-terminus


62

of the viral protein 2C (E325G and I324M). The results indicated that 2C protein

was a target for those inhibitors, however, the mechanism of inhibition remains

unknown.

1.5.5 Agents targeting 3A protein

Enviroxime was reported to inhibit viral replication for a number of

enteroviruses (e.g. EV71, PV1, CVB3, HRV2 and HRV14) (De Palma et al.

2009). The drug-resistant mutants were found to carry aa changes within the 3A

viral protein, which was considered as a key target of enviroxime and its

derivatives (Heinz & Vance 1995; Victor et al. 1997). However, additional

mutations observed within other viral genes (e.g. 3Dpol) and associated with an

increased level of enviroxime-resistance suggested that inhibition probably

occurs via targeting several viral proteins and/or cellular factors within the

replication complex (Brown-Augsburger et al. 1999).

GW5074 is a functional analogue of enviroxime. It exhibited a strong

inhibitory effect on enterovirus replication (EV71 and PV) with no cytotoxicity

in RD cells (Arita, Wakita & Shimizu 2008). It has been shown that GW5074

and enviroxime-resistant mutants have an identical mutation at the 70th aa-

position within the 3A protein (Arita, Wakita & Shimizu 2009). Additionally, it

has been recently reported that anti-enterovirus activity of GW5074 can be

associated with inhibition of host factors (e.g. phosphoinositide (PI) kinases)

required for viral replication (Arita et al. 2011).

A novel enviroxime-like compound, AN-12-H5, was shown to suppress

both viral replication (PV and EV71) and an early stage of infection (EV71),

following the binding step (Arita et al. 2010). The isolated mutants resistant to

AN-12-H5 carried several mutations both in the 3A and viral capsid proteins

(VP1 and VP3).


63

1.5.6 Agents targeting 3Dpol

DTriP-22, a nonnucleoside analogue, was shown to suppress viral

replication in vitro and in RD cells (Chen et al. 2009). Of particular interest was

the fact, that amounts of the compound required to inhibit EV71 in cell culture

were 100 times lower than those in vitro. It was suggested that DTriP-22 might

be accumulated by cells to a higher concentration, or undergo some

modifications within an intracellular environment which resulted in a higher

efficacy of the compound. Anti-EV71 activity of DTriP-22 was associated with a

reduction in the accumulation of both positive- and negative-strand RNA

synthesis via inhibition of the 3Dpol elongation activity. A single aa substitution

(R163K) within the ring finger domain of 3Dpol could render the virus drug

resistant.

Aurintricarboxylic acid (ATA) belonging to polyanionic compounds has

shown an anti-viral activity against several RNA viruses including HIV,

influenza, vaccinia and vesicular stomatitis virus (De Clercq 2002; Hung et al.

2009; Myskiw et al. 2007). Anti-EV71 activity was observed in Vero cells and

was most prominent during the early stage of the virus replication cycle (Hung et

al. 2010). It did not affect IRES-mediated viral translation and 2A or 3C protease

activity, but could inhibit RNA elongation directed by the 3Dpol in a

concentration-dependent manner.


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1.6 Vaccine development against EV71

There have been several examples where widespread vaccination has

significantly reduced the incidence of many diseases such as measles, mumps,

diphtheria, pertussis, poliomyelitis, Hepatitis B, rubella, etc

(http://www.cdc.gov/vaccines/vac-gen/whatifstop.htm). Considering the ability

of EV71 to cause severe neurological diseases, world-wide circulation of the

pathogen and numerous outbreaks observed in the past and even more recently,

development of a vaccine against EV71 remains a priority (Zhang, Lu & Lu

2010).

Seroepidemiological studies conducted with serum obtained before and

after the EV71 outbreak in Taiwan in 1998, showed that the disease prevalence

was associated with low seropositivity in patients 5 years old and younger, in

contrast to the adult population where about a half had antibodies against EV71

before the epidemic (Ho et al. 1999). Active immunization or passive transfer of

neutralizing antibodies could be valuable strategies to prevent EV71 outbreaks

and associated deaths in future.

The major strategies in developing a vaccine against EV71 which have

been attempted to date are as follows:

Whole-pathogen vaccines (inactivated or attenuated);

Epitope peptide vaccine (short synthetic peptides);

Subunit vaccine (purified or recombinant viral antigens);

Virus-like particle (VLP) vaccine;

DNA vaccine.

In order to succeed in vaccine design with any of those strategies, it is

important to understand and preserve the immunogenic determinants of the

pathogen. Whole virus particles have been shown to offer the most effective

protection against EV71 (Foo et al. 2007a; Wu et al. 2002; Yu et al. 2000). This

might be due to higher titres of neutralizing antibodies elicited by several

neutralizing epitopes, linear and conformational, located on the viral capsid, in


65

contrast to those represented, for instance, by short synthetic peptides (Foo et al.

2007a).

1.6.1 Immunogenic determinants of enteroviruses

Earlier studies on PVs have shown that the purified VP1 protein could

induce a neutralizing antibody response in mice, rat and rabbits (Blondel, Crainic

& Horodniceanu 1982; Chow & Baltimore 1982; Emini et al. 1982). Subsequent

findings uncovered other neutralizing epitopes from VP3 and VP4 (Emini et al.

1983).

van der Werf et al. (1983) described localization of poliovirus type 1

(PV1) neutralizing epitope in the viral capsid polypeptide VP1 by generating

deletions inside the VP1 sequence and analysing reactivity of the hybrid proteins

with a neutralizing monoclonal antibody C3. This monoclonal antibody was

produced by a hybridoma cell line after infection of mice with heat-inactivated

virions (Blondel et al. 1983). In addition to binding to the heat-denaturated and

native virus, the C3 antibody was found to immunoprecipitate the VP1 protein.

Deletion of both the C-terminal and central part of VP1 did not affect reactivity

of the fusion protein with C3 as long as the deletion did not reach the nucleotide

2,787 (amino acid 103 of VP1). The authors concluded that the C3 neutralizing

epitope was probably located in the domain of VP1 between amino-acids 95-110

(poliovirus nucleotides 2,754-2,806). The conclusion is strengthened by the

observation that the attenuated poliovirus Sabin 1 strain which had at least five

amino acid substitutions (positions 88, 90, 95, 98, and 106) in the proximity or

inside the region defined as the C3 neutralizing epitope showed much lower

reactivity with C3 antibody as compared to the wild-type Mahoney strain (van

der Werf et al. 1983).

A neutralizing epitope with similar location was identified in poliovirus

type 3 (PV3) by Minor et al. who found that mutants at amino acids 98, 99, and


66

100 in the VP1 region was resistant to neutralizing monoclonal antibodies

(Minor, Kew & Schild 1982).

In the case of EV71, Wu et al. (2002) first showed that the recombinant

VP1 protein expressed in E. coli was capable of inducing neutralizing antibody

response in mice.

Later, Sivasamugham et al. (2006) reported that the N-terminal part of the

VP1 protein contains a major antigenic region. In support to this conclusion, Tan

and Cardosa (2007) found that human cord sera with high titres of neutralizing

antibodies against EV71 reacts more strongly with the N-terminal half of VP1

protein (amino acids 1-135) rather than with its C-terminal fragment (amino

acids 134-297).

The N-terminus of VP1 capsid protein is shown to be very heterogeneous

both in length and sequence within the genus Enterovirus, but a highly conserved

region (amino acids 37 to 53 in VP1 of PV3 Sabin) remains in the N-terminal

part of the VP1 in all members (Hovi & Roivainen 1993). Besides being

immunogenic, the antibodies raised against this conserved sequence recognize a

wide range of enteroviruses. For instance, recombinant proteins carrying the first

100 N-terminal amino acids of the VP1 of EV71 induced strong antibody

responses in rabbits that reacted with the authentic EV71 and CAV16 when

analysed in an immunofluorescence assay (Sivasamugham et al. 2006).

Data at variance with the above has been generated by Damian G. W. Foo

et al. who have undertaken characterization of the linear neutralization epitopes

on the VP1 capsid protein of EV71 (Foo et al. 2007b). The study analysed mice

antiserum raised against 95 overlapping synthetic peptides, which were designed

based on the primary sequence of the VP1 gene of EV71 strain isolated from a

fatal case (5865/SIN/00009, genotype B4). Two of the peptides, designated SP55

and SP70, containing amino acid 163-177 and 208-222 of the VP1, respectively,

were capable of eliciting high titres of IgG1 sub-type neutralizing antibodies

against both homologous and heterologous EV71 strains. Both synthetic

peptides lie within the major hydrophilic regions in the C-terminal part of the


67

VP1 capsid protein. Hence, the authors suggested that its N-terminal moiety may

not be the best choice in vaccine development.

However, a very recent study conducted by Gao et al. (2012) mapped the

main human anti EV71 epitopes to the N-terminal part of VP1 protein. Sera

obtained from children in both acute and recovery stage was tested in ELISA

assay against 96 peptides spanning the VP1 sequence of BJ08 strain

(subgenotype C4). Strong positive reaction was observed with vp1-14 (amino

acids 40-51) and vp1-15 (amino acids 43-54) peptides against IgM and IgG,

respectively. Additionally, for the first time, human anti EV71 epitopes were

located in the VP2 and VP3 capsid proteins. Four peptides from the VP2 (amino

acids 16-27, 61-72, 118-129 and 148-159) and five peptides from the VP3

(amino acids 28-39, 34-45, 43-54, 70-81 and 223-234) region demonstrated

strong reaction with IgM.

The identification of human T-cell epitopes within the VP1 protein of

EV71 was undertaken by Damian G. W. Foo et al. (Foo et al. 2008b). By using

the ProPred algorithm, three regions (amino acids 66-77, 145-159 and 247-261)

within VP1 were predicted as potential human CD4+ T-cell epitopes. Synthetic

peptides spanning those regions were able to induce proliferation of CD4+ cells

obtained from EV71-positive volunteers. Additionally, the peptide-stimulated T-

cells produced increased levels of IL-2 and IFN-γ. The highest T-cell

proliferative response and cytokine production were triggered by amino acids

145-159.

1.6.2 Inactivated vaccine

An inactivated vaccine is constituted of a whole pathogen which is killed

by heat or an appropriate chemical method. Therefore, the main consideration in

this strategy is to assure that pathogen surface antigens remain intact after

inactivation. Analysing the folding pattern and interactions of the structural

subunits in the poliovirus particle, Hogle, JM et al. (1985) indicated that


68

inactivation of the virus can result in a substantial rearrangement of the virion

structure. It subsequently interferes with immunogenicity of the inactivated

vaccine.

Compared to natural viral infection, an inactivated virus is incapable of

entering the host cells and replication, therefore, the generated immune response

is usually weaker and limited to neutralizing antibodies. Inactivated virus is

quickly cleared by the host immune system and, therefore, multiple vaccine

boosters are required, in order to induce sufficient protective immunity. Chun-

Keung Yu et al. (2000) showed that ICR mice infected with a formalin-

inactivated EV71, developed neutralizing antibody titre of 1:64, while infection

with the native virus resulted in a doubled rise to that (1:128). Passive transfer of

serum with neutralizing antibodies titre 1:128 from actively immunized adult

mice could fully protect neonatal mice against an EV71 challenge of 4.6 x 109

PFU/ml. Additionally, pups derived and fed by EV71-infected dams with

elevated neutralizing antibody titres (1:128) were also resistant to EV71

challenge conducted 1 day after their birth. Maternal immunization with a

formalin-inactivated whole-virus vaccine showed only partial protection in pups

by delaying the mortality rate and increasing the survival rate compared to those

from non-vaccinated dams. It was suggested that the difference in the

neutralizing antibody response might be due to the dose of the vaccine, the

number of administrations, the type of adjuvant or the loss of B cell epitopes

during the vaccine preparation.

However, when compared to a VP1 DNA vaccine or recombinant VP1

protein, the heat-inactivated EV71 vaccine was more effective (Wu et al. 2002).

The ICR suckling mice born to dams immunized with inactivated virus (10 µg

protein/mouse) demonstrated 80% survival after administration to a challenge

dose of 2.3 x 103 LD50 virus/mouse, while the VP1 DNA vaccine (100 µg/mouse)

or recombinant VP1 protein (10 µg/mouse) failed to prevent mice death.

Following those promising results with an inactivated virus, the same research

group developed and characterized a laboratory-adapted EV71 strain, which

possessed several desirable features of an inactivated vaccine candidate (Lin et


69

al. 2002b). The strain was obtained from a clinical isolate during the 1998 EV71

outbreak in Taiwan by passaging the parent virus in Vero cells with the following

plaque cloning of a Vero cell-adapted lineage. The resulting strain, named YN3-

4a, had 14% nucleotide and 4% amino acid sequence differences within the VP1

gene compared to the original strain. It had a rapid growth rate in Vero cells

reaching titres of 1010 to 1011 TCID50 per ml. Similar titres were obtained when

propagated in serum-free medium. Mouse antibody elicited by YN3-4a could

neutralize a broad range of EV71 strains isolated from different geographic

regions (Malaysia and Taiwan) at different times (years 1997, 1998, 2000). The

YN3-4a strain exhibited a 2-fold higher heterologous neutralizing antibody titre

for 7 of the 15 viruses tested, than that which arose against the parent strain. The

YN3-4a remained genetically and phenotypically stable during 12 passages in

Vero cells. Amino acid sequences of the VP1 region of the YN3-4a strain and its

progeny from the subsequent passages, demonstrated 100% identity (99% at

nucleotide level). Nucleotide sequences of the 5′UTR and 3′UTR showed 98%

and 100% identity, respectively.

Two years later, a serum-free microcarrier Vero cell culture was

developed for large-scale EV71 production (up to 5.8 x 107 TCID50/ml) (Wu, Liu

& Lian 2004). Formalin-inactivated EV71 strains produced in a serum-free

microcarrier bioreactor showed slightly higher immunogenicity in mice than

those harvested from serum-supplemented cell cultures (Liu et al. 2007).

Additionally, the microcarrier cell culture facilitated the release of EV71

particles from the infected cells. This feature simplified the harvesting and

further processing of the virus.


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1.6.3 Attenuated vaccine

An attenuated vaccine is a weakened form of the pathogen. The

attenuation may be achieved by traditional passaging of the virus at sub-optimal

conditions in cell culture, or by site-directed mutagenesis. In both cases

attenuated mutants are usually limited in their ability to spread within the host

that results in the loss of pathogenicity. Since an attenuated virus is still capable

of replication within host cells, it can provide continual antigenic stimulation and

elicit cell-mediated immunity.

Attenuated vaccines are being successfully used against transmission of

the wild type PVs. Since 1988, when the PV eradication programme was

launched by the WHO, the number of polio cases decreased by over 99% and,

currently, only three countries are listed as polio-endemic (Afghanistan, Nigeria

and Pakistan) (http://www.who.int/mediacentre/factsheets/fs114/en/index.html).

Following the same strategy, it is possible that EV71 epidemics can be prevented

if an effective attenuated vaccine against EV71 is developed.

An understanding of the biological characteristics and natural circulation

of EV71 are required in order to succeed in this strategy. The attenuated vaccines

against PVs include all three PV serotypes. In the case of EV71, there have been

11 genogroups identified so far: A, B1-5 and C1-5 (Chan, Sam & AbuBakar

2010). Genotype A contains only the prototype strain, BrCr, which was isolated

in 1969 in California, and which has never been reported in the Asia-Pacific

region. Sub-genogroups within genetic lineages B and C are endemic in Asia.

Viral strains belonging to these genogroups have been shown to undergo rapid

evolutionary changes, leading to the emergence of new highly pathogenic strains

associated with large-scale outbreaks (McMinn 2002). In this situation,

prevention of new EV71 epidemics can be achieved with the development of an

attenuated EV71 vaccine strain with a broad spectrum of immunogenicity, or by

incorporating several attenuated strains derived from different genetic lineages

into an attenuated EV71 vaccine.


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1.6.3.1 Attenuated EV71 strains carrying the ts determinants of Sabin 1

To date, an effort to determine attenuation determinants in EV71 genome

has been attempted by Arita et al. (2008; 2007; 2005). Research was focused on

attenuation of the BrCr prototype strain of EV71, by introducing ts mutations

derived from the type 1 PV vaccine strain, Sabin 1 (Table 1-4). However, the

EV71 attenuated strain still showed mild neurovirulence in cynomolgus monkeys

(Arita et al. 2007). Sera derived from monkeys immunized with the attenuated

strain showed the highest neutralizing activity against the homologous strain

(genotype A), and significantly lower titres against heterologous strains in the

following order: A>B1>C4>B4>C2. Some large-scale outbreaks in the Asia-

Pacific region have been mostly associated with sub-genogroups B3, B4, C2 and

C4. Therefore, the BrCr prototype strain alone is probably not the best candidate

for developing the live-attenuated vaccine. Which strain or strains should be

incorporated in an anti-EV71 vaccine, in order to enable the cross sub-

genogroups protection, is the important question.

1.6.3.2 Attenuation by clustered charge-to-alanine mutagenesis

Based on the charge of the side chain and its interaction with the aqueous

environment, amino acids can be divided into several groups: charged, polar,

hydrophobic and neutral. The positive charged side chains are found in lysine

(Lys), arginine (Arg), and in some cases in histidine (His), whereas the negative

charged side chains are found in glutamine (Gln) and aspartate (Asp). Charged

residues and especially their clusters, which are strongly hydrophilic, are exposed

to the solvent and usually found on the outside of the folded protein whereas

hydrophobic aa-residues tend to be inside (Cunningham & Wells 1989;

Wertman, Drubin & Botstein 1992). It has been shown that hydrophobic

interactions in the folded protein make the largest contributions to its stability


72

(Alber et al. 1987). Residues with high solvent accessibility are much less

susceptible to destabilizing substitutions, but since they form hydrogen bonds

with other biomolecules or with the solvent, disruption of charged clusters might

interfere in these interactions, making them more thermosensitive (Alber 1989).

Indeed, it has been shown that the replacement of multiple charged

residues with hydrophobic alanine (Ala) in the receptor binding site of the human

growth hormone (hGH) disrupted interactions of the protein in vitro, but did not

severely alter its secondary structure (Cunningham & Wells 1989). Choice of a

replacement residue fell on Ala because:

“… it eliminates the side chain beyond the [beta] carbon yet does not

alter the main-chain conformation as can glycine or proline nor does it

impose extreme electrostatic or steric effects. Furthermore, alanine is the

most abundant amino acid and is found frequently in both buried and

exposed positions and all variety of secondary structures”

(Cunningham & Wells 1989).

The approach of site-directed mutagenesis to replace charged residue

clusters with Ala was applied to the actin gene (ACT1) of Saccharomyces

cerevisiae (Wertman, Drubin & Botstein 1992). A cluster was defined as a

stretch of 5 amino acids, in which 2 or more were charged residues. Examination

of mutant phenotypes in vivo revealed that 44% of the mutations (16/36) led to a

temperature-sensitivity. Comparable data was obtained in experiments on a

vaccinia virus gene G2R (Hassett & Condit 1994). As a result of the clustered

charge-to-alanine mutagenesis, 44% (4/9) of the mutants were ts to some degree

according to the plaque assay and 33% (3/9) displayed clear temperature

sensitivity in the single-step growth experiment.

Clustered charged-to-alanine mutagenesis of the 3Dpol of PV1 (Mahoney

strain) was used in order to generate a collection of ts mutant viruses (Diamond

& Kirkegaard 1994). Cluster definition was in accordance with the criteria used

by Wertman, Drubin et al. Each charged residue within such clusters was

replaced with alanine. Because of the size of the 3Dpol, it was difficult to

construct all possible mutations according to this algorithm. Therefore, only 27


73

mutants were generated and 12 of those were rescued in HeLa cells. Nine mutant

viruses (35%) displayed ts phenotypes and showed defects in RNA accumulation

and virus yield at the non-permissive temperature 39.5ºC. The mutations

responsible for the temperature sensitivity were mapped mainly to the N-terminal

third of the 3Dpol. Later, when crystal structure of the 3Dpol of PV was

determined, the N-terminus was found to be buried in a surface pocket of the

fingers domain (Thompson & Peersen 2004). Hydrogen bonds between the

amino group of the N-terminal glycine and carbonyls of the protein backbone

(residues 64, 239 and 241) stabilise the structure, and position the Asp238 in the

active site of the 3Dpol. Asp238 is essential in the polymerisation process as it

interacts with the 2′-hydroxyl group of the incoming rNTP. Therefore, mutations

within the N-terminus of the 3Dpol are likely to affect stability of the fingers

domain structure, and change the positioning of the Asp238 against the active

site. This in turn can result in a complete abolition or reduction of the polymerase

activity.

Parkin et al. (1996) applied the approach of clustered charged-to-alanine

mutagenesis to the polymerase subunit PB2 of the influenza virus A. It allowed

them to generate several vaccine candidates with ts and attenuated characteristics

in mice and ferrets. Clusters of 4 or 5 charged amino acids in a window of 5 were

targeted in site-directed mutagenesis (SDM). The increased stringency in their

definition of a “cluster” allowed them to identify five ts mutants of the six

viruses containing mutated PB2 genes. Additionally, growth of some mutants at

low temperature was also affected, as revealed by a reduction in plaque size.

Hanley et al. (2002) showed that the level of temperature sensitivity of

replication in vitro does not correlate with attenuation in vivo. Moreover, some of

the ts mutations can be dependent on the host cell line and referred as

temperature-sensitive host range (tshr) mutants. A collection of mutations was

generated in the nonstructural gene NS5 of the Dengue virus type 4 (DEN4). The

reason for selecting NS5 protein for mutagenesis was that it is the most highly

conserved protein among four DEN serotypes. Of the 32 recovered mutant

viruses containing a single pair of charge-to-alanine substitutions, 13 (41%) were


74

ts, both in Vero and HuH-7 cells. Eight mutants (25%) exhibited a tshr

phenotype: one was ts only in Vero cells, and seven were ts only in HuH-7 cells.

Fourteen (44%) mutant viruses showed restricted replication in the suckling

mouse brain, but there was no correlation with temperature sensitivity in either

Vero or HuH-7 cells. Additionally, there was no association between attenuation

in mouse brain and location of mutations in NS5 functional domains.

Furthermore, it was shown that a combination of two pairs of charge-to-alanine

mutations in one recombinant virus generates a mutant strain possessing some

characteristics different from parent viruses bearing a single pair of mutations.

For instance, one of the recombinant viruses was more ts in both cell lines, but

lost attenuation in vivo. In contrast, another recombinant virus was more ts in

Vero cells and more attenuated than either parent virus, but was not more ts in

HuH-7 cells.

The clustered charged-to-alanine mutagenesis is a promising method to

generate a collection of mutant viruses, including those with ts and/or attenuated

phenotype. Although temperature sensitivity is considered as a characteristic

related to an attenuated phenotype, some studies suggest that not every mutation

shows direct correlation of temperature sensitivity in vitro and attenuation in vivo

(Arita et al. 2005).

1.6.3.3 Approaches toward the development of a genetically stable

attenuated vaccine strain

The high mutation frequency of EV71 poses a challenge to the

development of an attenuated vaccine that would be genetically stable over time.

Viral replication directed by the 3Dpol is a error-prone process resulting in

approximately one miss-incorporation per 1 x 103–105 nucleotides synthesized

(Domingo & Holland 1997; Drake et al. 1998). As a result a viral population

consists of numerous related viral genomes, named quasispecies (Eigen 1993;

Holland, De La Torre & Steinhauer 1992). Some of the quasispecies variants


75

may possess mutations which will allow the virus to escape from the host

immune system, or will result in a highly virulent phenotype.

Attempts are being made towards the development of an attenuated

vaccine candidate with increased fidelity of replication. For PV, a single

mutation from Glycine to Serine (Gly64Ser) in the viral 3Dpol resulted in

reduction of mutation rate of the virus due to increased fidelity of RNA

replication (Pfeiffer & Kirkegaard 2003). The Gly64Ser mutant displayed an

attenuated phenotype in mice and did not have a growth defect in tissue culture

(Pfeiffer, Kirkegaard & Manchester 2005; Vignuzzi et al. 2006). The mutant

virus could not establish a productive infection in the CNS but replicated and

accumulated at wild-type levels in the spleens of infected mice (Vignuzzi, Wendt

& Andino 2008). According to the 3Dpol crystal structure, Gly64 participates in

positioning the active residue Asp238 within the catalytic site of the enzyme.

Thompson and Peersen (2004) suggested that Gly64Ser mutation might result in

an altered structure of the 3Dpol active site. It may increase the time of the

phosphoryl transfer reaction, and thus allow dissociation of incorrectly matched

rNTP-template complexes. High level of sequence similarity of the 3Dpol gene

among Picornaviruses, assumes possibility that Gly64Ser mutation will lead to

the same effect in EV71.

1.6.4 Synthetic peptides or epitope peptide vaccine

Synthetic peptides representing antigenic determinants of the virus are

able to stimulate specific immune response. However, immunogenicity of

synthetic peptides is generally lower than that of whole virus particles.

Protective effect of antiserum raised against the synthetic peptide SP70

spanning amino acids 208-222 of the VP1 capsid protein of EV71 (sub-

genogroup B4) was shown in vivo by Foo et al. (2007a). Passive transfer of anti-

SP70 antiserum with a neutralizing antibody titre of 1:32 one day after lethal

challenge (1 x 103 TCID50) with EV71 strains (sub-genogroup B2, B4 or B5),


76

provided 80% protection in suckling BALB/c mice throughout the experimental

period of 21 days. Upon lethal challenge with EV71 strains from the sub-

genogroup C2 or C4, the anti-SP70 antiserum showed 70% protective efficacy.

Compare to this data, anti-EV71 antiserum raised against heat-inactivated virus,

showed 100% protection after lethal challenge with EV71 if administrated at the

titre of 1:32.

Monoclonal antibodies generated in BALB/c mice against synthetic

peptides SP55 (amino acids 163-177) and SP70 (amino acids 208-222), were

demonstrated to possess neutralizing activity against EV71 (Fuyang strain, sub-

genogroup C4) in an in vitro neutralization assay (Li et al. 2009). Complete

protection of RD cells was observed with serum titres at 1:4 for SP55, 1:16 for

SP70 or 1:48 for heat-inactivated virus. Mouse anti-SP70 monoclonal antibody

could be a promising approach in anti-EV71 therapy after its humanization or

chimerization.

The main disadvantage of synthetic peptides is often poor immunogenicity

due to their small sizes and absence of strong T-helper determinants (Foo et al.

2007a). Therefore, an ideal synthetic peptide-based vaccine should contain both

B-cell epitope(s) and T-cell epitope(s) that are able to induce a protective

antibody response as well as a cytotoxic T-cell response. Multimerization of

peptides known as multiple antigen peptide (MAP) method, has been shown to

be a valuable approach in overcoming the poor immunogenicity of short peptides

by combining several synthetic peptides, representing both B and C-cell epitopes,

and attaching immunomodulating molecules for targeting and delivery (reviewed

by Tam 1996). Lipidation has been shown to allow peptide antigens to be

administrated orally, intragastrically and intranasally, without any extraneous

adjuvant. Such lipidated multiple epitopes MAPs have been reported to elicit

significantly higher mucosal and cell-mediated immune responses to HIV-1,

when compared to conventional peptide-protein conjugates or monoepitope

MAPs.


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1.6.5 Protein sub-unit vaccine

A sub-unit vaccine usually consists of a recombinant viral protein

expressed in prokaryotic or eukaryotic cells after their transformation with

recombinant plasmid DNA. The expressed protein must be purified prior to

administration by injection or, in those cases when transformed cells are non-

pathogenic and non-colonizing, they can be used as a delivery system via oral

route (Raha et al. 2005).

Protein sub-unit vaccines offer lower immunogenicity and their cytokine

production profiles are different from those of the whole pathogen. Comparative

studies showed that suckling mice born to dams immunized with a purified

recombinant VP1 protein expressed in E. coli, could survive at the same level

(80%) as those born to inactivated virus immunized dams only if they were

administrated to a 10 times lower challenge dose (230 LD50 virus/mouse against

2300 LD50 virus/mouse) (Wu et al. 2002). Proliferating spleen cells of mice

immunized with heat inactivated virus or recombinant VP1 protein produced

high levels of IL-4 or IL-10, respectively.

Transgenic tomato fruit expressing the VP1 protein of EV71 was

developed by Chen et al. (2006). BALB/c mice fed with transgenic tomatoes

developed mucosal IgA in feces and systemic IgG in serum at double levels of

those demonstrated by the control mice group on day 56 post the first challenge

(4 boosters in total). In an in vitro neutralization assay, serum obtained from the

experimental mice, showed neutralization titre of 1:16 while the positive control

serum (from human patients) exhibited the titre of 1:128.

Another study described EV71 VP1 expression and delivery by using an

attenuated Salmonella enterica serovar (Chiu et al. 2006). Oral immunization

(plus 2 boosters) of BALB/c mice with the Salmonella-based VP1 vaccines

resulted in eliciting the neutralization antibody with titres between 1:2 and 1:8. In

a lethal challenge assay (107 TCID50 per mouse) newborn ICR mice

demonstrated a 50-60% survival rate when born to VP1 vaccinated dams, or 92%

when born to dams immunised with an inactivated virus.


78

The first transgenic animal system expressing the VP1 capsid protein of

EV71 was generated in Taiwan (Chen et al. 2008a). Homozygous transgenic

mice (ICR) expressed and secreted recombinant VP1 protein into milk at

concentration levels of 2.51 ± 0.63 mg/ml. Four day old mice suckling milk from

the ICR or transgenic female were i.p. injected with EV71 (3.2 x 104 TCID50 per

mouse). EV71-related symptoms such as skin lesion and limb paralysis

developed accordingly in 80% and 70% of animals receiving wild-type milk. No

clinical manifestation was observed in mice suckling VP1-transgenic milk.

Serum obtained from those pups, at the age of 6-8 weeks, showed a significant

antiviral effect in an in vitro neutralization assay at serum dilutions 1:5 and 1:10.

One of the most successful sub-unit vaccine candidates developed recently

is a Baculovirus-based VP1 expression system. The VP1 gene of EV71-Fuyang

strain (sub-genogroup C4) was cloned under baculovirus polyhedron promoter

(ph) in Bac-Pph-gp64-VP1 construct, or under White Spot Syndrome Virus

(WSSV) iel promoter in Bac-Piel-gp61-VP1 construct (Meng et al. 2011). In

both cases, VP1 was expressed in insect cells (Sf9) after transfection with the

constructs. The recombinant viral particles displayed VP1 protein fused to the

baculovirus gp64 envelope protein, and induced anti-EV71 humoral immune

response in mice. After subcutaneous injection and an additional booster of 108

pfu of the purified recombinant baculovirus Bac-Pph-gp64-VP1 or Bac-Piel-

gp61-VP1, the immunized BALB/c mice serum reached 1:32 and 1:64

neutralization titres, respectively. The highest titres were observed on day 28

post-immunization, and remained at the same level until the end of the

experiment (56 days post-immunization).

1.6.6 Virus-like particle vaccine

The virus-like particle (VLP) consists of the outer coat of a virus, with no

viral genetic material inside, meaning it’s not infectious. The VLP is a self-

assembling particle formed by the viral structural proteins. As a result, it contains


79

more antibody-eliciting viral epitopes compared to short peptides or sub-unit

vaccines.

The first VLPs of EV71 were generated by Yu-Chen Hu et al. (2003).

VLP assembly was achieved in insect cells (Sf9) after co-infection with two

types of recombinant baculoviruses: Bac-P1 and Bac-3CD. Simultaneous

expression of the P1 and 3CD proteins of EV71 resulted in formation of the

particle aggregates similar to those formed by an authentic EV71. Later, the same

research group constructed the recombinant baculovirus vector carrying both P1

(under the Polyhedrin promoter, Pph) and 3CD (under p10 promoter) genome

regions of EV71 (Chung et al. 2006). Infections with a single construct, Bac-P1-

3CD, yielded higher VLP production than co-infections with two original

constructs. Intraperitoneal immunization of BALB/c mice with the purified VLPs

(10µg/mouse), and a following booster with the same dose in 4 weeks, elicited

anti-EV71 antibodies with a maximum neutralization titre of 213 in week 7

(Chung et al. 2008). Immunization with the denatured VLPs or heat-inactivated

EV71 resulted in significantly lower neutralization titres, 29.5 and 211,

respectively. The VLPs immunization induced both Th1 and Th2 immune

responses, which resulted in high levels of cytokines IFN-γ, IL-2 and IL-4. The

neonatal mice born to VLPs-immunized dams and administered to a lethal viral

challenge (1000 LD50) survived at a rate of 88.9%. In contrast, mice groups

immunized with the denatured VLPs and heat-inactivated EV71 showed 45.5%

and 57.9% survival rates, respectively. This data confirmed the importance of the

preservation of viral immunogenic epitopes in eliciting the sufficient protective

immunity. To reduce the competition from the 3CD expression and improve the

VLPs yield, the p10 promoter of the Bac-P1-3CD was replaced with a weaker

CMV promoter. Additional optimization of process parameters such as cell

density, percentage of air saturation, culturing in the bioreactor, etc., elevated the

VLP production up to 64.3mg/L which was 43-fold higher compared to the old

process (Chung et al. 2010). Altogether, this data demonstrated the potential of

the recombinant baculovirus system culture for industrial large-scale production

of EV71 VLPs vaccine.


80

1.6.7 DNA vaccine

A DNA vaccine is a genetically engineered circular double stranded DNA

molecule encoding defined proteins or immunogenic epitopes of the pathogen

with appropriate promoter for expression.

The first DNA vaccine against EV71 was developed by Wu et al. using

the VP1 gene inserted into plasmid DNA under the CMV promoter (Wu et al.

2002). Adult mice injected with 100 µg of plasmid DNA developed anti-EV71

antibody with neutralization titre similar to that of an inactivated virus and

recombinant VP1 protein. However, in vivo protection of suckling mice against

lethal challenge with EV71 was the lowest for the DNA vaccine.

Another anti-EV71 DNA vaccine based on the VP1 gene ligated into an

eukaryotic vector was constructed by Tung et al. (2007). BALB/c mice were

immunized with 100 µg of the construct and received two additional boosters on

day 14 and 28. The injected animals developed protective antibody, however, the

neutralization titre was significantly lower to that of the EV71-infected human

serum. The anti-VP1 IgG level was rising during 28 days post immunization, but

dropped significantly by day 41.

The anti-EV71 DNA vaccine remains a viable strategy. However, further

development of DNA vaccine candidates is required in order to increase their

immunogenicity and provide long-lasting protection against the virus.


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1.7 Project aims

Almost forty five years have passed since EV71 clinical isolates were first

recovered from stool and tissue specimens of patients with CNS complications

(Schmidt, Lennette & Ho 1974). Since that time certain progress has been made

towards understanding the biology of the pathogen and associated diseases.

Currently, a range of promising anti-EV71 inhibitors and vaccine candidates are

under development, however, there has been no specific anti-EV71 therapy

approved. Due to widespread circulation of EV71 and a high percentage of

associated neurological complications the development of preventive strategies

against new infections is a top priority. An attenuated vaccine would be the most

efficient approach to generate both humoral and cell-mediated immune response.

In order to succeed in this direction, comprehensive understanding of molecular

determinants of the virus pathogenicity and attenuation is required.

The major objective of this work is to identify molecular determinants

within the EV71 genome which can lead to inhibition of the virus, and which can

be potentially useful in an attenuated vaccine development. SDM will be

employed as a tool to generate EV71 mutants, whose growth properties are to be

studied in cell culture. Specific aims targeted in the project are as follows:

Construction of an infectious cDNA clone of EV71 which is to be used in

SDM. Two strategies are to be employed and assessed in their efficacy to

obtain an infectious virus progeny from EV71 cDNA constructs.

Analysis of the factors which contribute to infectivity of the in vitro generated

viral RNA upon its transfection into cell culture. Elimination of some steps

which do not contribute to the infectivity of the EV71 cDNA clone would

simplify the overall procedure. On the other hand some others factors might

be essential and need to be considered in order to rescue the virus in vitro.

Identification and analysis of the molecular basis of EV71 adaptation to an

increased temperature. Specific genetic determinants of EV71 and their


82

contribution to the adaptive phenotype will be under particular focus, as well

as, the mechanism of the virus adaptation.

Construction of EV71 mutants within the 5′UTR and assessment of their

growth properties in cell culture. SDM will be performed to alter the

secondary structure of the Vth and VIth domains in order to generate Sabin 3,

CAV8 and CAV16-like EV71 mutants.

Generating a collection of EV71 mutants within the VP1 capsid protein by

charged-to-alanine mutagenesis. Assessment of the in vitro inhibition level

offered by single VP1 mutations and by their combination with substitutions

within the 5′UTR.

Results of this work are expected to contribute to our knowledge of the

molecular biology of EV71 and understanding of the attenuated phenotype.

83

CHAPTER 2

CONSTRUCTION OF AN INFECTIOUS cDNA CLONE OF

EV71

CHAPTER 2 Construction of an infectious cDNA clone of EV71

84

2 CONSTRUCTION OF AN INFECTIOUS cDNA CLONE OF EV71

2.1 Introduction

Genetic manipulation of viruses cannot be achieved without a DNA clone

of the viral genome or the targeted genes. In the case of RNA viruses, reverse

genetics can be utilized in order to produce cDNA clones of the viral RNA.

Construction of the infectious cDNA clone poses a further challenge as such a

clone has to include the entire viral genome with both 5′ and 3′ terminus, and

poly(A) tail. The length of the viral genomes consisting of several kb often

makes it difficult to amplify the entire genome in a single RT-PCR and thus

requires subsequent cloning and ligation of the produced cDNA fragments. This

makes the procedure very laborious and time consuming. Constructed cDNA

clones are often non-infectious and require further sub cloning or correction of

the viral genome at inadvertently mutated positions.

2.2 Materials and methods

In this study, two approaches have been undertaken in order to produce an

infectious EV71 cDNA clone. The first strategy consisted of RT followed by 2

rounds of PCR. The first round amplified the 5′ and 3′ halves of the EV71

genome which were used as templates in the second round in order to produce a

full length genome. The second strategy utilized a long PCR DNA polymerase

which was able to amplify the entire genome in a single reaction (Figure 2-1).


85

A. Strategy 1 B. Strategy 2

Propagation of EV71 in Vero cells Propagation of EV71 in Vero cells

↓ ↓ Total RNA extraction

QIAamp Viral RNA Mini Kit (QIAGEN) Total RNA extraction

QIAamp Viral RNA Mini Kit (QIAGEN) ↓ ↓

Reverse Transcription Transcriptor High Fidelity cDNA Synthesis Kit

(Roche Applied Science)

Reverse Transcription SuperScript™ II Reverse Transcriptase

(Invitrogen)

↓ ↓ Removal of the RNA template

Ribonuclease H (Promega) Removal of the RNA template

Ribonuclease H (Promega) ↓ ↓

PCR amplification Expand Long Range dNTPack (Roche Applied

Science)

1st round: 25 cycles

PCR amplification iProof High-Fidelity DNA Pol (BioRad)

single round PCR: 20 cycles

↓

↓ Gel purification

Wizard® SV Gel and PCR Clean-Up System (Promega)

↓ 2nd round: 31 cycles

↓ Gel purification

Wizard® SV Gel and PCR Clean-Up System (Promega)

Gel purification Wizard® SV Gel and PCR Clean-Up System

(Promega)

↓ ↓

Addition of the 3´A overhangs GoTaq® Flexi DNA Polymerase (Promega)

↓ Cloning

TOPO® XL PCR Cloning Kit (Invitrogen) One Shot® Chemically Competent E. coli

(Invitrogen)

Cloning TOPO® XL PCR Cloning Kit (Invitrogen)

XL 10-Gold® Ultracompetent E. coli (Stratagene)

Figure 2-1: Construction of EV71 cDNA clones.


86

2.2.1 Cell culture and virus

The 6F/AUS/6/99 strain of EV71 (GenBank accession # DQ381846,

genotype C2) and African green monkey kidney (Vero) cell line were kind gifts

from Prof Peter McMinn (The University of Sidney). The original virus stock

was the fifth passage of EV71 in RD cells. Virus used in this study was obtained

after further propagation of the original virus in Vero cell culture.

Vero cells were maintained in Minimum Essential Medium (MEM) with

Earle's salts, L-glutamine and sodium bicarbonate (Sigma) supplemented with

5% fetal bovine serum (FBS, Invitrogen). The cells were cultured in 75 cm2

flasks at 37°C in the presence of 5% CO2. The cells were observed daily using a

microscope. When the confluence was at 90-100% level, the medium was

discarded and cells were rinsed with phosphate-buffered saline (PBS) and treated

with 0.05% Trypsin-EDTA (Invitrogen) at 37°C for 5 min. Fresh growth medium

was added to minimize cytotoxic effect of Trypsin, and an optimal volume of

cells was separated out for further propagation.

2.2.2 Total RNA extraction

Total RNA was extracted from EV71-infected Vero cell culture

supernatant with QIAamp Viral RNA Mini Kit (QIAGEN) following the

manufacturer's recommended protocol. Extracted RNA sample was eluted in 120

µl of milliQ H2O and stored at -80°C until use.


87

2.2.3 Reverse transcription of EV71 RNA

The reverse transcription (RT) was carried out using a MyCycler PCR

instrument (Bio-Rad).

The full-length EV71 cDNA was produced with a Transcriptor High

Fidelity cDNA Synthesis Kit (Roche Applied Science) or SuperScript™ II

Reverse Transcriptase (Invitrogen) at 45°C for 60 min or 42°C for 50 min,

respectively. The anchored-oligo (dT)18 primer (Roche Applied Science) was

used in reverse transcription at final concentration 2.5 µM. The RNase Inhibitor,

dNTPs mix, DTT and Reverse Transcriptase were used in accordance with the

manufacturer’s instructions.

Prior to PCR the RNA template complementary to cDNA was removed

from the RT reaction with Ribonuclease H (Promega). The endonuclease was

added directly to the RT product at final concentration of 1 U per 10 µl of

reaction volume. Reaction was incubated at 37°C for 20 min.

2.2.4 Design of EV71 gene specific primers

PCR primers used in full-length EV71 genome amplification were

designed based on the sequence of the strain 6F/AUS/6/99, GenBank accession

#DQ381846. The sense primer Ft7 contained the T7 promoter sequence at its 5′

end followed by the first 21 nucleotides of EV71. The anti-sense primer RMluI

consisted of a MluI restriction site followed by a stretch of 17 thymidine residues

and 25 nucleotides corresponding to the sequence of EV71 immediately before

the poly(A) tail. The anti-sense primer RMluI-30 contained a MluI restriction

site at its 5′ end followed by a stretch of 29 thymidine residues and 3 nucleotides

corresponding to the sequence of EV71 immediately before the poly(A) tail. In

order to stabilize the amplicons, both primers had two additional guanosine

residues at their 5′ ends (Boyer & Haenni 1994) (Table 2-1).


88

Primer name

Sequence (5′-3′)* Application(s)

Ft7 ggtaatacgactcactatagggttaaaacagcctgtgggttgc Full length genome amplification

RMluI ggacgcgttttttttttttttttttgctattctggttataacaaatttac Full length genome amplification

RMluI-30 ggatctacgcgttttttttttttttttttttttttttttttgct Full length genome amplification

F416 ggtgtgaagagcctattgag Sequencing the VP4 R644 caccggatggccaatcca Sequencing the 5′UTR,

colony PCR F795 ctcagctaccgagggttc Sequencing the VP2 F1632 tgcgacgccagtgatc Sequencing the VP3 R1779 tgctgagacgccatcgtc Sequencing the VP2 F2386 accatgaagttgtgcaagg Sequencing the VP1 R2551 gatggctacttacctgcgta Sequencing the VP3 R3411 gtcgttatgagtagcaagatgg Sequencing the VP1 F3821 gcatggggtttacagacgcag Amplification of the 3′-

half of the EV71 genome, sequencing the 2B and 2C

R3991 caagtgttgccgttaatgtgacca Amplification of the 5′-half of the EV71 genome, sequencing the 2A and 2B

F4556 acccagaccactttgacg Sequencing the 2C and 3A

F5222 gccacttaaacagagctgt Sequencing the 3B and 3C

F5851 ggtaaggtgattgggatc Sequencing the 3D F5987 tcaacggacctactcgcactaagc Sequencing the 3D F6013 gaaccaagtgtctttcacgatgtg Sequencing the 3D R6050 ttagtgccctcgaacaca Sequencing the 3B and

3C R6251 tctgtaccgtaacaagcatcctc Sequencing the 3B and

3C F6536 ccaatccaggtacagtca Sequencing the 3D,

3′UTR, colony PCR R6739 cttctatgagagacagtgc Sequencing the 3D R7380 accagtcattaacacgacc Sequencing the 3D

Table 2-1: Primers used in construction of EV71 cDNA clones, colony PCR and cycle

sequencing.

*Restriction sites are in Italic. T7 promoter sequence is underlined.


89

Primers used for cycle sequencing were designed with FastPCR© v5.2

software (http://www.biocenter.helsinki.fi/bi/programs/fastpcr.htm) based on the

sequence of 6F/AUS/6/99 strain. The EV71 genome was divided into 9

overlapping fragments which were sequenced with forward and reverse primers

located approximately 1 kb apart (Table 2-1).

2.2.5 RT-PCR amplification

The polymerase chain reaction was carried out using a MyCycler PCR

instrument (Bio-Rad).

Strategy 1:

Strategy 1 was based on the overlapping PCR and consisted of two

rounds. The 5′ and 3′ half-genome fragments of EV71 were amplified with

Expand Long Range dNTPack (Roche Applied Science) using Ft7/R3991and

F3821/RMluI primers, respectively (Figure 2-1). The 5′ and 3′ halves were gel-

purified and used as templates in full-length genome amplification with Ft7 and

RMluI primers. In the 1st round, the PCR mixture contained 1 x PCR buffer, 0.3

µM of each primer, 0.4 mM dNTPs mix and 3.15 U of Expand Long Template

enzyme mix. The cDNA product was added up to a total reaction volume of 45

µl. The PCR conditions were set up at 95°C for 2 min of initial denaturation,

followed by 10 cycles of 95°C for 10 sec, 60°C for 15 sec, 68°C for 3 min 50

sec, followed by 15 cycles of 95°C for 10 sec, 60°C for 15 sec, 68°C for 3 min

50 sec with a 10 sec increment in each cycle. Final elongation was carried out at

68°C for 7 min. The 2nd round PCR mixture contained 1 x PCR buffer, 0.3 µM of

Ft7 and RMluI primers, 0.4 mM dNTPs mix and 3.75 U of Expand Long

Template enzyme mix in a total reaction volume of 50 µl. Approximately 10 ng

of both 5′ and 3′ DNA were used as templates. The 2nd round included initial

denaturation at 95°C for 2 min, then 1 cycle at 95°C for 10 sec, 56°C for 15 sec,

68°C for 3 min 50 sec, followed by 10 cycles of 95°C for 10 sec, 56°C for 15

sec, 68°C for 7 min 30 sec, followed by 20 cycles of 95°C for 10 sec, 56°C for


90

15 sec, 68°C for 7 min 30 sec with a 10 sec increment in each cycle. Final

elongation was carried out at 68°C for 7 min.

Strategy 2:

Strategy 2 consisted of a single round PCR of 20 cycles. The PCR mixture

contained 1 x High-Fidelity PCR buffer, 0.5 µM of Ft7 and RMluI or RMluI-30

primers, 0.2 mM dNTP mix and 1 U of iProof High-Fidelity DNA Pol (BioRad)

in a total reaction volume of 50 µl. 2 µl of cDNA after reverse transcription with

SuperScript™ II RT (Invitrogen) were used as a template. The PCR was carried

out under the following conditions: initial denaturation at 98°C for 30 sec,

followed by 1 cycle at 98°C for 7 sec, 56°C for 20 sec, 72°C for 3 min 30 sec,

followed by 1 cycle at 98°C for 7 sec, 46°C for 20 sec, 72°C for 3 min 30 sec,

followed by 18 cycles at 98°C for 7 sec, 61°C for 20 sec and 72°C for 3 min 30

sec. Final elongation was carried out at 72°C for 5 min.

2.2.6 Agarose gel electrophoresis of DNA

0.7-2.0% agarose gel stained with ethidium bromide (EtBr) was used in

routine electrophoresis of DNA (estimating DNA concentration, visualisation of

PCR products, etc.). Agarose gel was prepared in 1 x Tris-acetate-EDTA (TAE)

buffer. EtBr was added to the gel at final concentration of 0.5 µg/ml.

Blue/Orange 6x Loading Dye (Promega) was used for loading DNA samples into

gel wells. The 100 bp or 1 kb DNA Ladders (Promega) were used to determine

the size of double-stranded DNA (dsDNA). When estimation of dsDNA

concentration was required the 100 bp or 1 kb DNA Ladders (New England

BioLabs) were used. DNA electrophoresis was run in 1 x TAE buffer at a

constant voltage of 5 V per 1 cm of the distance between the electrodes. When

separation of the DNA ladder was sufficient the DNA samples were visualized

with UV light.

When integrity of a DNA sample was required for further genetic

manipulation the DNA was run on agarose gel stained with crystal violet and


91

visualized under normal light. Crystal violet stock solution (1-2 mg/ml in water)

was added to the gel at final concentration of crystal violet 0.8-1.6 µg/ml.

Crystal violet 6 x Loading Dye (Appendix I) was used for loading DNA samples

into gel electrophoresis wells and visualizing the DNA. DNA electrophoresis was

run in 1 x TAE buffer at constant voltage of 5 V per 1 cm of the distance

between the electrodes.

2.2.7 Gel purification of DNA

Gel purification was used in cleanup of the full-length EV71 cDNA prior

to cloning and in vitro transcription reaction.

The PCR product containing the full-length EV71 genome was purified

with Gel Purification Reagents supplied with TOPO® XL PCR Cloning Kit

(Invitrogen). The DNA was excised from 0.7% agarose gel stained with crystal

violet and purification was performed in accordance with manufacturer’s

recommended protocol. DNA was eluted in 40 µl of milliQ H2O. Small aliquot of

the purified DNA was run on 0.7% agarose gel stained with EtBr in order to

estimate the DNA concentration.

Prior to in vitro transcription reaction, the DNA template was gel purified

with Wizard® SV Gel and PCR Clean-Up System (Promega). DNA was eluted

in 20 µl of milliQ H2O and concentration was estimated by agarose gel

electrophoresis. DNA was stored at -80°C until use.


92

2.2.8 Cloning of the full-length EV71 genome

Full length EV71 genome was ligated to pCR®-XL-TOPO® vector with

TOPO® XL PCR Cloning Kit (Invitrogen) (Figure 2-2).

Figure 2-2: pCR®-XL-TOPO® vector.

(Adapted from Catalog, Invitrogen Corp, CA, USA).


93

Prior to cloning, the DNA amplified with iProof High-Fidelity DNA Pol

(Strategy 2) was incubated with GoTaq® Flexi DNA Polymerase (Promega) at

72°C for 30 min in order to add 3′A overhangs. The reaction mixture contained

1x Colorless GoTaq® Flexi Buffer, 1.5 mM of MgCl2, 200 µM of dATP, 0.5 µg

of DNA and 10 U of the enzyme in 20 µl of total volume. 4 µl of this reaction or

50-100 ng of the purified PCR product from Strategy 1 were mixed with 1 µl of

the pCR®-XL-TOPO® vector (Invitrogen). The ligation mixture was incubated

for 5 min at room temperature, then 1 µl of the 6 x TOPO® Cloning stop solution

was added and tubes were placed on ice. Transformation was carried out using

the One Shot® Chemically Competent E. coli (Invitrogen) or XL 10-Gold®

Ultracompetent cells (Stratagene). The cloning reaction (2 µl) was added to the

competent cells and incubated on ice for 30 min. The cells were then subjected to

heat shock at 42°C for 30 sec, and immediately placed on ice for 2 min. SOC

medium (250 µl) or NZY+ broth (500 µl) were added to the One Shot or XL 10-

Gold competent cell suspension, respectively, followed by shaking at 37°C for 1

hour. After the 1 hour incubation, tubes were centrifuged at 0.2 rcf for 5 min.

Clear supernatant was discarded and the cell pellet was resuspended in the

remaining volume of medium and plated on LB agar supplemented with 50

µg/ml kanamycin sulphate (Invitrogen). Incubation was carried overnight at

37°C.

2.2.9 Screening the E.coli transformants

The presence and orientation of the insert was checked by colony PCR or

by restriction digestion of the plasmid DNAs with MluI or BamHI.

Prior to the PCR, a few colonies from each transformation were picked

and resuspended in 15 µl of milliQ H2O. The PCR mixture contained 1 x

MangoMix (BioLine), 0.1 µM of M13 Reverse Pimer (Invitrogen), 0.1 µM of

R644 primer or F6536 (Table 2-1) and 5 µl of the resuspended E.coli cells in a

total reaction volume of 15 µl. The PCR was carried out under the following


94

conditions: initial denaturation at 94°C for 10 min, followed by 25 cycles at 94°C

for 30 sec, 55°C for 30 sec and 72°C for 30 sec. Final elongation was carried out

at 72°C for 10 min. The PCR products were visualized by 2% agarose gel

electrophoresis.

2.2.10 Isolation of plasmid DNA

Plasmid DNA was isolated from 9 ml of overnight culture of E. coli with

Wizard® Plus SV Minipreps DNA Purification System (Promega) following the

manufacturer's recommended protocol. Plasmid DNA was eluted in 100 µl of

nuclease-free H2O and stored at -80°C until use. Concentration of the purified

plasmid DNA was estimated by 0.7% agarose gel electrophoresis.

2.2.11 DNA automated cycle sequencing and nucleotide sequence

analysis

DNA sequencing was performed at The University of Melbourne’s

Department of Pathology.

Purified plasmid DNA containing the full-length genome of EV71 was

used as a template in the sequencing reaction with EV71 specific primers (Table

2-1). Two viral clones, one of each from Strategy 1 and 2, were sequenced in

full. Additionally, a few clones from each strategy were sequenced within the

5′UTR, VP1, 3C, 3B and 3D genes.

Sequence alignment was performed using the BioEdit v7.0.5.3 software

(Hall 1999). Strain 6F/AUS/6/99 was used as a reference sequence.

Pairwise distances for EV71 cDNA clones were calculated with the

Kimura 2-Parameter model (for nt sequences) and p-distance algorithm (for aa

sequences) incorporated in MEGA3 software (Kumar, Tamura & Nei 2004).


95

2.2.12 Restriction endonuclease digestion of DNA

Restriction digestion of the plasmid DNA was used in:

a) Verification of the orientation of the insert and estimating the plasmid size

after TA cloning;

b) Excising the EV71 cDNA insert prior to in vitro transcription;

c) Construction of EV71 cDNA with authentic 3′ end.

a) Plasmid DNA purified from the E.coli transformants was digested with BamHI

or MluI (New England BioLabs Inc.). The reaction mixture contained 1 x

NEBuffer 3 supplemented with 0.1 µg/µl of Bovine Serum Albumin (BSA), 0.5-

1.0 µg of substrate DNA and 10 U of enzyme in a total volume of 15 µl.

Restriction digestion was performed at 37°C for 2 hours. Small aliquot of the

digestion reaction was run on 0.7% agarose gel stained with EtBr in order to

visualize the DNA fragments.

b) Prior to in vitro transcription reaction the plasmid DNA containing the full

length EV71 genome was linearized with MluI and EcoRI or NotI (New England

BioLabs Inc.) (Figure 2-2). Restriction digestion was carried out at 37°C for 4

hours and followed by enzyme inactivation at 65°C for 20 min. Digestion

reaction contained 1 x NEBuffer 3 and 2-10 U of each enzyme per 1 µg of DNA.

Total volume of enzyme(s) never exceeded 10% of the reaction volume.

Restriction digestion with NotI was supplemented with BSA at final

concentration of 0.1 µg/µl. Digested DNA was visualized on 0.7% agarose gel

stained with crystal violet and used in further gel purification.

c) Full-length EV71 cDNA obtained by PCR amplification with iProof

polymerase and RMluI or RMluI-30 reverse primers was digested with MluI (as

described above). Digested DNA was ethanol precipitated and polished with

Mung Bean Nuclease in accordance with the manufacture recommended protocol


96

(New England BioLabs Inc.). The reaction was terminated by addition of SDS to

0.01% of final concentration.

2.2.13 Ethanol precipitation of nucleic acids

If de-salting or concentrating a DNA sample was required it was ethanol

precipitated with sodium acetate. Prior to precipitating the sample volume was

adjusted to 100 µl with milliQ H2O. 10 µl of chilled 3M Sodium Acetate (pH

5.2) and 330 µl of chilled Absolute Ethanol were added to the sample and mixed

by inverting the tube. Then the sample was incubated on ice for 30 min and

centrifuged at 14,000 x g for 30 minutes at room temperature. Supernatant was

carefully removed and 1 ml of 70% Ethanol was added into the tube.

Centrifugation was repeated at 14,000 x g for 15 minutes at room temperature.

Supernatant was discarded and a DNA pellet was allowed to air dry for 30-60 sec

with the tube lid opened. Then the desired amount of milliQ H2O was added into

the tube in order to dissolve the DNA. Ethanol precipitated DNA was stored at -

80°C until use.

Sodium chloride at 0.2 M of final concentration was used instead of

sodium acetate for precipitation of DNA samples containing SDS, for example

DNA samples polished with Mung Bean Nuclease. Rest of the precipitation

protocol remained the same as described above.

2.2.14 in vitro transcription of EV71 cDNA clones

RiboMAX™ Large Scale RNA Production System-T7 (Promega) was

used in order to transcribe EV71 cDNA in vitro. The reaction was set up in

accordance with the recommended protocol in 20 µl of the total volume.

Reaction mixture was subjected to incubation at 37°C for 4 hours and followed

by a DNase treatment step for 30 minutes. RNase-Free DNase (Promega) was


97

added to the in vitro transcription reaction at final concentration of 1 U per 1 µg

of DNA template to be removed.

The Linear Control DNA supplied with RiboMAX™ Large Scale RNA

Production System-T7 was used as a template for production of RNA transcripts

of approximately 1.8 kb in length and served as a positive control of the in vitro

transcription reaction.

The in vitro transcribed RNA of EV71 was visualized by agarose gel

electrophoresis prior to being used in transfection of Vero cells.

2.2.15 RNA electrophoresis

In order to visualize and estimate concentration of the in vitro RNA

transcripts, the RNA samples were run on 1.5% native agarose gel stained with

EtBr. Autoclaved 1 x TAE buffer was used for gel preparation and

electrophoresis. The RiboRuler™ High Range RNA Ladder (Fermentas) was

used as a size/concentration reference for RNA. Additionally, 1 kb DNA Ladder

(Promega) was run on a gel next to in vitro transcribed RNA samples as a

reference for DNA template used in in vitro transcription. The 2 x RNA Loading

Dye (Fermentas) was used for loading RNA samples and the RNA ladder into gel

electrophoresis wells. Prior to loading, RNA samples mixed with 2 x RNA

Loading Dye were heated at 70°C for 10 min and then immediately chilled on

ice.

DNA electrophoresis was run in 1 x TAE buffer at constant voltage of 5 V

per 1 cm of the distance between the electrodes.


98

2.2.16 Transfection of Vero cells with in vitro RNA transcripts

Vero cells were seeded into a 24 well plate at concentration 2.5 x 105 cells

per well in 500 µl growth medium without antibiotics. The seeded plate was

incubated at 37°C in 5% CO2 incubator for 24 hours. After that, the growth

medium was replaced with Opti-MEM® I medium (Invitrogen) for another 24

hours. Vero cell culture showed 90-100% confluence on the day of transfection.

The entire in vitro transcription reaction was mixed with Opti-MEM® I

medium up to the total volume of 50 µl. In a separate tube, 2.5 µl of

Lipofectamine™ 2000 (Invitrogen) was mixed with 50 µl of Opti-MEM® I

medium and incubated at room temperature for 5-10 minutes. The premixed in

vitro transcription reaction and Lipofectamine were combined and added to the

cells. The plate was incubated at 37°C in 5% CO2 incubator for 5 hours. Negative

(transfection mixture without RNA) and positive (virus) controls were included

in each experiment.

In order to determine the cell culture growth conditions at which the

recovered virus reaches the highest titre, four different protocols of incubation

were used after transfection with in vitro RNA transcripts. Five hours after

transfection the OptiMEM medium was removed from designated wells and the

cells were maintained at the following growth conditions:

a) MEM supplemented with 5% FBS;

b) MEM supplemented with 10% FBS;

c) Cells were washed with PBS, treated with 200 µl of Trypsin,

0.05% EDTA (Invitrogen) and split into two wells in MEM

supplemented with 10% FBS;

d) One cell culture was retained in reduced serum medium containing

the RNA transcripts.

The cell cultures were incubated at 37°C in 5% CO2 incubator for 6 days.

The cytopathic effect was observed microscopically. The cells and supernatant

were harvested and stored at -80ºC.


99

In order to compare the growth kinetics of cell culture at different

experimental conditions, cells were collected and counted at 0, 5, 24, 48, 72, 96,

120 and 144 hours. Lipofectamine™ 2000 premixed with OptiMEM with no

RNA transcripts was added to the cells at 0 h and cells were maintained at the

same growth conditions as the cell cultures transfected with RNA.

2.2.17 Detection and quantitation of EV71 RNA

In order to monitor changes in EV71 RNA accumulation after transfection

of Vero cell culture with the in vitro RNA transcripts, the cells and supernatant

were collected at 5, 10, 24, 48, 72, 96, 120 and 144 hours post transfection and

used in RNA isolation followed by RT-Q-PCR. The QIAamp Viral RNA Mini

Kit (QIAGEN) was used in accordance with the manufacturer’s instructions. The

isolated RNA samples were subjected to a DNase (New England Biolabs)

treatment step at 37°C for 2 hours in order to prevent plasmid DNA carryover

from the in vitro transcription reaction. The DNase was inactivated by incubation

at 75°C for 10 minutes in the presence of 2 mM EGTA. Control PCR (no RT)

reactions with EV71 specific primers (F5987 and R6251), MangoMix™

(Bioline) and DNase treated RNA samples were performed to confirm the

complete digestion of the plasmid DNA.

Prior to real-time PCR, RT reactions were performed with GoScript™

Reverse Transcriptase (Promega) and Random Primers (Promega) in accordance

with the manufacturers recommended protocol with the exception that the

reaction mixture contained 9 µl of the RNA sample and 4.75 mM MgCl2. At the

end of the RT, the reverse transcriptase was heat inactivated for 15 min at 70ºC.

The Q-PCR was performed on the MyiQ PCR machine (BioRad) using SYBR®

Green I fluorescent dye. The real-time PCR conditions were optimised in order to

produce a single, well defined peak on the melting curve. Reaction volume of 20

µl consisted of 2 µl of the RT product previously diluted 1:100 with milliQ

water, 0.4 µM of each primer (F3821, R3991) and SensiMixPlus SYBR &


100

Fluorescein Mix (Quantace). The real-time PCR setting were as follows: 95ºC for

10 min, then 30 cycles of 95ºC for 15 sec, 60ºC for 30 sec and 72ºC for 30 sec,

followed by a final elongation at 72ºC for 10 min. The melting curve data

collection was set up from 95ºC for 15 sec with a decrease of the set point

temperature by 0.5ºC each cycle for 80 cycles. The amplicon size was 171 bp and

showed a single melting curve peak at 85.5-86.0ºC.

In order to monitor changes in EV71 RNA level inside the cells during the

first 24 hours after transfection, the supernatant was discarded at 5, 10, and 24 h

p.t., cells were washed with PBS and collected in the lysis buffer (Promega).

RNA isolation was performed with SV Total RNA Isolation System (Promega)

by following the recommended protocol. The DNase treatment step, control

PCR, RT and real-time PCR were performed as described above except that the

real-time PCR included 35 amplification cycles with 2 µl of the undiluted RT

product.

2.2.17.1 Preparation of EV71 DNA standards for Q-PCR

The standard curve method was used in order to quantify EV71 cDNA in

experimental samples. To prepare EV71 cDNA standards, the EV71 genome was

excised from the EV71 clone (plasmid DNA) and purified from the agarose gel.

The concentration and purity of the purified DNA were determined

spectrophotometrically on a Biowave II instrument (Biochrom Ltd). Purified

DNA was aliquoted and stored at -80ºC until use. Serial 10-fold dilutions of the

master stock were prepared prior to Q-PCR and used as external standards.

Quantitative data obtained in real-time PCR from the experimental samples were

analysed only if the DNA standard curve showed correlation coefficient of 0.998

or higher.


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2.2.18 Virus titration

Virus titres were determined by tissue culture inhibitory dose 50%

(TCID50) assay in Vero cells. Briefly, ninety-six well plates were seeded with 2 x

104 cells per well in MEM supplemented with 5% FBS and incubated for 24

hours prior to infection with the virus. Serial dilutions of the virus were prepared

in chilled MEM and inoculated into the 96-well plates, three wells per each

dilution. The inoculated plates were cultured at 37°C for 8-10 days and observed

for CPE. The TCID50 values were calculated according to the Reed-Muench

method (Reed & Muench 1938).

To estimate a number of plaque forming units (PFU) based on TCID50

titre, the Poisson distribution is commonly used. According to the calculations by

Bryan (1957), the PFU/ TCID50 ratio is ln(2), which gives 0.69. Therefore, 1 PFU

must be 0.69 TCID50. Although the obtained number was theoretically

calculated, it is proportional to the PFU (Wulff, Tzatzaris & Young 2012).


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2.3 Results

2.3.1 Amplification of the full-length genome of EV71

A full-length cDNA of the RNA genome of EV71 was successfully

obtained by both strategies (Figure 2-1). The first approach utilized an

overlapping PCR and consisted of two rounds. The first round PCRs produced

the 5′ and 3′-halves of EV71 genome with the expected product lengths of 4013

bp and 3614 bp, respectively (Figure 2-3).

A. B.

Figure 2-3: Agarose gel electrophoresis of the 5′ (A) and 3′-halves (B) of EV71

genome amplified in the 1st round PCR of Strategy 1.

Lane 1: the 5′-half of EV71 genome, 4013 bp in length; lane 2 and 3: DNA ladder; lane 4: the 3′-half of EV71 genome, 3614 bp in length.


103

The 5′-fragment was obtained at a lower concentration compared to the 3′-

fragment of the viral genome. That could be due to an incomplete extension of

the cDNA during reverse transcription step with the result that the majority of

cDNAs were shorter than an actual EV71 genome. In addition, Ft7 primer used

in PCR had a long non-complementary sequence at its 5′ end which could impair

efficient annealing of the primer to the cDNA template. The 5′ and 3′-halves

were gel purified and used in the 2nd PCR at approximately equal concentration.

The 2nd round PCR resulted in a specific PCR product of 7456 bp in length

comprising the full-length EV71 genome (Figure 2-4).

1 2

Figure 2-4: Agarose gel electrophoresis of the full-length genomic cDNA of EV71

obtained in overlapping PCR, Strategy 1.

Lane 1: cDNA of the full-length EV71 genome, 7456 bp in length; lane 2: DNA ladder.

10 kb 8 kb 6 kb


104

The 2nd approach consisted of a single round PCR with a highly

processive enzyme, iProof DNA polymerase, which allowed amplifying the full-

length EV71 genome within 20 cycles (Figure 2-5).

1 2

Figure 2-5: Agarose gel electrophoresis of the full-length genomic cDNA of EV71

obtained with iProof DNA polymerase.

Lane 1: cDNA of the full-length EV71 genome, 7456 bp in length; lane 2: DNA ladder.

10 kb 8 kb 6 kb


105

2.3.2 Characterization of the EV71 cDNA clones

The full-length EV71 cDNA was ligated into pCR®-XL-TOPO® vector

which was transformed into chemically competent E.coli. Several colonies were

screened by colony PCR to confirm transformation with the plasmid containing

the correct insert. Two orientations of the EV71 genome in a plasmid DNA were

possible due to TA cloning. PCR amplification with M13 Reverse primer, which

is complementary to plasmid DNA, and R644 or F6536 primers, which bind

within EV71 genome, showed both orientations present (Figure 2-6).


106

A. The EV71 positive cDNA strand cloned downstream of the M13 Rv priming site

B. The EV71 negative cDNA strand cloned downstream of the M13 Rv priming site

C. Screening the E.coli transformants in colony PCR with M13 Rv and R644

D. Screening the E.coli transformants in colony PCR with M13 Rv and F6536

1 2 3 4 5

1 2 3 4 5

Figure 2-6: Orientation of the EV71 cDNA after cloning into pCR®-XL-TOPO® vector. A, B: The cDNA of EV71 genome with the attached T7 promoter composes plasmid DNA bases between 336 and 7810, and marked in blue. Polarity of the EV71 cDNA strands marked as “+” and “-“. Positions of the T7 promoter, R644 and F6536, located within EV71 insert, are marked in blue. The pCR®-XL-TOPO® vector with M13 Rv and T7pol binding sites are marked in red. C: Results of the colony PCR with M13 Rv and R644 show DNA products of 798 bp in length in wells from 1 to 4 and confirm orientation of EV71 insert in accordance with scheme A. D: Results of the colony PCR with M13 Rv and F6536 show DNA products of 1047 bp in length in wells 2, 4 and 5 and confirm orientation of EV71 insert in accordance with scheme B.


107

Since the cDNA used in cloning had a T7 promoter inserted upstream of

the EV71 genome by the forward primer during PCR, a plasmid DNA with either

orientation was suitable for further in vitro transcription with T7 polymerase. The

T7 promoter derived from the vector was not considered for in vitro transcription

purpose due to a substantial piece (69 nt bases) of plasmid DNA between the

promoter and EV71 genome. That could impair the infectivity of EV71 RNA

transcripts in cell culture. Orientation of the insert was important in order to

determine restriction enzymes necessary to excise the EV71 genome prior to the

in vitro transcription. Even though, EcoRI could be used for this purpose with

both orientations (Figure 2-2) it did not give complete digestion in a single

reaction with MluI (a recognition site at the end of the poly(A) tail) (data not

shown). In pilot experiments, MluI alone or MluI/NotI combination was found to

be most effective in order to digest the cDNA clones with orientation A and B

(Figure 2-6), respectively.

The plasmid DNAs were purified from the E.coli transformants and

subjected to a restriction enzyme cleavage in order to verify the size of the EV71

cDNA clones (data not shown). One clone obtained with each strategy was

sequenced in full and one - within the 5′UTR, VP1, 3B, 3C, 3D and 3′UTR virus

genome regions. Mutations observed within the EV71 cDNA clones are

summarized in Appendix 2.

In order to compare the degree of genetic variation within EV71 cDNA

clones obtained in Strategies 1 and 2, the overall number of nucleotide and amino

acid differences within EV71 genome regions was calculated with MEGA3

(Table 2-2).


108

Genome region* Overall number of differences,

nt(aa)** Strategy 1 Strategy 2

5′UTR 5(N/A) 1(N/A) VP1 6(4) 2(2) 3D 3(0) 1(1)

Table 2-2: Genetic variation within cDNA clones of EV71.

* – Calculations were possible for those genome regions which had at least 2 sequences available within the same group, Strategy 1 or Strategy 2. ** – Calculations were performed at nucleotide and amino acid levels, nt(aa), except the 5′UTR, where amino acid sequences were not applicable (N/A).


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2.3.3 Infectivity of the EV71 cDNA clones in cell culture

The EV71 genome was excised from the cDNA clones and transcribed in

vitro by T7 polymerase. Integrity, size and concentration of the synthesized RNA

were checked by agarose gel electrophoresis (Figure 2-7).

1 2 3 4 5 6

Figure 2-7: Agarose gel electrophoresis of the EV71 in vitro RNA transcripts.

Lane 1: RiboRuler was loaded at concentration 30 ng per each band. Lanes 2, 3 and 4: the EV71 RNA loaded at 1, 0.1 and 0.01 μl per lane, respectively. Size of EV71 RNA transcripts was expected around 7.4 kb. Lane 5: Control RNA of approximately 1.8 kb long. Lane 6: 1 kb DNA ladder.

Spectrophotometric measurement showed that, concentration of the EV71

in vitro transcribed RNA after phenol-chloroform extraction and ethanol

precipitation reached 0.3-0.5 μg/μl with an A260/A280 ratio of 1.7-2.2.

The in vitro RNA transcripts were transfected into Vero cell culture in the

presence of Lipofectamine™ 2000. Monitoring the cell cultures under a

microscope showed increasing signs of CPE only after transfection with the RNA

transcripts derived from the cDNA clones obtained in Strategy 2 (Figure 2-8).

6 kb

2 kb 1.5 kb

0.75 kb

0.5 kb


110

in vitro transcribed

RNA, Strategy 1 in vitro transcribed RNA, Strategy 2

Positive control (virus)

Negative control (Opti-MEM)

0 h

48 h

72 h

96 h

120 h

144 h

168 h

Figure 2-8: CPE in Vero cell culture after transfection with the in vitro transcribed

RNA.

Signs of CPE can be observed both in the positive control wells (infected with the virus) and the experimental wells, which were transfected with the in vitro transcribed RNA obtained with Strategy 2. The negative control cell culture and the experimental wells transfected with the RNA transcripts obtained with Strategy 1 did not produce CPE but showed overgrowth after 72 hours of incubation.


111

Supernatant collected after transfection and used in infection of new Vero

cells was able to produce CPE only in Strategy 2. RNA isolated from that cell

culture showed positive results in RT-PCR conducted with EV71 specific

primers, F5987 and R6251. In contrast, both negative signs of CPE and negative

RT-PCR results were obtained after Strategy 1 (Figure 2-9).

Transfection of Vero cell culture was carried out with at least three

different cDNA clones from each strategy. Similar results to those presented

were obtained in all repeats.


112

A. B. C. D.

Supernatant

from Strategy 1

Supernatant

from Strategy 2

Cell culture infected

with EV71

Uninfected cell

culture

E. F. 1 2 3 4 5

6 7 8 9 10

Figure 2-9: RT-PCR of virus after second round infection from cotransfected RNA

showing viable virus from second strategy.

A, B: Vero cell cultures shown at 48 h after infection with supernatants obtained in Strategy 1 and 2, respectively. C: Cell culture infected with EV71 served as a Positive control. D: Uninfected cell culture served as a negative control. E, F: Results of RT-PCR on detection of EV71 RNA from the cell cultures. Lane 1: RT-PCR with RNA isolated from the positive control cell culture (C). Lane 2: RT-PCR with RNA isolated from the cell culture infected with the supernatant from Strategy 1 (A). Lane 3: RT-PCR with RNA isolated from the negative control cell culture (D). Lanes 4 and 9: negative controls of PCR with H2O used instead of cDNA template. Lane 5 and 10: 100 bp DNA ladder. Lanes 6: RT-PCR with RNA isolated from Vero cells infected with the supernatant obtained with Strategy 2 (B). Lane 7: PCR (no RT) with RNA isolated from cell culture B was performed in order to exclude a false positive PCR result due to possible DNA template carryover from the in vitro transcription reaction. Lane 8: positive control of PCR with full-length genome cDNA of EV71 as a template.

300 bp 200 bp

300 bp 200 bp


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2.3.4 Role of the poly(A) tail in infectivity of EV71 in vitro RNA

transcripts

The presence and length of the poly(A) tail can affect infectivity of the in

vitro transcribed RNA derived from cDNA clones of some viruses (Silvestri et al.

2006; Spagnolo & Hogue 2000). The EV71 cDNA clones produced in Strategy 1

had eighteen thymidine residues following the 3′UTR, whereas clones from

Strategy 2 had thirty. In order to check if a shortened poly(A) tail reduces the

infectivity of the in vitro RNA transcripts, the EV71 genome was amplified with

iProof polymerase using RMluI (18 thymidines) or RMluI-30 (30 thymidines)

reverse primers (Table 2-1). PCR products were digested with MluI and polished

with Mung Bean Nuclease. In total, six DNA templates of EV71 genome,

different in their 3′ ends, were obtained and used for in vitro RNA synthesis

(Figure 2-10).


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EV71 genome DNA 18++ GG-T7 -A(17)-A▼CGCGTCC

GG-T7 -T(17)-TGCGC▲AGG (PCR product with RMluI primer)

DNA 30++ GG-T7 -A(29)-A▼CGCGTAGATCC GG-T7 -T(29)-TGCGC▲ATCTAGG (PCR product with RMluI-30 primer)

DNA 18+ GG-T7 -A(18) GG-T7 -T(18)GCGC (MluI digestion of DNA 18++)

DNA 30+ GG-T7 -A(30) GG-T7 -T(30)GCGC (MluI digestion of DNA 30++)

DNA 18 GG-T7 -A(18) GG-T7 -T(18) (Mung Bean Nuclease polished DNA 18+)

DNA 30 GG-T7 -A(30) GG-T7 -T(30) (Mung Bean Nuclease polished DNA 30+)

Figure 2-10: Schematic diagram of the DNA templates used for in vitro RNA

synthesis.

All DNAs contained the T7 promoter followed by the full length EV71 genome. The 3′ ends of the constructs differed in their sequences as shown in the diagram. The MluI recognition site is underlined. Positions at which MluI digests the DNA strands are marked with▼ and ▲.


115

The in vitro produced RNAs derived from the DNA templates harbouring

different 3′ ends were used in transfection of Vero cell cultures at concentration 1

μg RNA per transfection. Supernatant and cells were collected at 72 hours post

transfection and used in TCID50 assay. According to TCID50 results, the titres of

the rescued viruses showed a very small variation (data not shown).

2.3.5 Role of the culturing conditions in rescue of EV71 from in vitro

RNA transcripts

Various conditions for culturing Vero cells were created by supplementing

the MEM with a different percentage of FBS and passaging the cells at 5 hours

after transfection with EV71 RNA transcripts harbouring the A(30)-tail. At

specific time points the supernatant and cells were collected and used in RNA

extraction followed by RT and Q-PCR with EV71 specific primers. Additionally,

TCID50 titres were estimated at the end of the incubation period. In parallel, non-

transfected cell cultures were grown at the same conditions and used to monitor

the growth kinetics by counting the cells at specified time points. Cell replication

continued for 72 hours and depended on concentration of FBS. Cells numbers

increased faster with higher FBS concentrations. The cell culture maintained in

OptiMEM medium showed the lowest number of cells during the whole period

of observation. The cell culture passaged and maintained in MEM supplemented

with 10% FBS continued to replicate during 144 hours of incubation (Figure 2-

11, A).


116

A.

B.

Figure 2-11: Role of the culturing conditions in rescue of EV71.

A. Growth kinetics of Vero cells under different conditions of culturing. 24-well plate was seeded with 2.5 x 105 cells per well in MEM supplemented with 5% FBS. In 24 hours, the medium was replaced with OptiMEM® I Reduced Serum Medium. Cell numbers after 24 hours incubation in OptiMEM corresponds to the cell culture immediately prior to transfection with RNA transcripts (designated as 0 h on the graph). Lipofectamine™2000 was added at this time point and cells were retained in OptiMEM-Lipofectamine medium for 5 h, then the medium was replaced (as indicated). Cells were collected and counted at the indicated times. B. Total number of PFU of EV71 rescued from the in vitro RNA transcripts under different cell culture conditions. Titres of the rescued viruses were estimated in Vero cell cultures collected at 144 hours post transfection.


117

The PFU titre of the rescued viruses differed between cell cultures

maintained at different growth conditions (Figure 2-11, B). Total virus

production in actively dividing cell culture was approximately 56 fold higher

when compared to that in Vero cells maintained in OptiMEM.

In order to examine the kinetics of the viral RNA synthesis in almost

quiescent (maintained in OptiMEM) or actively dividing (passaged and

maintained in MEM + 10% FBS) Vero cells, the levels of the viral RNA were

compared by real-time RT-PCR assay. Total RNA was isolated from the

supernatant and cells collected at 5, 10, 24, 48, 72, 96, 120 and 144 hours post

transfection. It was not possible to distinguish between the newly synthesized

viral RNA and in vitro RNA transcripts introduced during transfection. However,

in quiescent cells, where OptiMEM medium with the RNA transcripts were left

for a whole period of incubation, the levels of EV71 RNA showed a sharp

decrease during the first 10 h post transfection and then declined slowly until 144

h (Figure 2-12, A and Table 2-3, A). This reduction could indicate fast

degradation of the in vitro RNA transcripts in cell culture. There was no increase

in RNA levels observed upon transfection of the quiescent cells during 144 h of

incubation.


118

A.

B.

Figure 2-12: Comparative quantitation of EV71 RNA isolated from the supernatant and

cells after transfection of Vero cell culture with in vitro RNA transcripts.

Monolayers of Vero cells were transfected with the same amount of EV71 RNA transcripts. Five hours post transfection one cell culture (dark bars) was passaged and maintained in MEM supplemented with 10% FBS. The other cell culture (light bars) was retained in OptiMEM medium containing the RNA transcripts for a whole period of observation. Yields of EV71 RNA at each time point were normalized to the yield of the 5 h sample, which represents 100%. A. RNA was extracted from the pooled supernatant and cells at the times indicated. The 5 h sample was collected just prior to removing the medium and passaging the cells. B. RNA was extracted from the cells alone after discarding the supernatant.


119

A.

Growth conditions

Time post transfection Ct Mean Ct

Std. Dev. Copy # Mean Copy # Std. Dev. %

OptiMEM

5 h 12.1 N/A 3.73E+10 N/A 100.0 10 h 14.3 0.05 8.12E+09 2.79E+08 21.8 24 h 15.5 0.21 3.49E+09 5.13E+08 9.3 48 h 16.5 0.11 1.68E+09 1.31E+08 4.5 72 h 17.2 0.10 1.05E+09 7.43E+07 2.8 96 h 18.0 0.15 5.82E+08 6.04E+07 1.6

120 h 19.2 0.09 2.57E+08 1.60E+07 0.7 144 h 19.6 0.04 1.93E+08 4.82E+06 0.5

Passaged, MEM +

10% FBC

5 h 12.1 N/A 3.73E+10 N/A 100.0 10 h 18.2 0.15 5.12E+08 5.34E+07 1.4 24 h 19.0 0.15 2.82E+08 2.98E+07 0.8 48 h 18.9 0.05 3.11E+08 1.13E+07 0.8 72 h 18.3 0.12 4.70E+08 4.04E+07 1.3 96 h 16.7 0.06 1.42E+09 6.23E+07 3.8

120 h 15.3 0.12 3.99E+09 3.45E+08 10.7 144 h 14.9 0.07 5.37E+09 2.75E+08 14.4

B.

Growth conditions

Time post transfection Ct Mean Ct Std.

Dev Copy # Mean Copy # Std. Dev. %

OptiMEM

5 h 28.8 0.09 2.69E+04 1.65E+03 100.0

10 h 29.5 0.08 1.70E+04 9.28E+02 63.1

24 h 30.0 0.05 1.21E+04 3.72E+02 45.1

Passaged, MEM +

10% FBC

5 h 28.8 0.09 2.69E+04 1.65E+03 100.0

10 h 31.7 0.11 3.68E+03 2.63E+02 13.7

24 h 31.3 0.00 5.38E+03 1.44E+01 20.0

Table 2-3: EV71 RNA level after transfection of Vero cell culture with in vitro

RNA transcrips.

The number of copies of EV71 RNA was determined by real-time PCR. Conditions of the experiment and colouring scheme are identical to those described in Figure 2-12. RNA was extracted from the pooled supernatant and cells (A) or from the cells alone (B) at the times indicated post transfection. The number of copies of viral RNA per experimental well (150 µl – A, or 20000 cells seeded – B) is shown. Threshold Cycle (Ct) and Copy number (Copy #) Means with their Standard Deviations (Std. Dev.) were calculated from the sample duplicates (except 5 h sample in table A). N/A – not applicable.


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In actively dividing cells, the RNA transcripts were removed by passaging

the cells and changing the medium at 5 h post transfection. In accordance, the

EV71 RNA level showed a dramatic drop in the 10 h sample. However, a slow

increase in the RNA yield was already observed at 48-72 h post transfection

(Table 2-3, A) with continuing accumulation of viral RNA until 144 h of

incubation. This change could indicate the beginning of the viral replication in

actively dividing cells.

In order to compare EV71 RNA accumulation inside the transfected cells,

the total RNA was isolated from the cells alone at 5, 10 and 24 h post

transfection (Figure 2-12, B and Table 2-3, B). Cells incubated in OptiMEM with

RNA transcripts demonstrated a gradual decline (from 100% to 45%) in the

EV71 RNA level inside the cells during 24 h. This trend could be a result of the

reduction in RNA amount available from the supernatant, and/or a degradation of

the RNA transcripts by cellular nucleases inside the cells (Beelman & Parker

1995). The difference in RNA levels between two cell cultures at 10 h indicated

that cells, maintained in OptiMEM medium with the RNA transcripts, continued

to take in the RNA beyond five hours. Due to this fact it was impossible to

determine if viral replication took place in cells maintained in medium with RNA

transcripts. In the passaged cell culture, where RNA transcripts were removed, a

reduction in the intracellular EV71 RNA amount within the first five hours was

more obvious (from 100% to approximately 14%) (Table 2-3, B), and could

reflect the RNA degradation via intracellular pathways. However, at 24 h of

incubation this cell culture showed a small increase in intracellular EV71 RNA

accumulation (from 14% to 20%), which could indicate de novo RNA synthesis.


121

2.4 Discussion

In general, the construction of a full-length cDNA clone of the RNA

viruses consists of the following steps: the reverse transcription of the viral RNA

into a single-stranded complementary DNA (cDNA), the second strand DNA

synthesis by PCR, the cloning of the double-stranded cDNA into an appropriate

vector and, finally, the transformation of the vector into a bacterial host (Boyer &

Haenni 1994; Lai 2000). Replication of the plasmid with the viral cDNA insert in

it by the bacterial cells allows obtaining the multiple cDNA molecules of the

viral genome with the identical sequence, a requirement which is absolutely

necessary in some genetic manipulations. Despite the fact that the

aforementioned steps have been developed and successfully used as separate

methods during the last few decades, they still have some limitations when they

are applied for a production of infectious viral clones.

The first obstacle can be probably addressed to a strong secondary

structure of the viral RNA which makes it more difficult to produce the first

strand cDNA of the full length. Therefore, in some cases, obtaining of the entire

viral genome by long RT-PCR was unsuccessful and required several steps of

sub cloning (Yanagi et al. 1997). RNA templates with high GC content (up to

70%) may require an increase of RT temperature up to 65°C and usage of a

thermostable reverse transcriptase. In contrast, decreasing the RT temperature

and increasing the incubation time can result in an increase of the full-length

cDNA yield. The 6F/AUS/6/99 reference strain of EV71 exhibits GC content of

48%. Therefore, in both Strategies the reverse transcription was performed at the

lowest temperature within the range recommended by the manufacturers with

incubation time up to 1 hour.

Secondly, the introduction of unwanted deleterious mutations during the

RT step by a reverse transcriptase lacking the proof-reading activity becomes a

serious concern when transcribing a viral RNA genome of 7.4 kb in length. In

order to increase accuracy of the RNA transcription step, the Transcriptor High


122

Fidelity Reverse Transcriptase was used in the first approach. The enzyme

exhibits a 1.98 x 10-5 error rate alone or 3.88 x 10-5 when combined with a proof-

reading polymerase in a following PCR of 25 cycles according to the manual.

Unfortunately, no PCR product was observed after an attempt to amplify the full-

length EV71 genome from the cDNA with the Expand Long Range dNTPack in

a single round of 25 cycles. Therefore, the overlapping PCR with 5′ and 3′ halves

of EV71 cDNA was performed which resulted in an increase of the total number

of the PCR cycles and number of the mutations introduced. The second approach

utilized the SuperScript™ II RT which shows a mutation rate of 3.57 x 10-5

alone. In combination with the iProof High-Fidelity DNA polymerase it could

produce the full-length EV71 cDNA in a single round PCR. The error rate of the

iProof polymerase is claimed to be approximately 50-fold lower than that of Taq

pol, and 6-fold lower than that of Pfu pol. Additionally, with the reduction of the

PCR cycles number to twenty, amplification of the EV71 genome with a higher

fidelity was expected. Indeed, the overall number of mutations within the EV71

cDNA clones was lower in Strategy 2 (Table 2-2). Sequencing the entire EV71

genome from one of the cDNA clones obtained in Strategies 1 and 2 revealed

eleven and five nucleotide substitutions, respectively, when compared to the

6F/AUS/6/99 reference strain. After transfection of Vero cells with in vitro EV71

RNA transcripts only cDNA clones constructed in Strategy 2 produced the

infectious progeny. There have been several examples in obtaining non-

infectious cDNA clones of viruses (Arita et al. 2005; Moormann et al. 1996;

Tellier et al. 1996; Yanagi et al. 1997; Zibert et al. 1990). In all cases numerous

mutations within the viral genomes were observed and could account for the loss

of infectivity. In some cases replacing the mutated part with the same fragment

derived from the independent PCR product or cDNA clone could restore the

infectivity of the original non-infectious clone (Arita et al. 2005; Yanagi et al.

1997). Therefore, there is the possibility that lethal mutations were introduced by

RT-PCR into the viral genome which resulted in the lack of infectivity of the

EV71 cDNA clone obtained with Strategy 1.


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The RNA genome of the Picornaviridae does not require a cap structure

to be infectious (van der Werf et al. 1986). The 5′UTR contains the IRES

sequence which interacts with the host cell translation machinery and initiates

production of the viral proteins from the in vitro RNA transcripts upon

transfection into susceptible cell culture or animal system. Virus yield of the

rescued viruses is usually substantially lower when compared to that after

transfection with virion RNAs (van der Werf et al. 1986). Several factors have

been reported to have an impact on the infectivity of the in vitro RNA transcripts.

Incorporation of guanine residue(s) upstream of the viral genome by T7

polymerase or an additional non-viral sequence downstream of the poly(A) tract

was found to lower the yield of the rescued viruses. Non-viral nucleotides

upstream of the 5′ end were suggested to have a negative effect on the initiation

of the positive strand RNA synthesis directed from the 3′ end of the negative

strand RNA (Boyer & Haenni 1994). Inhibition of infectivity by the extra non-

viral nucleotides at the 3′ end was shown to depend on the length and nature of

the sequence (Sarnow 1989). Long heteropolymeric 3′ end extensions decreased

or abolished infectivity whereas short extra sequences were usually tolerated.

With some viruses, the biological activity of the in vitro RNA transcripts was

affected by the length of the poly(A) tail as well. Shortened poly(A) tail was

shown to decrease virus replication for a number of viruses (e.g. poliovirus,

hepatitis A virus, coronavirus) (Kusov, Gosert & Gauss-Muller 2005; Sarnow

1989; Silvestri et al. 2006). With poliovirus, a stretch of at least 20 A residues

was essential in order to approach level of the negative strand RNA synthesis

similar to that of the wild-type RNA transcripts (Silvestri et al. 2006). It was

suggested that poly(A) tail should be long enough in order to interact with the

PABP and initiate the VPg uridylation. Additionally, poly(A) tail probably plays

some role in the protection of the RNA transcripts in vivo against host cell

ribonucleases (Kusov, Gosert & Gauss-Muller 2005). Interestingly, with some

other viruses an absence of the poly(A) tail or an addition of the long non-viral

sequence downstream of the viral genome did not have any effect on infectivity

of the RNA transcripts (Eggen et al. 1989; Liu et al. 2008). Obtaining the RNA


124

transcripts harbouring the poly(A) tail with a minimum or no extra nucleotides

downstream is possible by incorporating a unique restriction site into a reverse

primer used during amplification. Subsequently, this site can be used in order to

linearize the plasmid DNA downstream of the poly(A) tail rather than using the

multiple restriction sites existing in a vector. In this study, the MluI restriction

enzyme was optimal for this purpose as it does not have a recognition site within

the EV71 genome and leaves an extension of only 4 nucleotides at the 5′ end of

the digested DNA. Those extra bases (GCGC) can be removed by a single-strand

nuclease in order to produce a template with no extraneous sequence downstream

of the poly(A) tail. To study the relationship between the poly(A) tract of the

various lengths with or without non-viral sequences downstream, six different

cDNA templates of the EV71 genome were constructed and used in the

production of the in vitro RNA transcripts (Figure 2-10). The cDNA templates

had the identical 5′ ends and differed only in their sequences downstream of the

3′UTR. Vero cell cultures were transfected with the same amount of the in vitro

RNA transcripts and viral titres of the rescued viruses were estimated in TCID50

assay. Results demonstrated small variations among the viral titres and between

repeats of the experiment (data not shown). This indicated that under

experimental conditions the infectivity of the EV71 RNA transcripts did not

depend on the length of the poly(A) tail.

Several studies, where sequence analysis of the rescued viruses was

conducted, demonstrated that after transfection of the RNA transcripts into cell

culture the authentic 3′ and 5′ end of the viral genomes were restored and poly(A)

tail length did not differ from that of the wild-type virus (Eggen et al. 1989;

Kusov, Gosert & Gauss-Muller 2005; Liu et al. 2008; Tacahashi & Uyeda 1999).

The restoration process was suggested to happen before replication and to depend

on the cell cycle and availability of host factors and enzymes required for

repairing the viral genome (Kusov, Gosert & Gauss-Muller 2005). Adenylating

enzymes which are most probably involved in the restoration of the shortened

poly(A) tail are more active in actively dividing cells (Kazazoglou, Tsiapalis &

Havredaki 1987). Therefore, infectivity of the in vitro-produced RNA transcripts


125

can be expected to be higher in an actively growing cell culture. Indeed, higher

titres of the rescued EV71 were obtained from actively dividing cells which were

passaged at 5 hours post transfection and maintained in MEM with 10% FBS.

Despite the removal of the in vitro RNA transcripts at five hours post transfection

the EV71 infection was established and an increase in EV71 RNA level in the

supernatant and cells was observed at 48-72 hours post transfection. The increase

in intracellular EV71 RNA was detected even earlier (24 hours post transfection),

and could indicate the beginning of viral replication. In contrast, the stationary

cell culture could not establish EV71 infection at a similar level. Interestingly,

that RNA uptake by cells continued beyond the 5 hours incubation period but did

not guarantee the higher viral titres in quiescent cells. Therefore, it can be

concluded that cell culture status plays an important role in rescuing of EV71

from the in vitro-produced RNA transcripts.

126

CHAPTER 3

TEMPERATURE RESISTANT MOLECULAR

DETERMINANTS OF EV71 UPON NATURAL SELECTION IN

CELL CULTURE

CHAPTER 3 Temperature resistant molecular determinants of EV71 upon natural selection in cell culture

127

3 TEMPERATURE RESISTANT MOLECULAR DETERMINANTS OF

EV71 UPON NATURAL SELECTION IN CELL CULTURE

3.1 Introduction

Virus growth in vitro at temperatures higher than those used for its

isolation is a phenotypic characteristic which often correlates with pathogenicity

of the virus, e.g. PV vaccine strains (Christodoulou et al. 1990; Macadam et al.

1989). Similar correlation between in vitro phenotype and neurovirulence was

observed with ts and TR variants of EV71 BrCr strain in monkeys (Hagiwara,

Yoneyama & Hashimoto 1983; Hashimoto & Hagiwara 1983). Resistance to

temperatures as high as 40ºC was also reported for some EV71 clinical isolates

during the outbreak in Taiwan in 1998 (Kung et al. 2007). The temperature

resistant strains were isolated from patients with CNS-involvement, in contrast to

the temperature sensitive viruses, isolated from patients with herpangina. It was

suggested that temperature resistance can be beneficial for the virus in

establishing the productive replication and resulting viremia in patients with

fever. The high viral load, in turn, triggers an abnormal release of pro-

inflammatory cytokines, which were reported to increase both the pulmonary

vascular and BBB permeability (Chen 2007; Wang et al. 2003). Pulmonary

oedema and brainstem encephalitis, which are likely results of the extensive

inflammatory response and virus entry into CNS, were reported as the leading

cause in EV71 associated deaths (Lin et al. 2002a; Wang et al. 1999). Fever, one

of the earliest symptoms of EV71 infection, can be the leading factor in the

natural selection of the TR phenotype among the existing variation of EV71

genomes in situ, known as quasispecies. Results of such selection within an

infected individual can be associated with further progression and outcome of the

disease. Studying these early steps of viral adaptation to increased body

temperatures in vivo is difficult and has never been attempted. However,


128

experiments in cell culture are easier to conduct and still can provide important

information on viral adaptation as well as contributing to our better

understanding of the pathogen. The aim of this study was to determine the

molecular determinants associated with the temperature resistant phenotype of

EV71 upon natural selection in cell culture during incubation at an elevated

temperature. The genetic determinants located both within the structural and non-

structural viral proteins and their contribution to the temperature resistant

phenotype are discussed.


129


3.2.1 Cell culture

African green monkey kidney (Vero) cells were used in those experiments

which required cell culture. Vero cells were maintained under the same growth

conditions described in Chapter 2.

3.2.2 Viruses and their cDNA clones

In order to study EV71 molecular determinants associated with ts or TR

phenotype due to natural selection in vitro, two viral stocks were prepared by

passaging the virus in Vero cells at a permissive or potentially restrictive

temperature:

a) The ts stock was obtained after multiple passages of EV71 at 37°C,

b) The TR stock was derived from the ts stock after 4 passages at

39°C for 72 h each.

Total RNA was extracted from the supernatants with QIAamp Viral RNA

Mini Kit (QIAGEN) in accordance with recommended protocol. All RNA

samples were stored at -80°C until use.

The cDNA clones from both viral stocks were constructed by following

the Strategy 2 protocol described in materials and methods in Chapter 2.


130

3.2.3 DNA automated cycle sequencing and nucleotide sequence analysis

Sequence of EV71genome was obtained in full for both viral stocks and

their cDNA clones. RT-PCR or PCR were performed with the extracted total

RNAs or purified plasmid DNAs, respectively. In order to reduce the number of

artificial mutations the PCR amplification was conducted with the iProof High-

Fidelity DNA Pol (BioRad) for no more than 20 cycles. The EV71 specific

primers used in amplification were designed in order to amplify and sequence the

overlapping fragments within the EV71 genome (Table 2-1).

Sequences were aligned in BioEdit v7.0.5.3 against the 6F/AUS/6/99

reference strain first, and then analysed in terms of nucleotide and amino acid

differences between the ts and TR strain only.

3.2.4 Structure modelling and protein interactions analysis

3.2.4.1 Retrieving the reference structures

Amino acid sequences of the viral proteins of the TR cDNA clone were

used in a BLAST search against the Protein Data Bank (PDB) (Altschul et al.

1997). The procedure included following steps: 1) the “protein blast” option was

used on the Blast query page (http://blast.ncbi.nlm.nih.gov/Blast.cgi), 2) the

amino acid sequence of the TR variant was uploaded from the FASTA file, 3) the

PDB option was chosen from the scroll-down list of the databases and 4) the

“blastp” algorithm was selected prior to initiating the BLAST search. A sequence

with the best score was chosen from the results list and the corresponding

structure was retrieved from the Molecular Modeling DataBase (MMDB). The

crystal structures selected from the MMDB and used in the modelling of the

EV71 mutations are summarized in Table 3-1.


131

PDB

accession # GenBank

accession # Description Reference

3VBF_A 378792527 Chain A, crystal structure of formaldehyde treated EV71

(Wang et al. 2012)

3VBF_C 378792529 Chain C, crystal structure of formaldehyde treated EV71

(Wang et al. 2012)

3N6M_A 340707630 Crystal structure of EV71 RDRP in complex with GTP

(Wu et al. 2010)

3OL6_A 315364538 Crystal structure of Poliovirus polymerase elongation complex, open conformation


3OL7_A 315364554 Crystal structure of Poliovirus polymerase elongation complex with CTP, closed conformation


Table 3-1: The MMDB crystal structures used in modelling the EV71 mutations.


132

3.2.4.2 Mapping the mutations on the 3d protein structures

The three-dimensional (3d) structures of the reference proteins were

retrieved from the MMDB and visualized in Cn3D 4.3 software available from

the National Center for Biotechnology Information (NCBI)

(http://www.ncbi.nlm.nih.gov/Structure/CN3D/cn3d.shtml). Amino acid

sequences obtained from the TR clone of EV71 were aligned with the

corresponding reference structures, and mutations observed within the TR clone

were mapped on to the reference structure in Cn3D 4.3 software.

3.2.4.3 Prediction of protein interactions and binding sites

Possible interactions for EV71 proteins of interest were predicted via the

NCBI Inferred Biomolecular Interactions Server (IBIS) (Shoemaker et al. 2012).

Briefly, each reference structure, listed in Table 3-1, was used as a query for a

related protein of EV71 on the IBIS query page

<http://www.ncbi.nlm.nih.gov/Structure/ibis/ibis.cgi>.

Predicted binding sites were analysed and mapped on the protein

structures viewed in Cn3D 4.3 software.

3.2.5 Site-directed mutagenesis

Site-directed mutagenesis was performed in order to introduce single

mutations observed in the ts variant into the corresponding positions of the TR

EV71 clone. Primers used in SDM were designed with PrimerX software

(http://www.bioinformatics.org/primerx/) and listed in Table 3-2.

http://www.bioinformatics.org/primerx/


133

Primer name* Sequence (5′-3′)**

F_VP3-317G GGGCCAGTTGTGCGGGTACTACACCCAATG

R_VP3-317G CATTGGGTGTAGTACCCGCACAACTGGCCC

F_VP1-275G CAGGATTAGTTGGAGGGATAGACCTTCCTC

R_VP1-275G GAGGAAGGTCTATCCCTCCAACTAATCCTG

F_VP1-293A GATAGACCTTCCTCTTAAAGGCACAACCAACCCG

R_VP1-293A CGGGTTGGTTGTGCCTTTAAGAGGAAGGTCTATC

F_VP1-658T CAAGCAGGAGAAAGACTTTGAATACGGGGCATGC

R_VP1-658T GCATGCCCCGTATTCAAAGTCTTTCTCCTGCTTG

F_VP1-724C CTGTAGGAACCTCGCAGTCCAAGTACCC

R_VP1-724C GGGTACTTGGACTGCGAGGTTCCTACAG

F_VP1-848C CAAATTATGCTGGCAACTCCATTAAACCAACTGGTGC

R_VP1-848C GCACCAGTTGGTTTAATGGAGTTGCCAGCATAATTTG

F_3D-895T CCTCCATTTTCAACTCGATGATCAACAAC

R_3D-895T GTTGTTGATCATCGAGTTGAAAATGGAGG

Table 3-2: List of primers used in SDM of the TR EV71 clone.

* Primer name indicates its orientation (F-forward, R-reverse), mutated gene (VP1, VP3 or 3D) and nucleotide position with introduced mutation (for example, G was introduced at position 317 within the VP3 gene). ** Position of the mutation within the primer’s sequence is underlined.


134

The SDM included three steps (Figure 3-1):

1. Mutant strand synthesis:

The PCR mixture contained 1 x High-Fidelity PCR buffer, 0.2 µM of both

forward and reverse mutagenic primers, 0.2 mM of dNTP mix, 0.5 U of iProof

High-Fidelity DNA Pol (BioRad) and 40-50 ng of the DNA template in a total

reaction volume of 25 µl. The PCR was carried out under the following

conditions: initial denaturation at 98°C for 2 min, followed by 18 cycles at 98°C

for 10 sec, 70°C for 20 sec and 72°C for 5 min. Final elongation was carried out

at 72°C for 5 min. Results of the PCR were visualized by running 2 µl aliquots

on 0.7% agarose gel stained with EtBr.

2. Digestion of the parental strand:

The parental DNA strand was digested with DpnI (Stratagene). 5U of the

restriction enzyme was added directly to the amplification reaction and gently

mixed by pipetting. Digestion was performed at 37°C for 2 hours.

3. Transformation of E.coli cells:

XL 10-Gold® Ultracompetent cells (Stratagene) were used in

transformation with mutant DNA molecules. Cells were gently thawed on ice and

aliquot in 45 µl per sample into prechilled 15-ml BD Falcon polypropylene

tubes. 5 µl of the DpnI-treated DNA samples were added to separate cells

aliquots. Transformation reactions were gently mixed and incubated on ice for 30

min. After the incubation, cells were subjected to a heat pulse at 42°C for 30 sec

and immediately returned on ice for 2 min. 0.5 ml of preheated (42°C) NZY+

broth (Appendix III) was added to the cells with following incubation at 37°C for

1 hour and shaking at 225 rpm. After the 1 hour incubation, the falcon tubes were

centrifuged at 0.2 rcf for 5 min. The clear supernatant was discarded and the cell

pellet was resuspended in the remaining volume of medium (approx. 150 µl).

The resuspended cells were spread on LB agar, supplemented with 50 µg/ml

kanamycin and incubated overnight at 37°C.

The presence of the correct mutation after SDM of EV71 was confirmed

by sequencing the purified plasmid DNA within the target gene.


135

Figure 3-1: Schematic representation of the SDM steps.

The diagram was adapted with modifications from the instruction manual for the QuikChange® II XL Site-Directed Mutagenesis Kit (Stratagene). x – Indicates positions of the mutated nucleotides both within the mutagenic primers and newly synthesised DNA.


136

3.2.6 Rescue of the EV71 mutants

The EV71 mutants were rescued in Vero cell culture from the in vitro

RNA transcripts as described in materials and methods in Chapter 2.

The cell culture containing the rescued virus was subjected to 3 cycles of

freezing/thawing followed by centrifugation to remove cell debris. Cleared

supernatant was aliquoted and stored at -80°C until use.

The titres of the rescued viruses were determined in TCID50 assay as

described in Chapter 2.

3.2.7 Temperature sensitivity assay

Temperature sensitivity of the viruses was examined at 37˚C, 38˚C 39˚C

and 40˚C on Vero cell monolayers. Briefly, 96-well plates were seeded with 2 x

104 cells per well and grown in MEM supplemented with 5% FBS in a CO2

incubator at 37°C for 24 hours. The medium was removed from the wells and

virus was added at an m.o.i of 0.1, in a total volume of 50 µl per well. Virus was

allowed to attach to the cells for 30 min at room temperature on a rocking

platform. Unbound virus was removed and wells were washed with 200 µl of

MEM. Then, 200 µl of fresh MEM supplemented with 5% FBS was added into

each well, and plates were incubated at each experimental temperature for 72 h.

At the end of the incubation, plates were frozen at -80°C and thawed at 37˚C 3

times, in order to release the intracellular virus. Cell debris was removed by

centrifugation and viral titres were determined in TCID50 assay as described in

materials and methods in Chapter 2.

Logarithmic difference of the arithmetic mean TCID50 values (∆TCID50)

obtained at 38˚C, 39˚C and 40˚C versus those obtained at 37˚C was plotted onto

graphs in Microsoft Excel Software. The “FORECAST” function of Microsoft

Excel was used to determine the shut-off temperature at which the TCID50 value


137

was reduced by 1.0 log10 when compared to the TCID50 at 37˚C (refer statistical

analysis).

3.2.8 Statistical analysis

All statistical calculations were performed in Microsoft Excel Software as

appropriate.

3.2.8.1 Arithmetic mean

The arithmetic mean (or average) was calculated for log10 TCID50 values

obtained from three temperature sensitivity assays conducted with a particular

virus and at a particular temperature.

3.2.8.2 Standard error of the mean

The standard error of the mean (SEM) was calculated for the arithmetic

mean of the log10 TCID50 values.

3.2.8.3 Shut-off temperature calculation

The shut-off temperature was defined as the temperature giving one log

(tenfold) decrease in viral titre obtained by either interpolation or extrapolation of

the temperature dependent reduction in viral titre calculated by linear regression.


138

3.2.8.4 Student’s t-test

To compare the effect of elevated incubation temperatures on virus

growth, the paired Student’s t-test was performed.

To compare the difference between the shut-off temperature of the query

virus and that of TRc, the unpaired version of Student’s t-test was used.


139

3.3 Results

3.3.1 Sequence analysis of the ts and TR EV71 viral stocks and their

cDNA clones

The TR viral stock was obtained by passaging the original ts virus at 39°C.

To map genetic differences between TR and ts EV71, the entire genome

sequences of both viral stocks and corresponding cDNA clones were determined.

To specify the difference between the viral stocks and viral clones, they were

designated as ts stock (tss), ts clone (tsc), TR stock (TRs) and TR clone (TRc). The

full-length genome sequence of the TRc was submitted to the GenBank

(accession #: JX025559, strain: LAZ60-TR).

Sequences obtained from the tss and TRs represented consensus of the

multiple viral genomes existing within the viral population. Therefore, where

well defined double peaks, similar to that illustrated in Figure 3-2, were observed

within the chromatograms, it was considered to be an existing variation of the

viral stock. All heterogenic nucleotide positions were confirmed by repeating the

sequencing reaction with an independent RT-PCR product amplified from the

same RNA sample.


140

A.

B.

Figure 3-2: Heterogeneity observed within the VP1 gene sequence of EV71 tss.

The illustrated chromatograms (A and B) are obtained from the sequencing of two independent RT-PCR products, originating from the tss RNA, and represent the same fragment within the EV71 genome. Position of the double peak M (C/A), indicated by the arrow, corresponds to the 293d nucleotide in the VP1 encoding region. Numbering above the peaks represents nt numbers within the chromatograms and is not related to EV71 genome.


141

In total, five nucleotide (nt) positions within the TRs sequence

corresponded to double peaks in the tss sequence and, therefore, were not

considered to be newly emerged mutations, but rather naturally selected from the

existing variation due to incubation at increased temperature. All five nt-

variations were located within the VP1 gene and resulted in amino acid (aa)

changes. Additionally, the EV71 sequence consensus obtained from the TRs had

four unique nt-changes when compared with the consensus of the tss. Among

those four newly identified nt-changes two were non-synonymous and resulted in

one aa-change within the VP3 capsid protein and one aa-change in the 3Dpol. All

nt- and aa-variations observed between the tss and TRs consensuses are

summarised in Table 3-3.

Genomic region Nucleotide position*

Nucleotide change**

Amino acid change**

VP2 333 G to A - VP3 234 A to G - VP3 317 G to A G106 to E VP1 275 A/G to A E/G92 to E VP1 293 C/A to C K/T98 to T VP1 658 C/T to C L/F220 to L VP1 724 A/C to A K/Q242 to K VP1 848 C/T to T S/F283 to F 3D 895 T to A S299 to T

Table 3-3: Mutations observed in the conversion of the tss isolate of EV71 to TRs.

* Nucleotide positions are numbered within the indicated genomic regions. Newly identified mutations are in bold. **Mutations are indicated as nucleotide or amino acid changes of the sequence consensus from the tss to TRs of EV71.


142

Full-length genome sequences obtained from one cDNA clone of each

variant (tsc and TRc) were compared to the consensus sequences of the

corresponding viral stocks (tss and TRs). The TRc sequence showed a 100%

identity to the TRs. One nucleotide difference (cytosine instead of thymidine) was

observed between the tsc and tss at position 682 within the 5′UTR region. This

mutation could be a result of natural variation represented by the minority of

EV71 genomes within the tss and, therefore, not detected as a double peak within

the tss consensus. It may however also represent an artificial mutation introduced

during the RT-PCR step prior to cDNA cloning. Therefore, position 682 was

excluded from the following analysis and SDM.

3.3.2 Structure modelling for EV71 mutants within the structural VP1

and VP3 proteins

The available 3d-structure of the capsid proteins of EV71, 3VBF, was

used in order to map mutations observed within the VP1 and VP3 proteins of the

TR variant of EV71. BLAST alignments of the TRc VP1 and VP3 sequences

against the corresponding chain A and C of the 3VBF strain are presented in

Figure 3-3.


143

A. 10 20 30 40 50 60 70 80

....*....|....*....|....*....|....*....|....*....|....*....|....*....|....*....|

TRc_VP1 1 GDRVADVIESSIGDSVSRALTRALPAPTGQDTQVSSHRLDTGKVPALQAAEIGASSNASDESMIETRCVLNSHSTAETTL 80

3VBF_A 1 GDRVADVIESSIGDSVSRALTHALPAPTGQNTQVSSHRLDTGKVPALQAAEIGASSNASDESMIETRCVLNSHSTAETTL 80

90 100 110 120 130 140 150 160

....*....|....*....|....*....|....*....|....*....|....*....|....*....|....*....|

TRc_VP1 81 DSFFSRAGLVGEIDLPLTGTTNPNGYANWDIDITGYAQMRRKVELFTYMRFDAEFTFVACTPTGEVVPQLLQYMFVPPGA 160

3VBF_A 81 DSFFSRAGLVGEIDLPLKGTTNPNGYANWDIDITGYAQMRRKVELFTYMRFDAEFTFVACTPTGEVVPQLLQYMFVPPGA 160

170 180 190 200 210 220 230 240

....*....|....*....|....*....|....*....|....*....|....*....|....*....|....*....|

TRc_VP1 161 PKPDSRESLVWQTATNPSVFVKLSDPPAQVSVPFMSPASAYQWFYDGYPTFGEHKQEKDLEYGACPNNMMGTFSVRTVGT 240

3VBF_A 161 PKPDSRESLAWQTATNPSVFVKLSDPPAQVSVPFMSPASAYQWFYDGYPTFGEHKQEKDLEYGAMPNNMMGTFSVRTVGT 240

250 260 270 280 290

....*....|....*....|....*....|....*....|....*....|....*..

TRc_VP1 241 SKSKYPLVIRIYMRMKHVRAWIPRPMRNQNYLFKANPNYAGNFIKPTGASRTAITTL 297

3VBF_A 241 SKSKYPLVVRIYMRMKHVRAWIPRPMRNQNYLFKANPNYAGNSIKPTGASRTAITTL 297

B. 10 20 30 40 50 60 70 80

....*....|....*....|....*....|....*....|....*....|....*....|....*....|....*....|

TRc_VP3 1 GFPTELKPGTNQFLTTDDGVSAPILPNFHPTPCIHIPGEVRNLLELCQVETILEVNNVPTNATSLMERLRFPVSAQAGKG 80

3VBF_C 1 GFPTELKPGTNQFLTTDDGVSAPILPNFHPTPCIHIPGEVRNLLELCQVETILEVNNVPTNATSLMERLRFPVSAQAGKG 80

90 100 110 120 130 140 150 160

....*....|....*....|....*....|....*....|....*....|....*....|....*....|....*....|

TRc_VP3 81 ELCAVFRADPGRSGPWQSTLLGQLCEYYTQWSGSLEVTFMFTGSFMATGKMLIAYTPPGGPLPKDRATAMLGTHVIWDFG 160

3VBF_C 81 ELCAVFRADPGRNGPWQSTLLGQLCGYYTQWSGSLEVTFMFTGSFMATGKMLIAYTPPGGPLPKDRATAMLGTHVIWDFG 160

170 180 190 200 210 220 230 240

....*....|....*....|....*....|....*....|....*....|....*....|....*....|....*....|

TRc_VP3 161 LQSSVTLVIPWISNTHYRAHARDGVFDYYTTGLVSIWYQTNYVVPIGAPNTAYIIALAAAQKNFTMKLCKDASDILQTGT 240

3VBF_C 161 LQSSVTLVIPWISNTHYRAHARDGVFDYYTTGLVSIWYQTNYVVPIGAPNTAYIIALAAAQKNFTMKLCKDASDILQTGT 240

..

TRc_VP3 241 IQ 242

3VBF_C 241 IQ 242

Figure 3-3: BLAST alignment of the VP1 (A) and VP3 (B) proteins of the EV71 TRc

against the reference, 3VBF.

The aligned residues are in capital letters. Identical residues are displayed on white background. Regions, which are aligned but not identical in their sequences, are on grey background. Mutations observed in the TRc when compared to the tsc are boxed.

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Search&doptcmdl=GenPept&db=Protein&term=3VBFA




http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Search&doptcmdl=GenPept&db=Protein&term=3VBFC





144

The protein BLAST alignment demonstrated 100% sequence coverage,

and 98% and 99% sequence homology between EV71 TRc and 3VBF reference

strain within VP1 and VP3 capsid proteins, respectively. The high score of the

alignment implies that amino acids of EV71 TRc would have their alpha carbons

superimposed in space on the corresponding positions of the reference structure.

Mutations within VP1

According to the reference structure, the VP1 protein folds into 14 strands,

one helix and several intermediate loops. Two amino acid changes, T98 and

K242, were mapped within two adjacent loops, exposed at the summit of the

five-fold axis peak of VP1 (Figure 3-4, A and B). Another two mutations, L220

and F283, were located on the rim of the canyon within VP1 loops interacting

with VP2 protein of the same subunit (Figure 3-4, C). The E92 mutation was the

only one which was mapped within strand 3 (composed of residues from 85 to

96) on the north wall of the canyon.


145

A.

B.


146

C.

Figure 3-4: Mutations within VP1 of EV71 TRc, mapped on 3VBF (Wang et al. 2012)

reference structure, (A) top, (B) side and (C) angle views.

A and B: The presented structure consists of 5 VP1 proteins constituting a central vertex of the pentamer (built up by five repeating subunits) of the viral capsid. The VP2, VP3 and VP4 proteins are omitted for better clarity. Each of the VP1 proteins is represented by a different colour (dark brown, pink, light brown, grey and green). Structural elements, helixes and strands, are represented by cylinders and flat boards, respectively. Mapped mutations are highlighted in yellow on the dark brown VP1 molecule. Star indicates the five-fold axis when the central vertex is being viewed from the top (A). Arrows indicate the five-fold axis and canyon when viewed from the side (B). C: Angle view of the central vertex. VP1 and VP2 proteins of one subunit are represented in dark brown and blue, respectively.


147

According to the IBIS prediction results, among 5 amino acid variations

within the VP1, only the 220th amino acid position was mapped within putative

binding sites of two inferred protein-protein interactions. One of those

interactions was an observed or native interaction with the VP2 protein within

EV71 capsid subunit. However, an alignment of the binding site residues from

the homologous enteroviruses did not reveal sequence conservation on position

220 (data not shown). The second predicted interaction was based on the

interaction between VP1 protein of PVs and the IGv domain of the PV receptor.

Again, there was no evolutionary sequence conservation observed on this

position between VP1 proteins of EV71 and PVs (data not shown). These results

indicate the existing variation of the 220th amino acid and its tolerance by the

VP1 structure.

Mutation within VP3

According to the folding pattern of 3VBF_C reference structure, the VP3

protein consists of 13 strands, 1 helix and several connecting loops. The G106E

mutation observed within the TR variant of EV71 was mapped to the C-terminal

amino acid of the helix. In the viral capsid, it is buried on the canyon floor

(Figure 3-5).


148

Figure 3-5: Mutation within VP3 of EV71 TRc, mapped on 3VBF (Wang et al. 2012)

reference structure, side view.

Five VP1 proteins constituting the central vertex of the pentamer are represented by different colours (dark brown, pink, light brown, grey and green). Structural elements, helixes and strands, are omitted for better clarity. The VP3 is in yellow, and E106 mutation is highlighted in red. Arrows indicate the five-fold axis and canyon.


149

The IBIS analysis of the VP3 protein structure did not predict any direct

interactions for the amino acid position 106. However, nearby positions 100, 103,

104, 107 and 108 were mapped within the binding sites involved in interactions

with the VP1 and VP2 proteins.

3.3.3 Structure modelling for EV71 mutant within the 3Dpol

The 3N6L and its neighbouring structures 3N6M and 3N6N are only

crystal structures available for the 3Dpol of EV71. The 3N6L represents the 3Dpol

itself, whereas 3N6M and 3N6N are the crystallized enzyme in complex with

guanosine triphosphate (GTP) and 5-bromouridine triphosphate (Br-UTP),

respectively. Unfortunately, none of those structures depict conformational

changes within the 3Dpol active site during RNA replication. Therefore, the 3OL6

structure representing a crystallized elongation complex of PV 3Dpol prior to the

NTP binding, and the 3OL7 structure depicting the catalysis/pre-RNA-

translocation step were used additionally to model the mutation observed in the

EV71 TR variant. BLAST alignment of the 3Dpol of the TRc showed 100%

coverage with both EV71 and PV reference structures. Sequence identity was

calculated at 93% and 68% when EV71 TRc was compared to

3N6L/3N6M/3N6N (EV71) or 3OL6/3OL7 (PV), respectively.

According to the reference protein structures the S299T mutation was

mapped within the C-end of motif B of the palm sub-domain. The C-portion of

motif B forms a part of the αH-helix (comprises amino acids between 294 and

313 in EV71 3Dpol) and is located within the enzyme active centre which

constitutes the GDD span and invariant residues D233 and D238 (Figure 3-6).


150

A. 10 20 30 40 50 60 70 80

....*....|....*....|....*....|....*....|....*....|....*....|....*....|....*....|

TRc_3Dpol

1 GEIQWMKPNKETGRLNINGPTRTKLEPSVFHDVFEGTKEPAVLTSKDPRLEVDFEQALFSKYVGNTLHEPDEFVKEAALH 80

3N6M_A 1 GEIQWVKPNKETGRLSINGPTRTKLEPSVFHDVFEGNKEPAVLHSKDPRLEVDFEQALFSKYVGNTLYEPDEYIKEAALH 80

3OL6_A 1 GEIQWMRPSKEVGYPIINAPSKTKLEPSAFHYVFEGVKEPAVLTKNDPRLKTDFEEAIFSKYVGNKITEVDEYMKEAVDH 80

90 100 110 120 130 140 150 160

....*....|....*....|....*....|....*....|....*....|....*....|....*....|....*....|

TRc_3Dpol

81 YANQLKQLDIKTTKMSMEDACYGTENLEAIDLHTSAGYPYSALGIKKRDILDPTTRDVSKMKSYMDKYGLDLPYSTYVKD 160

3N6M_A 81 YANQLKQLEINTSQMSMEEACYGTENLEAIDLHTSAGYPYSALGIKKRDILDPTTRDVSKMKFYMDKYGLDLPYSTYVKD 160

3OL6_A 81 YAGQLMSLDINTEQMCLEDAMYGTDGLEALDLSTSAGYPYVAMGKKKRDILNKQTRDTKEMQKLLDTYGINLPLVTYVKD 160

170 180 190 200 210 220 230 240

....*....|....*....|....*....|....*....|....*....|....*....|....*....|....*....|

TRc_3Dpol

161 ELRAIDKIKKGKSRLIEASSLNDSVYLRMTFGHLYEAFHANPGTVTGSAVGCNPDVFWSKLPILLPGSLFAFDYSGYDAS 240

3N6M_A 161 ELRSIDKIKKGKSRLIEASSLNDSVYLRMAFGHLYETFHANPGTITGSAVGCNPDTFWSKLPILLPGSLFAFDYSGYDAS 240

3OL6_A 161 ELRSKTKVEQGKSRLIEASSLNDSVAMRMAFGNLYAAFHKNPGVITGSAVGCDPDLFWSKIPVLMEEKLFAFDYTGYDAS 240

233 238

250 260 270 280 290 300 310 320

....*....|....*....|....*....|....*....|....*....|....*....|....*....|....*....|

TRc_3Dpol

241 LSPVWFRALEIVLREIGYSEDALSLIEGINHTHHVYRNKTYCVLGGMPSGCSGTSIFNTMINNIIIRTLLIKTFKGIDLD 320

3N6M_A 241 LSPVWFRALELVLREIGYSEGAISLIEGINHTHHVYRNKTYCVLGGMPSGCSGTSIFNSMINNIIIRALLIKTFKGIDLD 320

3OL6_A 241 LSPAWFEALKMVLEKIGFGD-RVDYIDYLNHSHHLYKNKTYCVKGGMPSGCSGTSIFNSMINNLIIRTLLLKTYKGIDLD 319

Motif B

330 340 350 360 370 380 390 400

....*....|....*....|....*....|....*....|....*....|....*....|....*....|....*....|

TRc_3Dpol

321 ELNMVAYGDDVLASYPFPIDCLELARTGKEYGLTMTPADKSPCFNEVTWENATFLKRGFLPDHQFPFLIHPTMPMREIHE 400

3N6M_A 321 ELNMVAYGDDVLASYPFPIDCLELAKTGKEYGLTMTPADKSPCFNEVNWDNATFLKRGFLPDEQFPFLIHPTMPMREIHE 400

3OL6_A 320 HLKMIAYGDDVIASYPHEVDASLLAQSGKDYGLTMTPADKSATFETVTWENVTFLKRFFRADEKYPFLIHPVMPMKEIHE 399

GDD span

410 420 430 440 450 460

....*....|....*....|....*....|....*....|....*....|....*....|..

TRc_3Dpol

401 SIRWTKDARSTQDHVRSLCLLAWHNGKEEYEKFVSTIRSVPIGKALAIPNFENLRRNWLELF 462

3N6M_A 401 SIRWTKDARNTQDHVRSLCLLAWHNGKQEYEKFVSTIRSVPVGRALAIPNYENLRRNWLELF 462

3OL6_A 400 SIRWTKDPRNTQDHVRSLCLLAWHNGEEEYNKFLAKIRSVPIGRALDLPEYSTLYRRWLDSF 461

B.

Figure 3-6: Sequence alignment and structure modelling of the 3Dpol of the EV71 TRc.

A. BLAST alignment of the TRc 3Dpol against the 3N6M and 3OL6 reference sequences. Colouring scheme is analogous to that of Figure 3-3. Motif B and GDD span, residues D233 and D238 are underlined. B. 3N6M (Wu et al. 2010) reference structure was used in modelling. The motifs and residues indicated within the sequence alignment (A) are highlighted in white and constitute the core of the enzyme. - The S299T mutation is located within the C-end of the motif B, N-terminal portion of the αH-helix.

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Search&doptcmdl=GenPept&db=Protein&term=3N6LA

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Search&doptcmdl=GenPept&db=Protein&term=3OL6A












151

In spite of the sequence conservation observed within the motif B of

3N6M and 3OL6 references (Figure 3-6, A), the N-terminal part (amino acid

residues 286-294) of the span occupies a notable different position when two

structures are merged (data not shown). In contrast, carbons of the C-terminal

portion (residues 295-300) constituting the αH-helix, are perfectly superimposed

in space which indicates structure conservation between EV71 and PV 3Dpol

within the active site.

Sequence variation within position 299 appears to be very limited between

RDRPs of different families of positive-strand RNA viruses. In fact, only Ser and

Thr were found when multiple BLAST alignment was performed with RDRP

sequences obtained from viruses belonging to the Picornaviridae, Caliciviridae

and Flaviviridae families (Figure 3-7).


152

A. Picornaviridae: ssRNA positive-strand viruses, no DNA stage

3N6M Human enterovirus 71

3OL6 Human poliovirus 1

3DDK Human coxsackievirus B3

1XR5 Human rhinovirus 14

1XR6 Human rhinovirus 1B

1XR7 Human rhinovirus 16

1U09 Foot-and-mouth disease virus C-S8c1

Caliciviridae: ssRNA positive-strand viruses, no DNA stage

1KHV Rabbit hemorrhagic disease virus

3NAH Murine norovirus 1

2CKW Sapporo virus

1SH0 Norwalk virus

Flaviviridae: ssRNA positive-strand viruses, no DNA stage

1S48 Bovine viral diarrhea virus 1

1NB7 Hepatitis C virus

2J7U Dengue virus

2HCS Kunjin virus

B. ** * *↓ 3N6M 251 L--VLREIGYSE--GAISLIEGINHTHHVY--RNKTYCV---------LGGMPSGCSGTSIFNSMINNIIIRALLIKT-- 313

3OL6 251 M--VLEKIGFGD--R-VDYIDYLNHSHHLY--KNKTYCV---------KGGMPSGCSGTSIFNSMINNLIIRTLLLKT-- 312

3DDK 251 M--ILEKLGYTH--KETNYIDYLCNSHHLY--RDKHYFV---------RGGMPSGCSGTSIFNSMINNIIIRTLMLKV-- 313

1XR5 251 K--VLTKLGFAG--S--SLIQSICNTHHIF--RDEIYVV---------EGGMPSGCSGTSIFNSMINNIIIRTLILDA-- 311

1XR6 252 K--VLENLSFQS--N---LIDRLCYSKHLF--KSTYYEV---------AGGVPSGCSGTSIFNTMINNIIIRTLVLDA-- 311

1XR7 252 M--VLDNLSFNP--T---LINRLCNSKHIF--KSTYYEV---------EGGVPSGCSGTSIFNSMINNIIIRTLVLDA-- 311

1U09 258 EEVFRTEFGFHP--NAEWILKTLVNTEHAY--ENKRITV---------EGGMPSGCSATSIINTILNNIYVLYALRRH-- 322

1KHV 268 D--ILADCCEQT--ELTKSVVLTLKSHPMTILDAMIVQT---------KRGLPSGMPFTSVINSICHWLLWSAAVYKS-- 332

3NAH 260 D--IMVRLSPEP--DLARVVMDDLLAPSLLDVGDYKIVV---------EEGLPSGCPCTTQLNSLAHWILTLCAMVEV-- 324

2CKW 260 A--ILERFAEPH--PIVSCAIEALSSPAEGYVNDIKFVT---------RGGLPSGMPFTSVVNSINHMIYVAAAILQA-- 324

1SH0 260 E--IMVKFSSEP--HLAQVVAEDLLSPSVVDVGDFTISI---------NEGLPSGVPCTSQWNSIAHWLLTLCALSEV-- 324

1S48 293 E--IQKYYYKKEWHKFIDTITDHXTEVPVITADGEVYIR---------NGQRGSGQPDTSAGNSXLNVLTXXYAFCESTG 361

1NB7 238 SIYQCCDLAPEARQAIRSLTERLYIGGPLTNSKGQNCGY---------RRCRASGVLTTSCGNTLTCYLKATAACRAA-- 306

2J7U 286 K--IIQQMDPEH-RQLANAIFKLTYQNKVVKVQRPTPTG-TVMDIISRKDQRGSGQVGTYGLNTFTNMEAQLVRQMEGEG 361

2HCS 244 K--VLELLDGEH-RRLARAIIELTYRHKVVKVMRPAADGRTVMDVISREDQRGSGQVVTYALNTFTNLAVQLVRMMEGEG 320

~~~~~~~~~~~~~~~~~~~~

Figure 3-7: Multiple BLAST alignment of RDRP of positive-strand RNA viruses.

A. PDB accession codes and virus descriptions for RDRPs sequences used in the structure-based alignment (B). B. Sequence alignment of RDRPs whose crystal structures are available in PDB. Only the section aligned with an EV71 3Dpol fragment between 251 and 313 is shown. Invariant residues are marked by asterisks. The 299th position in the 3Dpol sequence of EV71 is indicated by the arrow and Ser/Thr occupying the reciprocal position within RDRPs of different viruses is displayed with a grey background. Motif B is underlined. Amino acids within the αH-helix are marked by tildes.


153

Both amino acids (Ser and Thr) are hydroxyl-containing and differ only

by one methyl group replacing hydrogen at the β-carbon in threonine. This strict

limit on sequence diversity within position 299, which results in only either one

of two very similar residues, is probably related to a required structure

conservation of the core region of RDRPs. Residues within the active centre of

the 3Dpol form a number of interactions with the templating and nascent RNA

strands, an incoming NTP, two Mg2+ ions as well as several interactions within

the enzyme itself and are necessary for correct positioning of the catalytically

active amino acid residues. Amino acid positions constituting the binding sites of

EV71 3Dpol were predicted with IBIS and summarized in Table 3-4.


154

Amino acid positions of the predicted interactions within EV71 3Dpol

Predicted interactions are based on structure/virus

Interaction partner Identity to query, %

159*, 163*, 167*, 174*, 175*, 234*, 235, 236*, 237*, 238*, 289*, 290*, 329*

3N6M/EV71 Guanosine 5´-triphosphate

100

163*, 167*, 174*, 175*, 234*, 235, 236*, 237*, 238*, 289*, 329*, 360*

2IM0/PV1 Cytidine triphosphate

67

159*, 163*, 174*, 236*, 238*, 289*, 298*, 329*

3OLA/PV1 Deoxycytidine diphosphate

68

20*, 22, 24*, 43*, 108*, 111*, 114*, 115*, 121, 127*, 157*, 159*, 160*, 176*, 177*, 178*, 179*, 188*, 199*, 210*, 211*, 212*, 213, 289*, 290*, 291*, 292*, 293*, 327*, 413*, 416*, 420*, 457

3OL6/PV1 Template RNA 68

113, 128*, 133, 327*, 328*, 329*, 330*, 375*, 376*, 377*, 393*, 401*, 410, 413*, 414*, 417*, 418*, 421*

3OL6/PV1 Nascent RNA 68

233*, 329* 3OL7/PV1 Magnesium ion 68

Table 3-4: IBIS predicted interactions within the 3Dpol of EV71.

Amino acid positions discussed in Figure 3-8 are in bold. Those amino acids which are identical between the TR EV71 variant and reference structures of EV71 (3N6M) or PV1 (2IM0, 3OLA, 3OL6 and 3OL7) are marked with asterisks (*).


155

During the replication process the active site of the 3Dpol undergoes a

series of conformational changes. Every elongation cycle was proposed to consist

of six states (Gong & Peersen 2010). The prepositioning of the templating

nucleotide, the incoming NTP selection, recognition and binding, all occur when

the active site of the enzyme is in the open conformation, whereas the following

binding of two Mg2+ ions and subsequent catalysis take place after the active site

closure step (Figure 3-8). Interchange between open and closed conformations of

the enzyme is achieved via rearrangements of interactions within the palm sub-

domain. Structural alignment of the 3Dpol in both conformations demonstrates

clear structural changes within the interaction network formed around the

incoming NTP (positions 161-177, 238 and 289-296) and even more obvious

changes observed within the metal binding cluster (positions 233, 329 and 359).

Interestingly, that position of the invariant N298 remains unchanged after it

forms a hydrogen bond with the 2’-ribose hydroxyl of the NTP molecule. In fact,

the backbone of the αH-helix between amino acid residues 297 and 313 occupies

exactly the same position in both conformations (Figure 3-8, the 310th residue is

the last residue shown within the αH-helix). The fixed position of the αH-helix

within the catalytic site was previously observed in all four deferent types of

polymerase: RDRP (PV polymerase), RDDP (HIV1 reverse transcriptase),

DDDP (E.coli DNA polymerase I, Klenow fragment) and DDRP (T7

polymerase) (Hansen, Long & Schultz 1997). The structural alignment of the

3Dpol in open and closed conformation provides additional evidence that C-

terminal part of the motif B constituting the αH-helix remains fixed within the

core centre of the palm sub-domain during the polymerisation process. The fixed

position of the αH-helix and particularly N298 might be essential in NTP

recognition, followed by the active site closure and catalysis.


156

Figure 3-8: Interactions formed within the active centre of the 3Dpol during RNA

synthesis.

The figure is a result of the alignment of the crystal structures 3N6M (Wu et al. 2010), 3OL6 and 3OL7 (Gong & Peersen 2010). The 3N6M and 3OL6 polymerase backbones (chains A) represent the open conformation of the enzyme and occupy similar positions within the 3Dpol core region. Therefore, chain 3N6M_A was omitted for clarity and only the GTP molecule was left in further modelling. The 3OL7 structure consisting of the polymerase chain A, the templating and nascent RNA strands, two magnesium ions and a pyrophosphate anion (PPi) constitute the elongation complex in closed conformation. Both RNA strands have schematic representation within the elongation complex.


157

The right-bottom view is the 3Dpol backbone structure with RNA template (coloured in blue) threading through the enzyme and nascent RNA strand (coloured in brown) appearing from the catalytic centre. The main view represents the detailed structure of the polymerase core region (boxed in red on the right-bottom view). The RNA template strand and some residues within 3Dpol are omitted for clarity. The open state of the 3Dpol structure is coloured according to the element scheme, where carbon is grey, nitrogen is blue, oxygen is red and phosphorus is green. Structure in closed state is coloured in yellow. Amino acid residues forming interactions within the core region of the 3Dpol are highlighted in pink on the grey backbone. Interactions formed prior to the active site closure and after that are marked with dash lines in grey and yellow, respectively. The S299 is highlighted in turquoise. Position of the incoming NTP directly above the catalytic site (G328, D329 and D330) is coordinated by interactions with R163, K167, R174 and L175. In this position the incoming NTP is base-paired with the template nucleotide (Gong & Peersen 2010). The NTP recognition leads to formation of the extensive network of new interactions between the 2’and 3’-ribose hydroxyls and D238, S289 and N298 of the 3Dpol. As a result, the NTP is sinking towards the active site. These conformational changes position D233 for metal ion binding which, in turn, triggers the active site closure and catalysis.


158

3.3.4 Construction of EV71 mutants

The TR phenotype of EV71 resulted in seven amino acid changes of the

original ts variant (Table 3-3). In order to examine the contribution of each single

amino acid to the TR phenotype, the nucleotides responsible for the amino acid

changes were substituted within the TR cDNA clone with the corresponding

nucleotides of the tss. In total, seven mutant EV71 cDNA clones, each carrying a

single amino acid reversion from the TR to the ts sequence, were constructed

(Figure 3-9). Infectious progeny was rescued from all mutant clones after

transfection of Vero cells with the in vitro RNA transcripts.


159

Figure 3-9: Schematic representation of the EV71 mutant genomes.

Genome of the ts EV71 variant (tss) serves as a reference. The nucleotide changes identified within the TR EV71 variant are indicated with stars within the VP2, VP3, VP1 and 3D genome regions of the TR cDNA clone (TRc). The dark or white stars represent silent or non-silent mutations, respectively. The non-silent mutations within the TRc were substituted with the corresponding nucleotides of the tss. One mutant within the VP3, five mutants within the VP1 and one mutant within the 3D gene were constructed. Each of those mutants differed from the parent TRc by a single amino acid. Names of the EV71 mutants indicate the viral protein and amino acid change from the TR to the ts sequence.


160

3.3.5 Temperature sensitivity of the mutant viruses

Growth of the tsc, TRc and generated EV71 mutants was assayed by

measuring the TCID50 titre in Vero cell culture at 37˚C, 38˚C, 39˚C and 40˚C.

The results indicated that VP1 reversions within positions 98, 220, 242 and 283

of the TRc did not reduce the temperature resistance of the virus (Figure 3-10).

The shut-off temperature of those mutants fell within the region of the shut-off

temperature of the TRc (39.1˚C ± 0.1˚C variation) (Table 3-5). The VP1 reversion

on amino acid position 92 and the VP3 reversion on position 106 resulted in

0.5˚C and 0.7˚C decreases in their shut-off temperatures, respectively. However,

those differences were not statistically valid when compared to the TRc. In

contrast, the EV71 mutant within the 3Dpol demonstrated a statistically valid

reduction in viral replication at elevated temperatures. The shut-off temperature

of the 3D T299S mutant was similar to that of the tsc (37.4˚C and 37.3˚C,

respectively). The results indicated that Thr299 within the 3Dpol was mainly

responsible for the temperature resistant phenotype of EV71. The Glu106 (VP3)

and Glu92 (VP1) probably added further contribution to the temperature

resistance of the virus and might account for the difference observed between the

tsc and the 3D T299S mutant (Figure 3-10, C).


161

A.

B.

C.

Figure 3-10: Temperature dependent reduction in virus titres of EV71 mutants in Vero

cells.

Vero cells were infected with EV71 at an m.o.i of 0.1 and incubated at indicated temperatures for 72 h. The virus titres were determined by TCID50 assay. The mean log10 of the TCID50 from three experiments was used to calculate a reduction in virus titre (∆TCID50) at each temperature compared to the TCID50 at 37˚C (refer Table 3-5). The results obtained with EV71 mutants (A: within the VP3, B: within the VP1, and C: within the 3Dpol gene) were plotted on graphs against the data from the reference viruses: TR EV71 clone (TRc) and ts EV71 clone (tsc).


162

Virus name

Virus titre* ± SEM at: Reduction** in virus titre at: Shut-off (°C) *** 37°C 38°C 39°C 40°C 38°C 39°C 40°C

TRc 5.80 ±

0.14 5.55 ±

0.00 4.97 ±

0.08 1.62 ±

0.10 0.25

±0.14 0.83

±0.17a 4.18

±0.21a 39.1

tsc 5.55 ±

0.25 1.88 ±

0.08 1.30 ±

0.00 n/d 3.67 ±0.22a

4.25 ±0.25a n/d 37.3b

VP3 E106G 6.13 ±

0.33 5.30 ±

0.25 4.88 ±

0.33 n/d 0.83 ±0.3

1.25 ±0.58 n/d 38.4

VP1 E92G 5.72 ±

0.08 5.05 ±

0.38 4.47 ±

0.22 n/d 0.67 ±0.36

1.25 ±0.25a n/d 38.6

VP1 T98K 6.55 ±

0.25 5.97 ±

0.08 5.88 ±

0.08 2.22 ±

0.17 0.58

±0.17a 0.67

±0.22a 4.33

±0.42a 39.1

VP1 L220F 5.72 ± 0.22

5.38 ± 0.17

5.05 ± 0.14

2.88 ± 0.08

0.33 ±0.30

0.67 ±0.22a

2.83 ±0.17a 39.2

VP1 K242Q 5.30 ±

0.25 4.47 ±

0.22 4.38 ±

0.30 1.50 ±

0.05 0.83

±0.44 0.92

±0.08a 3.80

±0.23a 39.0 VP1 F283S 4.80 ±

0.00 4.22 ±

0.17 4.13 ±

0.22 1.45 ±

0.00 0.58

±0.17a 0.67

±0.22a 3.35

±0.00 39.1

3D T299S 5.47 ± 0.22

3.05 ± 0.00

2.13 ± 0.22 n/d 2.42

±0.22a 3.33

±0.08a n/d 37.4b

Table 3-5: Temperature sensitivity of EV71 mutants.

* Viruses were grown in Vero cells at the indicated temperatures for 72 h. Resulting virus titres were expressed as the mean ± standard error mean (SEM) of three experiments and represent log10 (TCID50) in 1 ml virus sample. The lower limit of virus detection was 1.45 log10 TCID50/ml. ** Reduction in virus titres is expressed as the mean ± SEM of the log10 differences between TCID50 values obtained at the indicated temperatures versus those at 37˚C from three experiments. *** Temperature at which TCID50 titre is reduced by 1.00 log10 when compared to viral titre at 37˚C. n/d – not determined. a – P<0.05 of the “paired” Student’s t-test indicates the statistical significance of the reduction in a virus titre against the virus titre at 37˚C. b – P<0.05 of the “unpaired” Student’s t-test indicates the statistical significance of the difference between the shut-off temperature of the query virus and that of TRc.


163

3.4 Discussion

In the poliomyelitis eradication era EV71 is a newly emerged human

pathogen which has been associated with numerous cases of neurological

diseases and fatalities in young children (Bible et al. 2007; Lin et al. 2002a).

Successful use of the poliomyelitis vaccines removed the need of further

development of some promising anti-picornaviral inhibitors which could be

useful against EV71 (Barnard 2006). As a result, to date there have been no

specific anti-EV71 compounds available and treatment of EV71 infections with

severe complications remains limited to symptomatic, immuno-modulating and

supportive therapies (Jan et al. 2010; Wang et al. 2006).

The molecular basis and mechanism determining the neuropathogenicity

of EV71 are not well understood. Recent studies on identification of EV71

receptors partially explained the mechanism of virus entry into human cells of

non-neural origin (Chen et al. 2012; Lin et al. 2009; Nishimura et al. 2009;

Yamayoshi et al. 2009; Yang, Chuang & Yang 2009). However, cellular

receptors facilitating the infection of neural cells remain to be discovered. Route

of inoculation is one of the main factors determining the progression and severity

of the disease in experimental animal models (Chen 2007; Khong et al. 2012).

Intraspinal inoculation of monkeys with EV71 strains isolated from HFMD or

fatal cases showed no difference in neurological manifestation or CNS lesions,

suggesting the same neurotropism of the EV71 strains irrespective of their

temperature sensitivity or isolation source (Nagata et al. 2002). However, natural

transmission of EV71 infection is limited to the faecal-oral and oral-oral (or

droplet) routes, which lead to initial replication of the virus in the enteric or

respiratory tract. Dissemination of viral particles to other organs, including CNS,

takes place during the next few days when the viral population starts facing

immune system pressure.

The earliest symptom associated with the immune response to EV71

infection is fever which is followed (in 1-2 days) by skin rash and herpes-like


164

mouth ulcers (Tran et al. 2011). At the beginning, the fever is usually mild

(around 38˚C) but the temperature can reach over 39˚C and become persistent in

patients with severe complications, such as pulmonary oedema, brainstem

encephalitis and meningitis (McMinn et al. 2001b; Ng et al. 2001; Wang et al.

2003; Xu et al. 2012). According to clinical observations, neurological symptoms

develop approximately 3 days after an increase in body temperature and an

appearance of skin lesions (Cho et al. 2010; Huang et al. 1999). Analysis of the

virus in CNS samples (Lum et al. 1998) several days after the onset of fever

indicates a possible role that the elevated body temperature may play in evolution

of the originally transmitted viral strain/s that may affect viral replication and

disease progression. Correlation between temperature resistance of the virus and

CNS-involvement has been reported for EV71 clinical isolates in Taiwan (Chang

et al. 2010; Kung et al. 2007; Kung et al. 2010).

Identification of the molecular determinants responsible for virus

adaptation to increased temperatures in field isolates is difficult due to the

original sequence variation between circulating viral strains. Additionally,

multiple selective forces applied upon the immune system response would direct

viral evolution in situ to emergence of adaptive mutations in response to a

number of factors. To eliminate these other factors and identify EV71 molecular

determinants, associated with temperature resistance of the virus, experiments

were conducted in vitro.

Sequence analysis of the original ts viral stock, propagated over multiple

passages at 37˚C, revealed five heterogeneous positions within the EV71

genome. All those positions were located within the VP1gene, which is accepted

to be the most variable region within the enterovirus genome (Kunkel & Schreier

2000; Oberste et al. 1999b). The increase in incubation temperature during the

next four passages resulted in a reduction of the genetic diversity of the virus

population. There were no nucleotide variations observed within the sequence of

the TR viral stock, and the cDNA clone obtained from that stock showed 100%

sequence identity to the TRs consensus. The reduced genetic variation of the TR

stock indicated that viral population had passed through a bottleneck, conditioned


165

by the temperature, and genomes with fitness lower than a certain threshold had

been eliminated from the population. This conclusion is an agreement with the

proposed quasispecies dynamics for RNA viruses (Domingo et al. 2001;

Manrubia et al. 2005).

Except for the overall decrease of the genetic diversity within the

population, four new mutations were identified within the TR EV71 variant, and

two of those mutations led to amino acid changes in the VP3 and 3D viral

proteins. It is difficult to conclude if some or all of those mutations pre-existed as

quasispecies prior to the increase in temperature. No variation within those VP3

or 3D nucleotide positions were identified by sequencing the ts stock or several ts

cDNA clones obtained from that stock (data not shown). Therefore, if some of

those mutations pre-existed they had to be represented by a relatively small

number of viral genomes. In contrast, all VP1 mutations were detected within the

ts cDNA clones (data not shown) prior to the change in the incubation

temperature.

Cell culture observation was that the 2nd passage of the virus at 39˚C

resulted in the development of well defined signs of CPE within the cell culture

monolayer. By the end of the 3rd and 4th passages the CPE was complete. This

observation indicates an extremely fast adaptation of the viral population to

replicate at an increased temperature. Rapid in situ selection of temperature

resistant variants after the onset of fever can be one of the leading factors

favouring productive replication and dissemination of viral particles. In

experimental animals, oral inoculation of EV71 was reported to result in early

and prominent viremia prior to the viral neuroinvasion (Chen 2007). Additional

factors, such as an abnormal increase in cytokine production and systemic

inflammatory response to viral replication may be responsible for pathogenesis

of the EV71 infections (Lin et al. 2009; Wang et al. 2003; Weng et al. 2010).

It has been previously reported that, both in in vitro and in vivo, selection

of EV71 variants resistant to new conditions occurred within 3-10 passages

(Arita, Wakita & Shimizu 2008; Chen et al. 2009; Chua et al. 2008; Wang et al.

2004). In all cases multiple mutations within the viral genome were observed,


166

but only one or two of those were proven to be adaptive. In this study, among

seven amino acid changes in the TR EV71, only Thr299 within the 3Dpol was

associated with a strong TR phenotype. Based on the results of structure

modelling, it can be hypothesized that this mutation may lead to stabilization of

the N-terminal part of the αH-helix within the catalytic centre of the enzyme. The

mutations detected in the capsid proteins contributed very little or did not

contribute anything at all to the temperature resistance of the virus. Those VP1

(Glu92) and VP3 (Glu106) mutations, which demonstrated a slight increase in

the shut-off temperature, were located at the bottom and north wall of the viral

capsid canyon, whereas mutations with no effect on TR phenotype were mapped

to VP1 loops exposed on the surface of the viral particle. Presence of multiple

non-significant (in terms of new phenotype) changes within the viral genome is

in accordance with the quasispecies dynamics during a bottleneck event, when all

mutations within the fittest variant become co-selected and accumulated within

new population, even if they don’t contribute to virus fitness (Manrubia et al.

2005). Therefore, based on this study and in agreement with previously published

data, it can be concluded that an adaptive phenotype can be successfully rendered

by a single mutation naturally selected within the viral genome. Level of the

acquired adaptation seems to be highly dependent on the strength of the applied

selective pressure. The TR EV71 variant obtained after propagation of the virus

at 39˚C demonstrated a sharp decrease in replication above that temperature. A

possible explanation of this phenomenon is that the first of the random mutations,

leading to a sufficient level of adaptation to new in-host conditions, releases the

viral genome from the selective pressure. The absence of the selective pressure

prevents a further increase in viral fitness and leads to accumulation of the

random mutations in viral population.

Temperature is only one of the multiple forces which lead viral adaptation

within the host. However, the same natural laws must apply to a range of

selective pressures in situ. Understanding the genetic determinants associated

with virus phenotype, and natural ways by which those determinants become

selected from the vast variety of existing viral quasispecies, have an important


167

implication in the development of effective strategies both in disease prevention

and treatment of viral infections.

168

CHAPTER 4

SITE-DIRECTED MUTAGENESIS OF EV71 WITHIN THE

5′UTR

CHAPTER 4 Site-directed mutagenesis of EV71 within the 5’UTR

169

4 SITE-DIRECTED MUTAGENESIS OF EV71 WITHIN THE 5′UTR

4.1 Introduction

The 5′UTR of the enterovirus genome is an essential region which

participates directly in viral RNA translation and replication (Fernandez-

Miragall, Lopez de Quinto & Martinez-Salas 2009; Kuhn, Luz & Beck 1990;

Meerovitch, Nicholson & Sonenberg 1991; Pelletier et al. 1988b). Structural

organisation of the 5′UTR results in the formation of several stem-loops. Their

functions were extensively studied, especially, on polioviruses (Martinez-Salas

2008; Wimmer, Hellen & Cao 1993). Mutations, deletions or insertions affecting

the secondary structure of the 5′UTR often resulted in non-infectious viruses or

mutants exhibiting severe defects in viral polyprotein synthesis of viral RNA

replication (Le et al. 1992; Malnou et al. 2002; Nicholson et al. 1991). In some

studies where revertants to the wt-phenotype were isolated, the mutations

resulting in the wt-sequence or compensatory substitutions restoring the

secondary structure within the 5′UTR were observed (Belov et al. 1995; Kuge,

Kawamura & Nomoto 1989). In other cases revertants possessed mutations,

which were not related to sequence or secondary structure restoration but were

involved in maintaining the tertiary structure interactions with host cell proteins

or intra-domain interactions within the 5′UTR (Malnou et al. 2002). The

secondary structure maintenance within some regions of the 5′UTR seems to be

more important than sequence conservation (Macadam et al. 1992).

The phylogenetic analysis of the 5′UTR of enteroviruses suggests that the

evolution of the region is being forced by the host cell environment. Interaction

of the structural elements of the 5′UTR with host cell specific proteins during

viral RNA translation/replication drives evolution of the region towards the virus

adaptation to different host species and results in clear clustering between the

human and animal enteroviruses (Belsham & Sonenberg 1996; Poyry et al. 1996;

Shiroki et al. 1997). Natural variation in the availability of the host cell proteins


170

within deferent tissues may enhance or restrict viral replication within the host

cells and partially explains bases in a tissue specificity of the virus (Guest et al.

2004; La Monica & Racaniello 1989). On the other hand, exchanging the 5′UTR

structural elements of one enterovirus with reciprocal 5′UTR structures derived

from the other enterovirus species can result in chimeric viruses with altered

requirements of the host cell factors, and different host cell range adaptation

(Gromeier, Alexander & Wimmer 1996; Gromeier et al. 1999).

To date, many studies have been aimed to identify molecular determinants

of attenuation within the 5′UTR of enteroviruses, with the majority of those

studies conducted on polioviruses (De Jesus et al. 2005; Malnou et al. 2002). The

main attenuating mutations of the Sabin vaccine strains were mapped within the

Vth domain of the IRES (Macadam et al. 1994; Ochs et al. 2003). Similarity

between the IRES of PV and EV71 allowed construction of the EV71 mutants

carrying the 5′UTR mutations of Sabin 1, 2 and 3 and exhibiting ts phenotypes in

cell culture (Arita et al. 2005). Combining the 5′UTR mutation of Sabin 1 with

the major ts determinants of Sabin 1 within the 3Dpol and 3′UTR allowed further

reduction in viral replication at elevated temperature and attenuated

neurovirulence in animals (Arita et al. 2008; Arita et al. 2005). The main concern

of Sabin vaccine strains is their low genetic stability resulting in quick reversion

of the 5′UTR to the wt-sequence (Sabin 3) or in acquiring the compensatory

mutations (Sabin 1 and 2) restoring the secondary structure of the Vth domain

(Macadam et al. 1989). Increased stability of the main attenuating determinant

within Sabin 3 5′UTR was shown to be possible via its pairing with the 537A

which creates a thermodynamically stable stem-loop structure and delays

reversion to a stronger CG pair of the wt-sequence (Macadam et al. 2006).

The VIth stem-loop of the PV1 5′UTR was mapped as another region

contributing to the neuropathogenic properties of the virus both in vitro and in

vivo (Gromeier et al. 1999). When replaced with the corresponding structure

derived from the non-neuropathogenic virus, such as HRV2, it resulted in

attenuated phenotype both in neural cell culture and experimental animals.


171

The aim of this study was to generate EV71 mutants within the Vth and

VIth domains of the 5′UTR. The 473 and 538 nucleotide positions lying within

the stem-loop V, and 602, 603, 606 and 607 within the stem loop VI were

targeted in SDM. The growth properties of the mutant viruses were studied in

Vero and SK-N-SH cells. Stability of the EV71 mutant on position 473

(equivalent to 472 of PV3) was studied during serial passages in cell culture.


172


4.2.1 Cell culture

African green monkey kidney (Vero) cell line was used in virus

propagation, rescuing the 5′UTR mutants from the in vitro RNA transcripts,

TCID50 assay, binding assay and growth kinetics assay. The stability of the

5′UTR mutants and viral load accumulation were also conducted on Vero cells.

The cell line was maintained in accordance with growth conditions described in

Chapter 2.

Human neuroblastoma (SK-N-SH) cell line was only used in growth

kinetics of the parent and mutant viruses. The SK-N-SH cells were maintained in

MEM Eagle with Earle’s salts, L-glutamine and sodium bicarbonate (Sigma)

supplemented with 15% FBS (Invitrogen). The cells were grown in 75 cm2 flasks

in a 37˚C incubator with 5% CO2 supply. When compared to Vero cells, the

neuroblastoma cell line was difficult in achieving the confluent stage in culturing

flasks. Therefore, SK-N-SH cells were passaged only approximately once a week

at 50-60% confluence. To maintain the cell growth between passages the growth

medium was replaced with a fresh one every second day.


173

4.2.2 Design of mutagenic primers

Primers used in SDM were designed with the PrimerX software

(http://www.bioinformatics.org/primerx/) based on the DNA sequence of the

5′UTR of the temperature resistant EV71 clone. The following parameters were

specified as required primer features:

a) melting temperature: between 75-85°C,

b) GC content: between 40-60%,

c) length of the primers: between 25-45 bp,

d) 5′ and 3′ flanking regions: between 11-21 bp,

e) primers must terminate in G or C,

f) mutation site must be at the centre of the primer.

Primer pair (F_s3 and R_s3) used in PCR/Restriction enzyme cleavage

was designed in order to amplify a cDNA fragment between 443rd and 569th

nucleotide positions within EV71 5′UTR. The forward primer, F_s3, had a

mismatching G at its 3′-end in order to introduce a Sau3AI recognition site into

EV71 wt-sequence. The reverse primer, R_s3, was designed complementary to

the EV71 genome with consideration of melting temperature and sequence

composition of the forward primer in order to avoid dimers formation. The

FastPCR v5.2 software was used, in order to check the primers compatibility

(<http://www.biocenter.helsinki.fi/bi/programs/fastpcr.htm>) (Kalendar, Lee &

Schulman 2011).

Selected primers were synthesized and reverse-phase cartridge (RPC)

purified by Sigma. The list of primers and their sequences are summarized in

Table 4-1.

http://www.bioinformatics.org/primerx/


174

Primer name Sequence (5′-3′)*

F_SL6-CAV8 GCTGCTTATGGTGACAATTGAAGAATTGTTACCATATAGC

R_SL6-CAV8 GCTATATGGTAACAATTCTTCAATTGTCACCATAAGCAGC

F_SL6-CAV16 GCTGCTTATGGTGACAATTGAAAGATTGTTACCATATAGC

R_SL6-CAV16 GCTATATGGTAACAATCTTTCAATTGTCACCATAAGCAGC

F_C473T CTGAATGCGGCTAATTCTAACTGCGGAGCAC

R_C473T GTGCTCCGCAGTTAGAATTAGCCGCATTCAG

F_5UTR-538A GCAACTCTGCAGCGAAACCGACTACTTTG

R_5UTR-538A CAAAGTAGTCGGTTTCGCTGCAGAGTTGC

F_s3 GTAGTCCTCCGGCCCCTGAATGCGGCTGAT

R_s3 AAGGAAACACGGACACCCAAAGTAGTCGG

Table 4-1: Primers used in SDM and PCR/Restriction enzyme cleavage of EV71

5′UTR.

* Position of the mutated nucleotide within the primer’s sequence is underlined.


175

4.2.3 Construction and recovery of mutant viruses.

Principle:

SDM was conducted on the TR EV71 cDNA clone in order to introduce

mutations within the 5′UTR. The mutations were intended to change the

secondary structure of EV71 RNA genome within the 5′UTR Vth and VIth stem-

loops (Figure 4-1, A).

Two mutant viruses, clones 473U and 473U/538A, carried nucleotide

substitutions within the Vth domain which is located within the IRES. The 473U

mutation corresponded to the 472U attenuating determinant of Sabin 3, whereas

the 473U/538A mutant carried both the Sabin 3 attenuating determinant and its

compensatory substitution on position 538th. The SL6-CAV8 and SL6-CAV16

mutants possessed the VIth domain of the CAV8 and CAV16, respectively.

The RNA secondary structures of the parent and mutant 5′UTR stem-

loops were predicted with the UNAFold software (available on the mfold web

server <http://www.bioinfo.rpi.edu/applications/mfold>) (Zuker 2003) (Figure 4-

1, B and C).


176

A.

B.

C.

Figure 4-1: Predicted secondary structure of EV71 RNA within the 5′UTR.

A. Schematic diagram of the EV71 5′UTR. Adapted with modifications from Thompson and Sarnow (2003). Stem-loops I-VI are numbered. Positions targeted in SDM are indicated with stars and numbered. B. Secondary structure of stem-loop V of the parent virus, 473U and 473U/538A mutants. Mutated nucleotides are boxed. C. Secondary structure of stem-loop VI of the parent virus, SL6-CAV8 and SL6-CAV16 mutants. Mutated nucleotides are boxed and numbered.


177

Methodology:

SDM was performed with mutagenic primers listed in Table 4-1 and

following the protocol described in materials and methods in Chapter 3. The

5′UTR region of selected EV71 cDNA mutant clones was sequenced by the dye-

terminator sequencing method to confirm the presence of the introduced

mutations. The F416 and R644 primers (Table 2-1) were used in sequencing

reaction with purified plasmid DNA as a template.

The EV71 mutant viruses were rescued from the in vitro RNA transcripts

after transfection of Vero cell culture as described in materials and methods in

Chapter 2. On the 6th day after transfection the cell culture was subjected to three

freeze-thaw cycles to release the intracellular virus. Cell debris was removed by

centrifugation, supernatant was aliquoted and stored at -80˚C, until use.

The parent and mutant viruses were passaged once in Vero cell culture in

order to increase volume of the viral stocks used in further experiments. Titres of

the rescued viruses were determined by the TCID50 method as described in

Chapter 2.

4.2.4 Growth kinetics of the parent and mutant viruses

Growth kinetics assay was performed on Vero and SK-N-SH cell

monolayers at 37.0°C and 39.5°C. Prior to the assay, Vero cells were seeded into

96-well plates at a density of 2 x 104 cells per well and grown in 100 µl of MEM

supplemented with 5% FBS in a CO2 incubator at 37.0°C for 24 hours. SK-N-SH

cells were seeded at a density of 1.3 x 104 cells per well in MEM with 15% FBS

and incubated at the same conditions. On the day of inoculation the medium was

removed from the wells and 103.6-4.0 TCID50 of the virus in 50 µl volume was

added to each well. Plates were incubated at 37.0°C or 39.5°C for 1 h to allow

virus adsorption. After 1 h incubation, inoculated wells were washed with MEM

and 200 µl of fresh MEM plus 5% or 15% FBS was added into each well with

Vero or SK-N-SH cell culture, respectively. Plates were returned for further


178

incubation at 37.0°C or 39.5°C and harvested at indicated time points (1, 6, 12,

18, 24, 48, 96 h post infection). Plates were frozen and thawed three times in

order to release the intracellular virus. Cell debris was removed by centrifugation

and viral titres were determined in TCID50 assay as described in materials and

methods in Chapter 2. The TCID50/1ml values were used to create growth

kinetics graphs in the Microsoft Excel 2007 software.

4.2.5 Stability of the mutant viruses

Stability of the 473U and 473U/538A mutations was examined during

serial passages of the mutant viruses in Vero cell culture. Vero cells were seeded

in 500 µl of MEM with 5% FBS at 2.25 x 105 cells per well in a 24-well plate.

The plate was incubated at 37°C in a 5% CO2 incubator for 24 hours. Then

growth medium was removed and 150 µl of supernatant containing 103.6-4.0

TCID50 of the mutant virus were added per well. The plate was incubated at room

temperature on a rocking platform for 60 min. After the adsorption, the

inoculated virus was removed and 500 µl of fresh MEM with 5% FBS were

added per well. The plate was incubated at 39°C in a 5% CO2 incubator for 72

hours. The plate was frozen and thawed three times and its well content was

centrifuged to remove the cell debris. 150 µl of the cleared supernatant was used

for the next passage of the virus by following the same protocol. The experiment

with each of two mutants, 473U and 473U/538A, was performed in triplicates for

10 passages. Stability of the introduced mutations was assessed by the

PCR/Restriction enzyme cleavage assay, and viral load accumulation was

determined in TCID50 assay.


179

4.2.5.1 PCR/Restriction enzyme cleavage assay

Principle:

The PCR/Restriction enzyme cleavage assay allows determining the

presence of the particular sequence within a heterogeneous DNA sample. When

DNA template used in the restriction enzyme digestion is labelled (radiochemical

or biotin labelling), the assay provides data for a quantitative analysis

(Chumakov et al. 1991; Hahn et al. 1995; Macadam et al. 2006). Post digestion

staining of DNA samples with SYBR Green I was used in a similar quantitative

method named as the Melting curve Single Nucleotide Polymorphism (McSNP)

analysis (Ye et al. 2002). Conventional agarose gel electrophoresis after the

restriction enzyme digestion can also provide quantitative data if an appropriate

Imaging system and software are employed. However, results obtained in this

case are shown to be less accurate (Macadam et al. 2006).

The PCR/Restriction enzyme cleavage is a multi-step procedure. When

starting material is a viral RNA genome, the first step is a total RNA extraction

from a viral sample with following reverse transcription of the RNA molecules

into cDNA copies. Obtained cDNA is amplified with two primers; one of those

introduces a recognition site for the restriction enzyme cleavage into the wt-

sequence only. Digestion of the PCR product with an appropriate restriction

enzyme allows detection of the wt-sequence within the heterogeneous viral

population. A schematic diagram of the PCR/Restriction enzyme cleavage assay

in application to the 473U and 473U/538A EV71 mutant viruses examined in this

study is presented in Figure 4-2.


180

473U and 473U/538A mutant viruses Revertant to the wt-sequence

----GCUAAUUCUAACU----------

473 viral RNA ----GCUAAUCCUAACU----------

473

↓ Reverse transcription with random hexamers

----CGATTAAGATTGA---------- cDNA ----CGATTAGGATTGA----------

↓ PCR amplification with F_s3 and R_s3 primers

443

|---GCTGAT→

----CGATTAAGATTGA----------

443

|---GCTGAT→

----CGATTAGGATTGA---------

↓ 443

|---GCTGATTCTAACT----------

|---CGATTAAGATTGA----------

443

|---GCTGATCCTAACT----------

|---CGATTAGGATTGA----------

↓

443

|---GCTGATTCTAACT----------

←----| 569

443

|---GCTGATCCTAACT----------

←----| 569

↓

443

|---GCTGATTCTAACT--------|

|---CGACTAAGATTGA--------|

569

PCR product

443

|---GCTGATCCTAACT--------|

|---CGACTAGGATTGA--------|

Sau3AI 569

↓ Restriction enzyme cleavage, agarose gel electrophoresis

No digestion: DNA fragment of 127bp Positive digestion:

DNA fragments of 100bp and 27bp

Figure 4-2: Scheme of the PCR/Restriction enzyme cleavage assay used for detection

of the wt-sequence revertants within the 473U and 473U/538A mutants.

RNA sequence of the mutant EV71 genome fragment containing the Sabin 3 attenuating determinant or the wt-sequence targeted in the restriction digest are shown. The Sabin 3 mutation (473U) and wt-nucleotide (C473) are boxed. The forward PCR primer, F_s3, has a mismatching nucleotide (underlined) at its 3′ end. The reverse PCR primer, R_s3 (sequence is not shown), is complementary to the DNA template. The 5′-terminal nucleotide position of the PCR primers are indicated as 443 (forward) and 569 (reverse). PCR amplification with F_s3 and R_s3 generates the Sau3AI recognition site (highlighted by a grey background) within the wt-sequence only. Restriction digestion of the PCR product allows detection of the wt-sequence if such sequence was presented in the original RNA sample.


181

Methodology:

Total RNA was isolated from Vero cell culture supernatant obtained from

the 1st, 5th and 10th passage of the 443U and 443U/538A EV71 mutants. RNA

extraction was performed with QIAamp Viral RNA Mini Kit (QIAGEN) in

accordance with the manufacturer's recommended protocol. Extracted RNA was

eluted in 80 ul of milliQ H2O and stored at -80°C until use. Total RNA extracted

from the cell culture infected with the TR EV71 variant (wt-5′UTR sequence)

served as a positive control in a following PCR/Restriction enzyme cleavage

assay.

The first strand cDNA synthesis was performed with Random hexamers

(Promega) and GoScript™ Reverse Transcriptase (Promega) in accordance with

the manufacturer's recommended protocol. The MgCl2 final concentration was

adjusted to 4.75 mM in order to increase yield of short cDNA fragments. The

RT-reaction was incubated in a MyCycler PCR instrument (Bio-Rad) at 42°C for

60 min and followed by the reverse transcriptase inactivation step at 70°C for 15

min. The cDNA samples were stored at -20°C until use.

PCR amplification was performed with 3 µl of the RT product. The PCR

mixture contained 1 x OneTaq Standard Reaction Buffer, 0.4 µM of each of the

F_s3 and R_s3 primers, 0.2 mM dNTP mix and 1.25 U of OneTaq® Hot Start

DNA Polymerase (New England BioLabs) in a total reaction volume of 50 µl.

The PCR was carried out under the following conditions: initial denaturation at

94°C for 2 min, followed by 30 cycles at 94°C for 15 sec, 60°C for 30 sec, 68°C

for 45 sec, followed by the final elongation at 68°C for 5 min.

The PCR product was ethanol precipitated with 3M sodium acetate (pH

5.2) by following the protocol described in Chapter 2, except for small

modifications. The incubation on ice was increased up to 4 h and following

centrifugation was at room temperature for 50 min. The second centrifugation

with 70% ethanol was performed at the same conditions for 20 min. The DNA

pellet was dissolved in 25 µl of milliQ H2O. The concentration of the DNA was

estimated by the 3%-agarose gel electrophoresis in 0.5% TBE buffer against the

50 bp DNA ladder (New England BioLabs).


182

Approximately 100-150 ng of the precipitated DNA was used as a

substrate in restriction digestion with Sau3AI enzyme (Promega). The reaction

mixture contained 1 x NEBuffer B supplemented with 0.1 µg/µl of BSA and 1.5

µl (4.5-15 U) of the enzyme in a total volume of 20 µl. Restriction digestion was

performed at 37°C for 8 hours. The digested DNA products were separated on

3% agarose gel stained with EtBr. Size of the DNA fragments was estimated

against the 50 bp DNA ladder (New England BioLabs). The amount of the

restriction enzyme needed for digestion of 1 µg DNA of 127 bp in size was

calculated based on the Sau3AI specifications and in accordance with the

recommendations of the supplier (<http://www.promega.com/resources/product-

guides-and-selectors/restriction-enzyme-resource/applications-and-reaction-

conditions-for-restriction-enzymes/>) (Table 4-2).

DNA substrate

Base pairs

Molecular weight*

Picomoles in 1 µg

Cut sites Picomoles cut sites

Units needed

Unit definition (lambda)

48,502 31,526,300 0.0317 116 3.6772 1.0

PCR product

127 82,550 12.1139 1 12.1139 3.3

Table 4-2: Correlation between substrate recognition sites density and amount of

Sau3AI needed to digest 1 µg of the DNA substrate in 1 hour.

* Molecular weight of the DNA substrates was calculated based on the average molecular weight of a nucleotide pair of 650 Da.


183

4.2.5.2 DNA automated cycle sequencing

The cDNA samples used in PCR/Restriction enzyme cleavage assay were

additionally analysed by sequencing.

The PCR amplification was performed with 2 µl of the cDNA, 1 x HF

PCR Buffer, 0.5 µM of each of the Ft7 and R1779 primers (Table 2-1), 0.2 mM

dNTP mix and 1 U of iProof High-Fidelity DNA Pol (BioRad) in a total reaction

volume of 50 µl. The PCR was carried out under the following conditions: initial

denaturation at 98°C for 30 sec, followed by 25 cycles at 98°C for 7 sec, 57°C

for 20 sec, 72°C for 60 sec, followed by the final elongation at 72°C for 5 min.

The PCR product was gel purified with Wizard® SV Gel and PCR Clean-Up

System (Promega).

The DNA BDT labelling reaction and capillary separation was performed

at the AGRF Sequencing Service. The R644 primer (Table 2-1) was used in the

sequencing reaction.

4.2.5.3 Viral load accumulation

The cleared supernatant from each passage of the mutant viruses, 473U

and 473U/538A, was used to estimate the viral titre by the TCID50 method as

described in materials and methods of Chapter 2. The calculated TCID50 values

were plotted on a graph in Microsoft Office Excel 2007 software.


184

4.3 Results

4.3.1 In vitro replication of the parental and 5′UTR mutant EV71

All of the constructed EV71 5′UTR mutants were infectious upon

transfection of Vero cells with the in vitro RNA transcripts. The TCID50 titres of

the rescued viruses were similar to that of the parental strain and ranged between

3.75-4.25 log10 in 100 µl of the cleared supernatant. The growth kinetics of the

parental and mutant viruses was studied in vitro after infection of Vero and SK-

N-SH cells with the 1st passage of the rescued viruses in Vero cells.

Mutations within the IRES Vth domain

Previous studies reported that Sabin 3 (472U) substitution within the Vth

domain of the PV2 IRES had a negative effect on viral replication in cells of

neuronal origin, such as SH-SY5Y (La Monica & Racaniello 1989). To study if

the reciprocal position within EV71 genome has a similar effect on viral

replication, the growth kinetics of the 473U EV71 mutant was determined both in

SK-N-SH and Vero cells. The growth properties of the 473U/538A EV71 mutant

were of an additional interest as that virus possessed the wt secondary RNA

structure within the domain Vth but contained a weakened U-A base pairing

between the complementary positions 473rd and 538th. The U-A base pair was

shown to revert to the wt strong C-G pair more slowly than U-G of Sabin 3

(Macadam et al. 2006). The TCID50 titres of the parental and mutant strains were

estimated at 0, 6, 12, 24, 48 and 96 h p.i. The replication curves obtained from

the mutant viruses in Vero and SK-N-SH cells at 37.0˚C and 39.5˚C were

compared to those of the parental virus. The growth curve analysis showed that

mutant viruses had a very similar pattern of replication to that of the parent strain

in both cell types. A slight difference in viral growth was observed in Vero cells

at 39.5˚C where the parental strain showed an increase in replication between 6


185

and 12 h p.i. and reached TCID50 titre of approximately 1 log10 higher when

compared to the mutant viruses (Figure 4-3, A).

Replication in SK-N-SH cells was totally suppressed at both temperatures

in all viruses including the parental strain (Figure 4-3, B). The 473U or

473U/538A mutations within the IRES domain Vth did not contribute further to

the observed phenotype of the mutant viruses. The low replication rate of the

parental virus in SK-N-SH cells might be explained by the fact that the viral

stock used in construction of the original EV71 cDNA clone was obtained after

multiple passages in Vero cell culture and as a result developed host cell specific

substitutions which were not beneficial for viral growth in another cell types.


186

A.

B.

Figure 4-3: In vitro replication of the parental and mutant (473U, 473U/538A) EV71

viruses at 37.0˚C and 39.5˚C in Vero (A) and SK-N-SH (B) cells.

The plotted TCID50 values are the means of virus titres from three (A) and two (B) experiments. The standard deviation values were omitted for clarity and can be found in Appendix IV.


187

Mutations within the VIth domain of the 5′UTR

The upper part of the domain VI was maped as a region which is

responsible for the attenuated phenotype of the chimeric PV1(R6) both in human

neuroblastoma SK-N-MC cells and CD155 tg mice (Gromeier et al. 1999).

Raplacing the upper loop of the 5′UTR VIth domain of the neuropathogenic PV1

with the corresponding fragment of HRV2 resulted in an attenuated phenotype of

the chimeric virus.

The sequence difference between the stem-loop VI of EV71 and CAV16

lies only within the upper part of the domain and mapped only to four nucleotide

positions: 602, 603, 606 and 607 (Figure 4-1). That sequence similarity allowed

generating the CAV16-like stem-loop VI within the EV71 5′UTR with two

mutagenic primers spanning the target sequence. The SL6-CAV8 was an

additional interest as it is an intermediate mutant on the VIth domain from EV71

to CAV16.

When studied in vitro, the growth properties of the EV71 mutants, SL6-

CAV8 and SL6-CAV16, were not at all affected by the replacment of the stem-

loop VI. Under the experimental conditions, both mutant viruses grew with the

same potential as the parental strain. In fact, the SL6-CAV8 virus reached a

slightly higher titre than that of the parental strain by 96 h of incubation in Vero

cells at 39.5˚C (Figure 4-4).


188

A.

B.

Figure 4-4: In vitro replication of the parental and mutant (SL6-CAV8, SL6-CAV16)

EV71 viruses at 37.0˚C and 39.5˚C in Vero (A) and SK-N-SH (B) cells.

The plotted TCID50 values are the means of virus titres from three (A) and two (B) experiments. The standard deviation values were omitted for clarity and can be found in Appendix V.


189

4.3.2 Stability of the V stem-loop EV71 mutants in vitro

The Sabin 3 attenuated phenotype is partly determined by the nucleotide

change from C to U on position 472 (Westrop et al. 1989). The position is known

to be very unstable during viral replication both in vivo and in vitro (Dunn et al.

1990; Evans et al. 1985; Macadam et al. 2006). Vaccine-derived viruses exhibit

the U472C reversion at a very early stage after administering the Sabin 3 vaccine

(Contreras et al. 1992). Stability of the Sabin 3 attenuating determinant was

shown to be increased via pairing the 472U with 537A located on the other side

of the stem within domain VI (Macadam et al. 2006). In EV71 genome those

positions are equivalent to 473 and 538, respectively. To study the stability of the

473U mutation within EV71 genome and the effect of the nucleotide change

from G to A in position 538, the mutant viruses were passaged 10 times in Vero

cell culture at a growth restricting temperature of 39˚C. The replication kinetics

of the 473U and 473U/538A mutants during passaging was monitored by

estimating the TCID50 titres. The genetic stability of the mutant viruses was

assessed by the PCR/Restriction enzyme cleavage assay.

4.3.2.1 Viral yield accumulation during serial passaging

The Vero cell monolayers were inoculated with the same amount of the

473U or 473U/538A mutant virus. The viral production of the 1st passage was

lower in the 473U infected cells (Figure 4-5).


190

Figure 4-5: Dynamics of the virus accumulation during serial passaging in cell culture.

The 473U and 473U/538A mutant viruses were passaged at 39˚C on Vero cell monolayers 10 times. Viral titres were estimated from the supernatant and cells collected after 72 h of incubation. The experiment was performed in triplicates. The log10 TCID50 curves obtained from three triplicates of two EV71 mutants are designated as -1, -2 and -3 following the virus name.


191

However, by the 3rd passage the 473U viral yield already increased

significantly and two of three cell cultures exceeded the viral titres of the

473U/538A mutant. The increase in viral load accumulation of the 473U

followed the same pattern until passage 6, when according to microscopic

observation approximately 80-90% cells showed CPE. In the following 4

passages the CPE tended to be complete prior to the end of the incubation period

and as a result, viral accumulation became limited by the total number of Vero

cells available for virus to replicate. To observe the further increase in viral yield

of the 473U mutant would require to increase the cell seeded area or to decrease

the volume of the inoculum. However, the experimental protocol was kept

unchanged in order to maintain the same conditions throughout of the experiment

for both viruses.

The viral yield of the 473U/538A mutant did not increase during the first

half of the experiment and by the last passage gained the TCID50 titre on average

equal to that of the 473U mutant of passage 4. Under the experimental

conditions the difference in viral titres obtained from the 473U and 473U/538A

mutants in the last passage ranged only from 0.5 to 2.0 log10 of TCID50 (Figure 4-

5). However, it should be taken into account that the experimental conditions did

not support the 473U accumulation after passage 6. Therefore, the actual

difference in the final TCID50 titres between two viruses can be expected at a

significantly higher level if the experimental conditions support viral replication

of both viruses. When compared to the same virus, the ∆TCID50 between the 1st

and 10th passage reached 2.92 and 0.67 log10 on average for the 473U and

473U/538A mutant, respectively. Again, the ∆TCID50 of the 473U virus is

probably significantly underestimated due to the same reason mentioned above.


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4.3.2.2 Stability of the 473U mutation during serial passaging

The PCR/Restriction enzyme cleavage assay was employed in order to

detect revertants to the wt-sequence within the population of the 473U and

473U/538A mutant viruses. The parental EV71 strain carrying the wt-sequence

within the 5′UTR was used as a positive control during the assay. The RT-PCR

amplification of the positive control RNA resulted in a PCR product of 127 bp.

The following digestion of the ethanol precipitated PCR product with Sau3AI

restriction enzyme generated two new DNA fragments of 100 bp and 27 bp in

size. The DNA band size in the “mock” digestion of the wt-sample remained

similar to that observed with the original RT-PCR product used in the restriction

enzyme cleavage (Figure 4-6).

Figure 4-6: Results of the PCR/Restriction enzyme cleavage assay conducted with the

viral RNA of the wt-5′UTR sequence.

Lane 1: The RT-PCR product of the wt-sample. Lane 2: the 50 bp DNA ladder. The band size from the bottom: 50 bp, 100 bp, 150 bp, 200 bp, etc. Lane 3: The “mock” digestion of the wt RT-PCR product. Lane 4: The wt RT-PCR product digested with Sau3AI.

← 27 bp

← 100 bp 127 bp →


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In the next step, viral RNA was isolated from each of three cell culture

replicates of the 473U and 473U/538A mutant viruses of passage 1, 5 and 10.

The RT-PCR of the isolated RNA samples resulted in a DNA product of the

same size as observed in the positive control (data not shown). The Sau3AI

restriction enzyme cleavage showed positive digestion in passages 5 and 10 of

the samples obtained from the 473U infected cell cultures (Figure 4-7, A). No

digestion was achieved with the 1st passage of the 473U mutant and with all

samples obtained from the 473U/538A infected cells (Figure 4-7, B). The results

of the PCR/Restriction enzyme cleavage assay clearly indicated that under the

experimental conditions the EV71 473U mutant could revert to the wt-sequence

within the first five passages in vitro. According to the agarose gel image, the 5th

and 10th passages of the 473U mutant (Figure 4-7, A) contained some undigested

DNA product, which could indicate the presence of the 473U mutation within the

viral population. This would imply that the viral population achieved the certain

equilibrium between the mutant and wt-revertant sequences. However, the

restriction digestion of the control wt-5′UTR sequence also did not result in

100% cleavage (Figure 4-6, lane 4) indicating the possible problems with the

efficacy of the reaction. Therefore, in order to verify those results, the sequencing

of the cDNA samples obtained from one of the cell culture triplicates was

employed. According to the sequencing chromatogram the 1st passage of the

473U virus already contained a mixed population (473U and 473C). By the 5th

passage the reversion to the wt-sequence was complete and the viral population

remained homogeneous in this position until the end of the experiment.

Based on results from both assays, stability of the 473U mutation was

increased by the base pairing with 538A and resulted in no revertants detected

during ten passages of the 473U/538A virus in cell culture (Figure 4-7, B).


194

A.

B.

Figure 4-7: Comparative results of the PCR/Restriction enzyme cleavage assay and

sequencing conducted with the EV71 mutant viruses upon serial passaging in Vero

cells.

Agarose gel electrophoresis was performed after the Sau3AI digestion of the RT-PCR product obtained from the 473U (A.) or 473U/538A (B.) infected cell culture triplicates (refer Figure 4-5). The cDNA sample from one of the triplicates (473U-1 and 473U/538A-1) was used in sequencing (A and B, chromatograms). In both agarose gel images: the 50 bp DNA ladder was used as a DNA size reference. The band size from the bottom: 50 bp, 100 bp, 150 bp, 200 bp, etc. The virus triplicates of the 1st, 5th and 10th passage are indicated. In both chromatograms: nucleotides at the mutation sites (473 and 473/538) are boxed and numbered. Emergence of the wt-C473 can be observed within the 1st passage of the 473U EV71 mutant. The reversion is complete by the 5th passage of the virus (A, chromatogram). No reversion of the 473U was detected in the 473U/538A EV71 mutant during 10 passages (B, chromatogram).


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4.4 Discussion

The 5′UTR region of the Picornaviridae forms a highly organized

structure within the viral genome (Wimmer, Hellen & Cao 1993). The cloverleaf

element and IRES are the main structural features of the 5′UTR. The 5′-terminal

cloverleaf, also known as the oriL cis-acting element, is required for initiation of

the positive RNA strand synthesis, whereas the IRES was shown to initiate viral

polyprotein production in a cap-independent manner (Andino, Rieckhof &

Baltimore 1990; Pelletier et al. 1988b). The IRES elements of picornaviruses are

classified into four types: type I (genus Enterovirus and Rhinovirus), type II

(genus Aphthovirus and Cardiovirus), type III (genus Hepatovirus) and HCV-

like type (genus Teschovirus) (Fernandez-Miragall, Lopez de Quinto &

Martinez-Salas 2009).

The IRES element of EV71 belongs to type I IRES (Thompson & Sarnow

2003). It consists of stem-loop II, IV and V according to the nomenclature used

by Wimmer, Hellen et al. (1993). In PV, the stem-loop V was identified to carry

the main attenuating determinants of Sabin vaccine strains 1, 2 and 3, a fact

which has made this domain of increased scientific interest. Many studies aimed

at the attenuation of picornaviruses carrying the type I IRES element via SDM of

the stem-loop V (Ben M'hadheb-Gharbi et al. 2006; M'Hadheb-Gharbi et al.

2007; M'hadheb-Gharbi et al. 2008; Malnou et al. 2002). In case of Sabin 3

vaccine strain, the mechanism of the attenuation was linked to the structure-

dependent interaction of the polypyrimidine tract, a nucleotide stretch adjacent to

the stem-loop V, with host cell proteins, such as PTBP, required for efficient

IRES-mediated translation of viral RNA (Guest et al. 2004). In vivo, the defect of

interaction between the PTBP and the polypyrimidine tract couples with the

PTBP-deficiency in the CNS and results in the CNS-specific attenuation. When

propagated in vitro, the attenuated mutants exhibit a ts-phenotype. The

phenotypic differences between the wt and attenuated viruses become more


196

evident in cell cultures of the neural origin (Agol et al. 1989; La Monica &

Racaniello 1989).

The structure similarity between IRES elements of PV and EV71 implies

a possibility that Sabin attenuating determinants within the stem-loop V will have

a similar effect on phenotype and neuropathogenic properties of EV71. Indeed,

the 5′UTR mutation of the Sabin vaccine strains introduced into the reciprocal

positions of EV71 BrCr strain were shown to lead to temperature sensitivity in

Vero cells (Arita et al. 2005). The BrCr strain, the only member of genotype A,

was isolated in the USA and has never been reported in the Asia-Pacific region.

The recent EV71 outbreaks in the region were associated with sub-genogroups

B3 (Malaysia, 1997), B4 (Singapore and Taiwan, 2000), C2 (Taiwan, 1998 and

Australia, 1999) and C4 (China, 2008) (Bible et al. 2007; Mao et al. 2010;

McMinn et al. 2001a). Therefore, in this study a cDNA clone of the EV71 strain

belonging to sub-genotype C2, and originated from a local EV71 isolate

(Australia, 1999), was used in SDM. The reciprocal position to the Sabin 3

attenuating determinant was used as a target in the SDM of EV71. The Sabin 3

like viruses previously demonstrated a higher reduction in viral growth when

compared to Sabin 1 or Sabin 2 like mutants (Arita et al. 2005; Ben M'hadheb-

Gharbi et al. 2006). The growth kinetics results obtained in this study were

generally in agreement with the data reported by other research groups. The

replication of the 473U EV71 mutant was reduced in Vero cell culture at an

elevated temperature. However, the reduction in viral titre (∆37/39.5˚C TCID50 =

1 log10) was less significant in this study than that (∆36/39˚C TCID50 = 2.25

log10) reported by Arita et al. for the BrCr S3 mutant (Arita et al. 2005). The

discrepancy can be possibly explained by different experimental protocols used

in the growth kinetics assay. In this study, the ∆TCID50 titres were calculated

from viral replication at 37 and 39.5˚C (∆˚C=2.5), whereas Arita et al. evaluated

viral growth at 36 and 39˚C (∆˚C=3.0). Additional factors, such as the amount of

the inoculated virus or the incubation time, could also account for the difference.

On the other hand, the effect of the Sabin 3 mutation may not be equal upon

introduction into EV71 strains originally distinct in their sequences and growth


197

properties. The fact that even very closely related viruses can differ in their

growth kinetics in vitro was previously reported for the Mahoney, Sabin 1 and

Sabin 2-related cDNA clone derivatives obtained from different sources (Agol et

al. 1989).

When studied in SK-N-SH cells both the parental and Sabin 3-like EV71

mutant (473U) exhibited a restricted growth which was declined even further at

an elevated temperature. The experimental data indicated that replication of the

parental strain was not supported by the human cell culture of the neuronal

origin. Therefore, it is impossible to conclude whether Sabin 3 mutation within

EV71 has or has no effect on virus growth in neural cells. Even though, the

original 6F/AUS/6/99 EV71 strain was isolated from the cerebrospinal fluid of

the patient with brainstem encephalitis/myelitis (McMinn et al. 2001a), the virus

was passaged multiple times in Vero cell culture prior to cloning and could

develop the host range mutations adaptive to monkey cells. The cDNA clone

constructed in this study carried several mutations when compared to the original

6F/AUS/6/99 EV71 strain (Appendix VI). The adaptation to the cell culture host

in highly passaged EV71 laboratory strains has been previously highlighted

(Nagata et al. 2002). According to those results, the Nagoya/Japan73 strain,

isolated from a HFMD case and passaged more than 10 times in Vero cells, was

more neurovirulent in monkeys than the C7/Osaka/Japan97 or SK-

EV006/Malaysia97 strains, isolated from the fatal encephalitis and passaged no

more than five times. Therefore, both the results of this study and previously

published data can lead to the conclusion that highly passaged laboratory strains

demonstrate host specific adaptation to the cultured cells (monkey versus human)

irrespective of the original organ tissue specificity (CNS versus kidney).

The genetic instability of the Sabin 3 attenuated strain is one of the main

concerns of the OPV use (Contreras et al. 1992). The 5′UTR attenuating

determinant of Sabin 3 reverts to the wt sequence more rapidly than those of the

type 2 and type 1 PV strains (Dunn et al. 1990). The natural reversion occurs

only at position 472 and results in establishing the wt base pair (C472-G537)

within the stem of the domain Vth. The C-G base pair is thermodynamically


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stronger than U-A pair, a fact which can explain the results of natural selection

within the viral population in this particular case. With the PV genome as an

experimental model, Macadam et al. proved that Sabin 3 attenuating determinant,

472U, can be stabilized by the complimentary mutation, G537A (Macadam et al.

2006). In that study, the in vitro temperature sensitivity and in vivo

neurovirulence of the U-A mutant was at the middle level when compared to

those of the wt C-G (highest) and Sabin 3 U-G (lowest) viruses. Results obtained

with EV71 mutants (473U and 473U/538A) in this study supported data of

Macadam et al. Even if there was no evident difference in growth kinetics

(Figure 4-3) between two mutant viruses, the temperature sensitivity assay (data

presented in Chapter 5) revealed that shut-off temperature for the 473U/538A

mutant falls within the intermediate temperature range observed between the

parental (wt C-G pair) strain and 473U (Sabin 3-like U-G pair) mutant.

Genetic stability of the Sabin 3 attenuating determinant within the EV71

genome was significantly increased when it was base paired with adenine at the

538th nucleotide (473U/538A mutant). Therefore, in terms of long incubation the

growth kinetics of the 473U/538A EV71 was more restricted than that of the

473U EV71 (Sabin 3-like mutant) (Figure 4-5). It was not possible to determine

the exact proportion of the revertants within the viral population. Even though,

the non-radioactive PCR/Restriction enzyme cleavage assay has been previously

used as a quantitative method, in this study it was not reliable enough to provide

the quantitative data on the composition of the viral population. The sequence

analysis of the 1st passage of the 473U EV71 mutant revealed the emergence of

the wt-sequence revertants. Sequences obtained from the 5th and 10th passages

demonstrated 100% homogeneous wt population at position 473. The

PCR/Restriction enzyme cleavage assay could not detect the wt-sequence within

the 1st passage of the virus, and showed only approximately 70% reversion in the

passage 5 and 10 (Figure 4-7, A). Accuracy of the assay was most likely affected

by the incomplete digestion of the DNA by Sau3AI enzyme. Several reasons,

such as ethanol or salt carryover from the precipitation step, high density of the

recognition site or loss of the enzyme activity could account for the partial


199

digestion. Therefore, prior to digestion of the experimental samples, the PCR

product obtained from the positive control was column purified or used directly

in the restriction enzyme cleavage. However, Sau3AI digestion of those DNA

samples was even more inhibited than with the precipitated DNA (data not

shown). The high number of the recognition sites per weight unit (1 µg) of the

substrate DNA is the result of the short length of the PCR product used for

digestion. Although, the amount of the enzyme was calculated in accordance

with the DNA size, and, based on calculations, was deemed to be sufficient

(Table 4-2), the complete digestion was not achieved. Decreasing the DNA input

into reaction could probably increase the efficacy of the cleavage but would have

also resulted in reduced intensity of the DNA bands on the agarose gel and

difficulties in detection due to low sensitivity of the UV-light based imaging

system. Therefore, in this study the PCR/Restriction enzyme cleavage assay

could be used only as a detection method without quantification of the digested

DNA products. However, mutation analysis by the standard PCR/Restriction

enzyme cleavage assay (MAPREC) employing the radio-labelling step is proven

to be a more sensitive and accurate method, which was suggested to be employed

as an in vitro screening test with the Sabin OPV lots (Dorsam et al. 1998). It has

been well documented that results obtained with the MAPREC test correlated

with those of the neurovirulence test in monkeys (Chumakov et al. 1991; Dorsam

et al. 2000). As a future step it would be worth to examine other non-radioactive

approaches, such as pyrosequencing and real-time PCR, in order to determine the

proportion of EV71 revertants within the viral population. These methods have

been already successfully evaluated in their sensitivity and specificity to identify

YMDD mutants of HBV both in artificially made plasmid DNA mixtures and in

patients with chronic hepatitis B (Wen et al. 2008; Yang et al. 2006).

Pyrosequencing is one of the next-generation sequencing methods (Liu et al.

2012). Even though it is more expensive than the conventional dideoxynucleotide

sequencing (the Sanger method), it provides quantitative data and has already

given very promising results in detection of HIV1 minor sequence variants from

clinical samples (Wang et al. 2007a).


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The VIth domain of the 5′UTR of PV was previously shown to enhance the

efficiency of viral RNA translation in vitro (Meerovitch, Nicholson & Sonenberg

1991). The removal of the domain VI resulted in decreased neurovirulence in

mice and monkeys (Iizuka et al. 1989; Slobodskaya et al. 1996). Exchanging the

entire domain VI of PV1 with that of HRV2 abrogated neurovirulence in tg mice

(Gromeier et al. 1999). The region responsible for neurovirulence was identified

within the upper part of the stem-loop VI. In vivo experiments with chimeric

PV/HRV2 constructs indicated that the level of neurovirulence is determined by

a synergy between upper regions of the stem-loop V and VI. In this study the

upper loop of the domain VI of EV71 was replaced with the similar structure

derived from CAV8 (SL6-CAV8 EV71 mutant) or CAV16 (SL6-CAV16 EV71

mutant). Due to the fact that the stem region of EV71 domain VI is identical to

those of CAV8 and CAV16, the upper part replacement resulted in generating the

CAV8- or CAV16-like EV71 mutants on the entire domain VI structure (Figure

4-1). The growth kinetics and temperature sensitivity of both mutant viruses were

not affected in Vero cell culture both at 37˚C and 39.5˚C. Unfortunately,

infection of the SK-N-SH cells did not give conclusive results as viral growth

was inhibited both for the parental and mutant strains. In order to make final

conclusions on the contribution of the VIth domain of EV71 5′UTR to the virus

neurovirulence further experiments would be required.

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CHAPTER 5

SCANNING THE VP1 PROTEIN OF EV71 FOR

ts-DETERMINANTS BY CHARGED-TO-ALANINE

MUTAGENESIS

CHAPTER 5 Scanning the VP1 protein of EV71 for ts-determinants by charged-to-alanine mutagenesis

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5 SCANNING THE VP1 PROTEIN OF EV71 FOR ts-DETERMINANTS

BY CHARGED-TO-ALANINE MUTAGENESIS

5.1 Introduction

Like other members of the Picornaviridae family, EV71 is a non-

enveloped virus possessing a single stranded RNA genome which is packed into

a capsid built of viral structural proteins. Being exposed to the environment, viral

capsid not only protects virus genetic material, but also plays an essential role in

the early steps of EV71 infection, such as interaction with host cell receptors and

penetration of viral RNA into the cells. The enterovirus capsid displays the

icosahedral symmetry and consists of the repeating structural units or 60

protomers organized further into 12 pentamers (Hogle, Chow & Filman 1985;

Wang et al. 2012). Each protomer is formed by a single copy of four structural

proteins, VP1, VP2, VP3 and VP4, and each pentamer is composed of five

protomers with VP1 proteins centred on the five-fold axis. The VP1 protein is

known to be present in several structural futures of the viral capsid, such as the

five-fold vertex, canyon and hydrophobic pocket. The BC loop (residues 97-102)

of the VP1, exposed on the surface of the capsid near the five-fold axis, has been

mapped as a possible EV71 immunogenic site, based on the structural homology

to other enteroviruses (Ranganathan et al. 2002). Another study confirmed that a

synthetic peptide spanning 94-108 amino acids of VP1 showed immunoreactivity

with human anti-EV71 IgG in western blot (Foo et al. 2008a). The VP1 segments

between residues 163-177 and 208-222 were identified as neutralizing epitopes,

eliciting the protective antibodies in mouse models (Foo et al. 2007a; Foo et al.

2007b). Recently, another segment (amino acids from 40 to 51) of the VP1 was

shown to be an antigenic epitope with human anti-EV71 IgM (Gao et al. 2012).

The depression (called the canyon) on the outer surface of the viral capsid,

and surrounding the five-fold vertex, was identified as a receptor binding site for


203

picornaviruses (Chapman & Rossmann 1993; Colonno et al. 1988; Rossmann,

He & Kuhn 2002). Additionally, the neutralization antigenic site of the VP1 (the

BC loop) and VP2 (the EF loop) were shown to contribute to the receptor-virion

docking process and lead to serotype-specific differences in binding assays

(Harber et al. 1995). Some of the determinants of EV71 host specificity and

neurovirulence in animal models also were mapped within the VP1 protein

(145E: the mouse virulence determinant; 116Y: slight attenuated neurovirulence

in monkeys) (Arita et al. 2008; Arita et al. 2005; Chua et al. 2008).

Knowledge of the molecular bases of EV71 immunogenicity, virulence

and host-range phenotype, etc. is important in the development of an effective

vaccine. The VP1 protein is undoubtedly the major capsid protein which has

been associated with all those properties. However, there has been no study

conducted which would explore the VP1 for regions or molecular determinants

leading to EV71 temperature sensitivity. Even though some studies indicate that

there is not always a direct correlation between the level of temperature

sensitivity in vitro and attenuation in vivo (Bouchard, Lam & Racaniello 1995;

Georgescu et al. 1995; Hanley et al. 2002; Parkin, Chiu & Coelingh 1996), the

temperature sensitivity often serves as a phenotypic marker of attenuation (Arita

et al. 2005).

The charged-residue-to-alanine mutagenesis has been a useful tool in the

construction of temperature-dependant defective mutant viruses (Diamond &

Kirkegaard 1994; Hanley et al. 2002; Hassett & Condit 1994; Parkin, Chiu &

Coelingh 1996). Clusters of charged amino acids are expected to reside on the

surface of a folded protein (Chothia 1976; Wertman, Drubin & Botstein 1992).

Substituting the charged amino acids with alanine is likely to reduce affinity of

protein interactions with the solvent or other biomolecules, and make the protein

deficient in those interactions and, therefore, more temperature sensitive (ts)

(Diamond & Kirkegaard 1994).

In this study the approach of charged-to-alanine mutagenesis was applied

in order to scan the VP1 capsid protein of EV71 for possible ts determinants. Six

clusters of charged amino acids were targeted by SDM. The in vitro infectivity of


204

the constructed mutant cDNA clones correlated well with the structure-related

locations of the alanine substitutions. Combination of ts determinants from two

different clusters and, in addition, with the 5′UTR mutations led to further

increase in temperature sensitivity of EV71. Cooperative effect from attenuating

determinants residing in different genes of EV71 was shown to be essential for

attenuation of virus neurovirulence (Arita et al. 2008). Additionally, combination

of several mutations should facilitate the genetic stability of the mutant strain, the

issue which must be considered in the development of attenuated vaccines.


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5.2.1 Site-directed mutagenesis

SDM within the VP1:

Charged-to-alanine mutagenesis was used in order substitute a single

amino acid within the charged clusters of the VP1 protein. The cluster was

defined as a stretch of 4 to 9 amino acids with 50% of them charged and

separated within the cluster, at most, by 1 hydrophobic amino acid. The

mutagenic primers were designed with the PrimerX software based on the DNA

sequence of the VP1 gene of the TR EV71 cDNA clone, TRc (refer Chapter 3).

Parameters used in primer design were identical to those outlined in materials

and methods in Chapter 4. Mutagenic primers were synthesized and RPC-

purified by Sigma.

Site-directed mutagenesis was performed in accordance with the protocol

described in materials and methods in Chapter 3. Synthesis of the mutant DNA

strand was conducted with the mutagenic primers at annealing temperature (Tan)

listed in Table 5-1.


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Primer name* Sequence (5′-3′)** Tan, ˚C F_VP1_37A CGCAGGTAAGTAGCGCTCGATTGGATACTG 65 R_VP1_37A CCAGTATCCAATCGAGCGCTACTTACCTGC F_VP1_38A GCAGGTAAGTAGCCATGCATTGGATACTGGTAAAG 59 R_VP1_38A CTTTACCAGTATCCAATGCATGGCTACTTACCTGC F_VP1_40A GTAGCCATCGATTGGCGACTGGTAAAGTTCC 59 R_VP1_40A GGAACTTTACCAGTCGCCAATCGATGGCTAC F_VP1_43A CATCGATTGGATACTGGTGCAGTTCCAGCACTCCA

AG 72

R_VP1_43A CTTGGAGTGCTGGAACTGCACCAGTATCCAATCGATG

F_VP1_120A GGTTACGCGCAAATGGCTAGAAAGGTGGAGTTG 60 R_VP1_120A CAACTCCACCTTTCTAGCCATTTGCGCGTAACC F_VP1_121A GGTTACGCGCAAATGCGTGCAAAGGTGGAGTTGTT

CAC 65

R_VP1_121A GTGAACAACTCCACCTTTGCACGCATTTGCGCGTAACC

F_VP1_122A GCGCAAATGCGTAGAGCGGTGGAGTTGTTCAC 63 R_VP1_122A GTGAACAACTCCACCGCTCTACGCATTTGCGC F_VP1_124A GCGTAGAAAGGTGGCGTTGTTCACCTAC 59 R_VP1_124A GTAGGTGAACAACGCCACCTTTCTACGC F_VP1_130A GAGTTGTTCACCTACATGGCTTTTGACGCAGAGTTC

AC 62

R_VP1_130A GTGAACTCTGCGTCAAAAGCCATGTAGGTGAACAACTC

F_VP1_132A CCTACATGCGTTTTGCGGCAGAGTTCACCTTTG 61 R_VP1_132A CAAAGGTGAACTCTGCCGCAAAACGCATGTAGG F_VP1_134A GCGTTTTGACGCAGCGTTCACCTTTGTTG 60 R_VP1_134A CAACAAAGGTGAACGCTGCGTCAAAACGC F_VP1_162A GTACCACCCGGAGCCCCCGCTCCAGACTCCAGAGA

ATC 65

R_VP1_162A GATTCTCTGGAGTCTGGAGCGGGGGCTCCGGGTGGTAC

F_VP1_164A CCCCCAAACCAGCCTCCAGAGAATC 65 R_VP1_164A GATTCTCTGGAGGCTGGTTTGGGGG F_VP1_166A CCCCAAACCAGACTCCGCAGAATCTCTCGTATG 65 R_VP1_166A CATACGAGAGATTCTGCGGAGTCTGGTTTGGGG F_VP1_167A CAAACCAGACTCCAGAGCATCTCTCGTATGGCAG 59 R_VP1_167A CTGCCATACGAGAGATGCTCTGGAGTCTGGTTTG F_VP1_213A GTATCCCACATTCGGTGCACACAAGCAGGAGAAAG 60 R_VP1_213A CTTTCTCCTGCTTGTGTGCACCGAATGTGGGATAC


207

F_VP1_214A GTATCCCACATTCGGTGAAGCCAAGCAGGAGAAAGACCTTG

62

R_VP1_214A CAAGGTCTTTCTCCTGCTTGGCTTCACCGAATGTGGGATAC

F_VP1_215A CACATTCGGTGAACACGCGCAGGAGAAAGACCTTG 61

R_VP1_215A CAAGGTCTTTCTCCTGCGCGTGTTCACCGAATGTG F_VP1_217A GGTGAACACAAGCAGGCGAAAGACCTTGAATACG 61 R_VP1_217A CGTATTCAAGGTCTTTCGCCTGCTTGTGTTCACC F_VP1_218A GTGAACACAAGCAGGAGGCAGACCTTGAATACGG 61 R_VP1_218A CCGTATTCAAGGTCTGCCTCCTGCTTGTGTTCAC F_VP1_219A CAAGCAGGAGAAAGCCCTTGAATACGGGGC 61 R_VP1_219A GCCCCGTATTCAAGGGCTTTCTCCTGCTTG F_VP1_221A GGAGAAAGACCTTGCATACGGGGCATG 60 R_VP1_221A CATGCCCCGTATGCAAGGTCTTTCTCC F_VP1_254A GTGATCAGGATTTACATGGCGATGAAGCATGTCAG

GG 61

R_VP1_254A CCCTGACATGCTTCATCGCCATGTAAATCCTGATCAC

F_VP1_256A GATTTACATGAGGATGGCGCATGTCAGGGCGTG 61

R_VP1_256A CACGCCCTGACATGCGCCATCCTCATGTAAATC F_VP1_257A GATTTACATGAGGATGAAGGCTGTCAGGGCGTGGA

TACC 61

R_VP1_257A GGTATCCACGCCCTGACAGCCTTCATCCTCATGTAAATC

F_VP1_259A GATGAAGCATGTCGCGGCGTGGATACCTC 61

R_VP1_259A GAGGTATCCACGCCGCGACATGCTTCATC Table 5-1: List of primers used in SDM within the VP1 encoding region of EV71.

* Primer name indicates its orientation (F-forward, R-reverse) and position of an amino acid replaced with alanine (A). ** The mutated nucleotides within the primer’s sequence are underlined. Tan – annealing temperature used in SDM.


208

Combining the VP1 and 5′UTR mutations:

The cDNA clone of the 5′UTR EV71 mutant (the 473U/538A strain, refer

Chapter 4) was used in SDM to introduce additional mutations within the VP1

region. SDM was performed in accordance with the protocol described in

materials and methods in Chapter 3, except small modifications. The PCR with

mutagenic primers was carried out only for ten cycles in order to reduce the

number of PCR-associated mistakes during the multiple step procedure (Figure

5-1).

EV71 cDNA clone SDM steps

473U/538A

↓ SDM with F_VP1_164A/ R_VP1_164A

473U/538A/164A

↓ SDM with F_VP1_213A/ R_VP1_213A

473U/538A/164A/213A

Figure 5-1: Schematic representation of the SDM steps used to combine the 5′UTR and

VP1 mutations within the EV71 genome.


209

5.2.2 DNA automated cycle sequencing and nucleotide sequence analysis

of EV71 mutants

All mutant viruses were sequenced within the targeted gene to confirm

presence of the correct mutation. The DNA BDT labelling reaction and capillary

separation was performed at The University of Melbourne’s Department of

Pathology or at the AGRF Sequencing Service. The F2386 and R3411 primers

(Table 2-1) were used in the sequencing reaction of the VP1 gene.

5.2.3 Rescue of the EV71 mutants

The EV71 mutant viruses were rescued in Vero cell culture from the in

vitro RNA transcripts as described in materials and methods in Chapter 2. On

day 6 after transfection, the cell culture was subjected to three freeze-thaw cycles

to release the intracellular virus. Cell debris was removed by centrifugation,

supernatant was aliquoted and stored at -80˚C, until use.

The mutant viruses were passaged once in Vero cell culture in order to

increase volume of the viral stocks used in further experiments. Titres of the

rescued viruses were determined by the TCID50 method as described in Chapter

2.

5.2.4 Cell culture assays

All cell culture experiments were conducted with Vero cell line. Cells

were maintained in accordance with growth conditions described in materials and

methods in Chapter 2.

Detailed protocols of the temperature sensitivity and growth kinetics

assays are described in Chapter 3 and 4, respectively.


210

The TCID50 titres obtained in the growth kinetic experiments were used to

calculate the efficiency of plating, by dividing the virus titre at 39.5˚C by the

virus titre at 37.0˚C (Bouchard, Lam & Racaniello 1995).

5.2.4.1 Virus binding assay

Vero cells were seeded into a 24-well plate at a density of 2.25 x 105 cells

per well and grown in 500 µl of MEM supplemented with 5% FBS in a CO2

incubator at 37°C for 24 hours. On the day of inoculation the medium was

removed from the wells and cell monolayers were washed once with PBS (pH of

7.4, room temperature). 102-2.5 TCID50 of the virus in 150 µl volume was added

per well. Plates were incubated at room temperature on a rocking platform for 30

min to allow virus adsorption. After 30 min incubation, the inoculums were

collected from the wells and subjected to quick centrifugation to remove

detached cells, if such existed. The TCID50 viral titres were estimated for the

original viral samples and the supernatants collected after the binding assay. The

difference between two TCID50 values (∆TCID50 = “original TCID50” – “TCID50

after”) was used to calculate the percentage of the bound virus. The experiment

was performed in triplicates. The TCID50 assay was performed as described in

materials and methods in Chapter 2.

5.2.5 Mapping the VP1 mutations on the 3d structure

The 3VBF_A crystal structure of the EV71 VP1 capsid protein (Wang et

al. 2012) was retrieved from the MMDB. Amino acid positions targeted in

charged-to-alanine mutagenesis were mapped on the 3VBF_A subunit structure

in Cn3D 4.3 software available from the NCBI web server

(http://www.ncbi.nlm.nih.gov/Structure/CN3D/cn3d.shtml).


211

Prediction of interactions and binding sites within the VP1 capsid protein

was carried out with the IBIS (Shoemaker, Zhang 2012). The 3VBF_A reference

structure was used as a query.

5.2.6 Statistical analysis

The average mean, SEM and P-values of the Student’s t-test were

calculated in Microsoft Excel software as described in materials and methods in

Chapter 3.


212

5.3 Results

5.3.1 Effect of the VP1 mutations on virus infectivity

The VP1 mutant viruses were obtained by SDM of the parental TR EV71

cDNA clone. In total, six charged amino acid clusters were identified within the

EV71 VP1 sequence. Each of the charged amino acids (D, E, R, H and K) within

those clusters was replaced with alanine (A) (Figure 5-2).

10 20 30 40 50 60 70 80

....*....|....*....|....*....|....*....|....*....|....*....|....*....|....*....|

TRc_VP1 1 GDRVADVIESSIGDSVSRALTRALPAPTGQDTQVSSHRLDTGKVPALQAAEIGASSNASDESMIETRCVLNSHSTAETTL 80

cluster 1

90 100 110 120 130 140 150 160

....*....|....*....|....*....|....*....|....*....|....*....|....*....|....*....|

TRc_VP1 81 DSFFSRAGLVGEIDLPLTGTTNPNGYANWDIDITGYAQMRRKVELFTYMRFDAEFTFVACTPTGEVVPQLLQYMFVPPGA 160

cluster 2 cluster 3

170 180 190 200 210 220 230 240

....*....|....*....|....*....|....*....|....*....|....*....|....*....|....*....|

TRc_VP1 161 PKPDSRESLVWQTATNPSVFVKLSDPPAQVSVPFMSPASAYQWFYDGYPTFGEHKQEKDLEYGACPNNMMGTFSVRTVGT 240

cluster 4 cluster 5

250 260 270 280 290

....*....|....*....|....*....|....*....|....*....|....*..

TRc_VP1 241 SKSKYPLVIRIYMRMKHVRAWIPRPMRNQNYLFKANPNYAGNFIKPTGASRTAITTL 297

cluster 6

Figure 5-2: Clusters of charged amino acids within the VP1. The cluster was defined as a stretch of 4 to 9 amino acids with 50% of them charged and separated within the cluster, at most, by 1 hydrophobic amino acid (except cluster 1, with 2 hydrophobic amino acids in a row). Cluster 1 included amino acids from 37 to 43, cluster 2: from 120 to 124, cluster 3: from 130 to 134, cluster 4: from 162 to 167, cluster 5: from 213 to 221, and cluster 6: from 254 to 259. Amino acids with charged side chains are underlined. Amino acids substituted with alanine are highlighted with grey background.


213

As a result, twenty six mutant EV71 cDNA clones, constructed in vitro,

differed from the parental strain by one substitution with A. Out of those twenty

six clones, eleven (42%) resulted in infectious viruses, which were rescued after

transfection of Vero cells with the in vitro RNA transcripts. Additionally, two

EV71 mutants carrying a combination of the 5′UTR and VP1 mutations

(473U/538A/164A and 473U/538A/164A/213A) were also infectious. No

infectious progeny was obtained from cDNA clones of the clusters 2, 3 and 6

(Table 5-2). Rescued viruses showed transfection was reproducible. Therefore,

fifteen VP1 mutations, where no infectious viruses were obtained, can be

considered “lethal”. Where EV71 mutants were rescued, their titres fell into a

range of 104.25-105.50 TCID50/ml, with the exception of the

473U/538A/164A/213A strain, which only reached 102.8 TCID50/ml.


214

EV71 genome region Mutant Infectivity

VP1, cluster 1 37A ni

38A i

40A i

43A i


121A ni

122A ni

124A ni


132A ni

134A ni

VP1, cluster 4 162A i

164A i

166A ni

167A i

VP1, cluster 5 213A i

214A ni

215A i

217A i

218A i

219A i

221A ni


256A ni

257A ni

259A ni

5′UTR and VP1 473U/538A/164A i

473U/538A/164A/213A i

Table 5-2: Infectivity of the EV71 mutants in vitro.

The in vitro RNA transcripts obtained from the mutant cDNA clones were used to transfect Vero cells. If CPE developed after the transfection and/or within the next passage in vitro, the cDNA clone was considered infectious (i). Where no CPE was observed, the cDNA clone was considered non-infectious (ni) and eliminated from further experiments.


215

In order to examine if location of the amino acid changes within the VP1

molecule correlates with virus infectivity, all amino acids targeted in SDM with

alanine were mapped onto the VP1 crystal structure. The results of the modelling

revealed that all infectious viruses (100%) contained alanine mutations (positions

38, 40, 43, 162, 164, 167, 213, 215, 217, 218 and 219) within the protein loops

(Figure 5-3, A). In contrast, the majority (73%) of the “lethal” alanine mutations

were located within the centre of the folded VP1 molecule and constituted its

structural elements: the α-helix (cluster 2: positions 120, 121, 122 and 124) or β-

sheets (cluster 3: positions 130, 132, 134, and cluster 6: positions 254, 256, 257

and 259) (Figure 5-3, B). Additionally, results of the putative VP1 binding sites

prediction demonstrated that the central amino acids within the cluster 2

(positions 121 and 122) interact with the VP3 capsid protein, whereas amino

acids from the cluster 3 (position 132) and 6 (position 256 and 257) form

interactions with the VP4.


216

A.

B.

Figure 5-3: Location of the alanines substituting charged amino acids within the VP1

capsid protein.

The VP1 molecule is viewed from the outside, with a 5-fold axis at the top. Substitutions with alanine resulting in infectious or non-infectious mutant viruses are mapped onto the VP1 structure in diagram A and B, respectively. Amino acid positions are numbered and highlighted in grey on the backbone of the VP1 molecule.


217

5.3.2 Growth kinetics of the VP1 mutant viruses

Growth kinetics of the EV71 mutants carrying single alanine substitutions

within the VP1 was studied in Vero cells at 37.0˚C and 39.5˚C. Even though an

attempt was made to inoculate all strains at the same TCID50, the titration of the

viral stocks on the day of the experiment revealed some variation between the

original virus titres. The differences fell into a range of (log10 values) 0.75

TCID50/ml from the titre of the parental strain, except for the 164A (1.35

TCID50/ml), 217A (1.30 TCID50/ml) and 218A (0.85 TCID50/ml) mutants.

Therefore, in order to compare the effect of the incubation temperature on growth

properties between the strains, the efficiency of plating (EOP) was calculated as

the ratio of the virus tire at 39.5˚C and the virus titre at 37.0˚C (Bouchard, Lam

& Racaniello 1995). The EOP was obtained from the TCID50 data at 12 h (single-

cycle growth) and 96 h (multiple-cycle growth) post infection. The Student’s t-

test was performed for statistical validation of the observed differences

(Appendix VIII).

The results indicated that mutant viruses of the cluster 1 replicated

approximately at the same rate as the parental strain (Figure 5-4, A; Appendix

VIII, A). The 38A mutant showed a decrease in the growth kinetics during first

12 h. However, that difference was not statistically significant. By the end of the

incubation period all mutant viruses of the cluster 1 reached TCID50 titres similar

to that of the original strain.

Within the cluster 4, two (162A and 164A) of three mutant viruses

showed a statistically significant reduction in replication at 39.5˚C, both during

the single- and multiple-cycle growth (Figure 5-4, B; Appendix VIII, B).


218

A.

B.

C.

Figure 5-4: In vitro replication of the parental and VP1 mutant viruses at 37.0˚C and

39.5˚C in Vero cells.

The growth kinetic curves of EV71 mutants within the cluster 1, 4 and 5 presented by the graph A, B and C, respectively. The plotted TCID50 values are the means of virus titres from three experiments. The standard deviation values were omitted for clarity and can be found in Appendix VII. Reduction in virus growth at 39.5˚C was estimated at 12 h and 96 h (Appendix VIII). *- Difference in the growth reduction between the parental and indicated mutant viruses was considered statistically significant (P<0.05).


219

The substitutions with alanine within the cluster 5 resulted in one mutant

(213A) which demonstrated a statistically valid reduction in the single-cycle

kinetics at 39.5˚C (Figure 5-4, C; Appendix VIII, C). Longer incubation up to 96

h revealed that four mutant viruses (213A, 217A, 218A and 219A) were inhibited

by the temperature, with the most pronounced decrease in replication of the 213A

and 217A strains. Even though log10 TCID50 means of mutants 217A, 218A and

219A at 12 h were similar to those at 96 h (Figure 5-4, C), the variation between

experiments was higher at 12 h, and that resulted in statistically invalid

difference in the single-cycle growth between the parental strain and those

mutants (Appendix VIII, C).

5.3.3 Effect from the combination of the 5′UTR and VP1 mutations on

temperature sensitivity of EV71

To examine if combination of the 5′UTR and VP1 mutations leads to an

enhanced ts phenotype, the shut-off temperatures of the parental strain (TRc) and

mutant viruses (the 473U, 473U/538A, 164A, 213A, 473U/538A/164A and

473U/538A/164A/213A) were evaluated by the temperature sensitivity assay

conducted in Vero cells.

The shut-off temperature of the 5′UTR EV71 mutants (473U and

473U/538A) was only slightly reduced when compared to that of the parental TRc

strain (Figure 5-5, A; Table 5-3). Temperature sensitivity of the 473U/538A virus

was intermediate (38.9˚C) between EV71 with the wt-IRES secondary structure,

TRc (39.1˚C), and the 473U mutant (38.7˚C), possessing the Sabin 3-like domain

Vth.

Within the VP1 protein, single amino acid substitutions with alanine, at

position 164 and 213 resulted in a ts phenotype (38.7˚C) comparable to that of

the 473U virus. Due to very little inhibitory effect, derived from either single

mutation (473U, 164A and 213A), and a variation in the virus titre between three


220

experiments, only one (164A) of those mutants was statistically different from

the TRc (Table 5-3).


221

A. B. C.

Figure 5-5: Temperature dependent reduction in virus titres of EV71 mutants in Vero

cells.

Vero cells were infected with EV71 at an m.o.i of 0.1 and incubated at indicated temperatures for 72 h. The virus titres were determined by TCID50 assay. The mean log10 of the TCID50 from three experiments was used to calculate a reduction in virus titre (∆TCID50) at each temperature compared to the TCID50 at 37˚C (refer Table 5-3). The results obtained with EV71 mutants (A: within the 5′UTR, B: within the VP1, and C: within both the 5′UTR and VP1 gene) were plotted on graphs against the data from the parental virus: TR EV71 clone (TRc).


222

Virus name

Virus titre* ± SEM at: Reduction** in virus titre at: Shut-off

(°C) *** 37°C 38°C 39°C 40°C 38°C 39°C 40°C

TRc 5.80 ±

0.14 5.55 ±

0.00 4.97 ±

0.08 1.62 ±

0.10 0.25 ±

0.14 0.83 ± 0.17a

4.18 ±0.21a 39.1

473U 5.55 ± 0.00

5.30 ± 0.25

4.22 ± 0.17 n/d 0.25 ±

0.25 1.33 ± 0.17a n/d 38.7

473U/538A 4.97 ±

0.08 4.72 ±

0.08 3.88 ±

0.08 n/d 0.25 ± 0.00a

1.08 ± 0.08a n/d 38.9

164A 5.05 ± 0.00

4.80 ± 0.00

3.63 ± 0.08 n/d 0.25 ±

0.00a 1.42 ± 0.08a n/d 38.7b

213A 5.22 ±

0.17 5.22 ±

0.17 3.80 ±

0.00 n/d 0.00 ± 0.29

1.42 ± 0.17a n/d 38.7

473U/538A/ 164A

4.72 ± 0.08

4.30 ± 0.25

2.47 ± 0.22 n/d 0.42 ±

0.30 2.25 ± 0.29a n/d 38.3bc

473U/538A/ 164A/213A

2.88 ± 0.08

2.63 ± 0.08 < 1.45 n/d 0.25 ±

0.14 > 1.43 n/d −

Table 5-3: Temperature sensitivity of EV71 carrying mutations within the 5′UTR, VP1

or both genes.

* Viruses were grown in Vero cells at the indicated temperatures for 72 h. Resulting virus titres were expressed as the mean ± standard error mean (SEM) of three experiments and represent log10 (TCID50) in 1 ml virus sample. The lower limit of virus detection was 1.45 log10 TCID50/ml. The symbol “<” indicates that virus titre was below the detection limit and therefore the SEM was not applicable. ** Reduction in virus titres is expressed as the mean ± SEM of the log10 differences between TCID50 values obtained at the indicated temperatures versus those at 37˚C from three experiments. The symbol “>” indicates that a reduction in virus titre is greater than the indicated value, due to the fact that virus titre at 39˚C was below the detection limit. There is no SEM for that value. *** Temperature at which TCID50 titre is reduced by 1.00 log10 when compared to viral titre at 37˚C. n/d – not determined. a – P<0.05 of the “paired” Student’s t-test indicates the statistical significance of the reduction in a virus titre against the virus titre at 37˚C. b – P<0.05 of the “unpaired” Student’s t-test indicates the statistical significance of the difference between the shut-off temperature of the query virus and that of TRc. c – P<0.05 of the “unpaired” Student’s t-test indicates the statistical significance of the difference between the shut-off temperature of the 473U/538A/164A mutant and that of its parental strain (473U/538A mutant).


223

The degree of temperature sensitivity was increased by combination of the

5′UTR and VP1 mutations (Figure 5-5, C). The introduction of the 164A

substitution into the 473U/538A EV71 clone resulted in a mutant virus with a

more pronounced ts phenotype, which was statistically different from that of the

parental 5′UTR mutant and the original TRc. Temperature sensitivity of the virus

carrying combined mutations was stronger (∆Shut-off = 0.8˚C) than could be

expected from a simple summary of the 473U/538A (∆Shut-off = 0.2˚C) and

164A (∆Shut-off = 0.4˚C) phenotypes. In a similar manner, an additional alanine-

substitution of the 213rd amino acid in the VP1 further enhanced temperature

sensitivity of the 473U/538A/164A/213A EV71 mutant. Its growth was already

restricted at 37˚C, and at 39˚C the virus titre was below the detection level in the

TCID50 assay. Therefore, at these experimental conditions it was not possible to

estimate the shut-off temperature for the 473U/538A/164A/213A mutant (Table

5-3).

5.3.4 Binding activity of the mutant viruses

According to the crystal structure of the EV71 capsid, the 164th and 213th

amino acids of the VP1 protein reside close to the surface of the viral particle

(Figure 5-6). The 164th amino acid lies at the interface of the adjacent protomers,

within the EF-loop oriented towards the 5-fold axis, whereas the 213th amino acid

is situated on the rim of the canyon. The external location of both amino acid

residues, allows an assumption that mutations at these positions might interfere

with virus attachment during EV71 infection.


224

Figure 5-6: Location of the 164th and 213th amino acids of VP1 within the viral capsid.

The schematic of the viral capsid with two VP1 proteins highlighted in pink and brown, one VP2 – in blue and one VP3 – in yellow. The portion of the crystal structure (PDB accession #: 3VBF) consists of the same highlighted units. The 164th and 213th amino acids are indicated with arrows and coloured in turquoise on the “pink” VP1 molecule.


225

The effect of 164- and 213-alanine substitutions on the binding activity of

EV71was analysed in Vero cells. Results indicated that the amount of virus

bound was slightly reduced in EV71 carrying single VP1 mutations (Figure 5-7).

In fact, statistical significance of the observed small differences was conferred

only on alanine-164. Two mutant viruses (the 164A and 473U/538A/164A) with

alanine-164 had similar binding activity irrespective to the 5′UTR sequence

(82% and 76%, P=0.19).

The effect from alanine-213 alone was not statistically significant when

compared to the parental TRc strain (95% versus 100%, P=0.26). However,

unpredictably, its combination with alanine-164 resulted in a further decrease in

the binding activity observed with the 473U/538A/164A/213A mutant virus

(31%). The reduction was statistically significant when compared both to the TRc

(P=0.01) and the parental 473U/538A/164A (P=0.02) strain.


226

Figure 5-7: Effect of substitution of VP1 amino acids at positions 164 and 213 with

alanine on binding of EV71 to Vero cells.

The parental (TRc) or mutant (164A, 213A, 473U/538A/164A and 473U/538A/164A/213A) EV71 was incubated with Vero cells at room temperature for 30 min. The amount of virus bound to Vero cells was determined by the TCID50 assay. The binding activity of the TRc was taken as 100%. The error bar shows standard deviation of the triplicates. * P<0.05 of the “unpaired” Student’s t-test indicates the statistical significance of the difference between the TRc and mutant EV71.


227

5.4 Discussion

In this study, the scanning mutagenesis of the VP1 capsid protein of EV71

was performed. Substitutions with alanine were introduced within the clusters of

charged amino acids. By defining a cluster as a sequence of 4 to 9 residues with

50% of them charged and separated, at most, by only 1 hydrophobic residue, a

total of six clusters were identified and twenty six mutant EV71 cDNA clones

were generated. After transfection of Vero cells with the in vitro RNA transcripts

eleven mutant viruses were successfully rescued, which led to 42% of the

infectivity among the total number of mutant VP1 cDNA clones. These results

are similar to previously reported data, when a portion of rescued mutants fell

between 37-44% (Diamond & Kirkegaard 1994; Hanley et al. 2002) with the

exception of the influenza A virus mutants, whose efficiency of rescue reached

75% (Parkin, Chiu & Coelingh 1996). Variations observed between the studies

are most likely due to the nature of those proteins targeted by the charged-to-

alanine mutagenesis. Different levels of the sequence conservation and different

patterns of the secondary/tertiary structure interactions would impose certain

restrictions on the number of viable clones harbouring the mutant protein.

Indeed, analysis of the 3d structure of EV71 VP1 confirmed that the majority

(73%) of the lethal phenotypes were associated with mutations located within the

centre of the folded VP1 molecule and within its structural elements, α-helix and

β-strands. It is very likely, that even a single substitution with alanine within the

core region of the VP1 affects the proper folding of the protein. Importance of

structural conservation within the cores of the capsid proteins was previously

emphasised for PV (Filman et al. 1989). Sequence differences within the β-

strands could be accommodated only through the compensatory changes leading

to local adjustments in the protein structure. According to the structural analysis,

all infectious viruses rescued in this study, carried alanine substitutions within

the protein loops. Out of those eleven mutants, six (162A, 164A, 213A, 217A,

218A and 219A) showed statistically valid reduction in their growth at 39.5˚C.


228

Interestingly, two (162A and 164A) of the ts mutations were mapped

immediately next to a highly conserved sequence motif, PxGzP (Appendix IX)

(Wien et al. 1997). Similar to PV and HRV, in EV71 sequences the “x” position

is occupied by proline, and “z” - by alanine. The motif resides within the EF loop

which is located between two adjacent protomers of the viral capsid (Figure 5-6)

and next to a tightly packed hydrophobic pocket. It has been shown that

substituting the valine-160 (PV1/Mahoney) for an isoleucine which possesses a

bulky side chain resulted in a disruption of the tight packing and increased

flexibility within the region. As a consequence, conversion from the 160S to

135S particle was more efficient in the mutant virus than in the wt (Wien et al.

1997). The isoleucine-160 was in fact one of the adaptive mutations allowing

viral growth on cells expressing both the wt and mutant PVR (Colston &

Racaniello 1995). In the present study, alanine-substitutions for K162 and D164

might result in the opposite effect, when the hydrophobic alanine with a small

side chain leads to a tighter packing of the PxGzP motif and EF loop towards the

hydrophobic pocket. In turn, the stabilization of the capsid structure might result

in a decreased efficiency of the conformational transitions upon the capsid-

receptor interaction in the alanine mutants. This hypothesis is, generally, in

accordance with the proposed capsid dynamics of the Picornaviridae (Lewis et

al. 1998; Reisdorph et al. 2003). Besides the possible defect in the uncoating

step, both mutants demonstrated a slightly impaired binding to Vero cells. When

compared to the parental strain, the 162A and 164A viruses displayed

approximately 76% ± 5% SEM (data not shown) and 82% ± 5% SEM (Figure 5-

7) of the wt-efficiency. Interestingly, the PV1/Mahoney VP1 mutant at the amino

acid positions 166-169 (equivalent to 162-165 in EV71 VP1) also demonstrated a

reduction in virus binding assay (Harber et al. 1995). However, inhibition with

the mutant PV1 was more pronounced (≤ 10%), possibly, due to multiple

mutations within the region.

As could be expected, combining the 164A with the 5′UTR mutations

(473U/538A) did not change binding phenotype of the 164A virus (Figure 5-7).

In contrast, when the 164A was combined with the 213A (VP1), the resulting


229

473U/538A/164A/213A mutant displayed a stronger inhibition in the binding

assay. Due to the fact, that 213A alone had no an evident effect on virus binding,

the phenotype resulting from the 164A/213A combination was not a simple

summary of two single VP1 mutations. Again, the mechanism behind this

phenomenon is most likely based on structural interactions within the protein.

Due to the flexibility within the folded protein molecule and its motion towards

the thermodynamically stable state the effect from a newly introduced amino acid

change into the wt or mutant protein might be to some degree different,

especially if those proteins already differ in their structure and properties.

In general, the decrease in binding efficiency of mutant EV71 due to SDM

at the 164 or 164/213 positions was very little, if compared to the effect observed

with a naturally selected host-adaptive mutation, reported by Arita, Ami et al.

(2008). In their study, a single amino acid change (G145E) associated with virus

adaptation to mice, resulted in 98% reduction of binding activity in human cell

line. Therefore, it is unlikely that alanine at positions 164, 164/213 interfered

directly with the cell receptors’ recognition. Although, structure-specific capsid-

receptor interactions contribute to the infectivity of the virus, it may utilize

alternative ways to enter the cells (Arita et al. 1998).

In order to give strong support towards any hypothesis of how specific

alanine substitutions alter virus phenotype, further experiments would be

required. This study was not designed to reveal the exact mechanism of the

temperature sensitivity of the generated VP1 mutants, but was mainly aimed to

scan the VP1 protein for possible ts region/determinants.

The single-cycle growth kinetic experiments demonstrated that 162A,

164A and 213A mutant viruses were restricted in their growth at 39.5˚C (Figure

5-4). Since the VP1 is a structural protein, the alanine mutations should not have

a direct effect on virus replication. However, they might somehow affect the

maturation step or virion assembly.

An additional interest of this study was to examine if the ts determinants

derived from the structural VP1 lead to further inhibition of virus replication,

when combined with the IRES mutations within the 5′UTR. A cumulative effect


230

from the Sabin 1 5′UTR, 3D and 3′UTR ts molecular determinants introduced

into the TR EV71 was previously reported (Arita et al. 2008; Arita et al. 2005).

Results of this study demonstrated that the EV71 mutant harbouring the 164A

(VP1) and 473U/538A (5′UTR) showed stronger ts phenotype, compared to the

phenotypes caused by the single mutations (Figure 5-5, Table 5-3). The

introduction of the 213A in the next step inhibited virus production both at

39.5˚C and 37.0˚C. Therefore, the results demonstrated that combination of the

IRES 5′UTR mutations and charged-to-alanine substitutions within the VP1

structural protein was essential for stronger inhibition of EV71 in cell culture.

In this study, temperature sensitivity was used as a marker of virus

inhibition in vitro. However, this characteristic cannot be an absolute indicator of

the virus attenuation (Arita et al. 2005). Experiments in an animal model would

be required to give a conclusive answer if those mutations are associated with an

attenuated EV71 phenotype. Nevertheless, results of this study extend our

knowledge of EV71 as a pathogen. The identified ts molecular determinants

within the VP1 protein can be potentially useful in the development of an

attenuated vaccine strain.

231

CHAPTER 6

FINAL DISCUSSION AND CONCLUSIONS

CHAPTER 6 Final discussion and conclusions

232

6 FINAL DISCUSSION AND CONCLUSIONS

6.1 Contributions of this work

Since it was first time reported almost forty five years ago, EV71 has been

identified as a causative agent of HFMD in numerous outbreaks around the

world. Symptoms of HFMD, which can be caused by a number of human

enteroviruses, are usually mild and self-limited. However, EV71 associated cases

of HFMD have often been complicated with severe neurological disorders, which

led to a long-term recovery or a lethal outcome. Therefore, in the polio

eradication era EV71 has become a new challenge to human health. Many studies

have been aimed to discover the molecular basis and mechanisms underlying the

pathogenicity of EV71. Since DNA sequencing became a routine method in

many research laboratories, numerous clinical isolates of EV71 both from

uncomplicated and severe cases have been sequenced in full or in part. In some

studies efforts were made to reveal a possible connection between the EV71

genome and its virulence. However, our knowledge in this area remains very

limited and mechanisms of the virus pathogenicity are still to be determined.

The aim of this work was to explore the EV71 genome in regards to

molecular basis of the virus inhibition. The SDM in vitro was employed as the

main experimental approach and temperature sensitivity of the virus was used as

criteria to evaluate the difference between the parental and mutant strains. A

prerequisite to this study was a construction of an infectious EV71 cDNA clone.

Although, full-length genome cDNA clones have been obtained for many

enteroviruses including EV71, these constructs are not available commercially.

The success of full-length genome cloning is complicated by some issues

discussed in detail in Chapter 2. In this study several methodological approaches

have been utilized, in order to reveal the contribution of some factors to

infectivity of cDNA clones of EV71. Finally, an attempt was made to explore the


233

mechanism of viral adaptation to an elevated temperature in vitro. Experimental

investigations into the genes of EV71 revealed their roles to the virus adaptation.

The main contributions of this work are as follows:

Examination of the role that the cell culture status plays in rescuing the

infectious progeny of EV71 from its in vitro RNA transcripts (Chapter 2).

Practically, this work has led to the optimization of the cell culture transfection

protocol with in vitro RNAs. These results were presented at the ASBMB's

Special Symposium "Recent Advances in Pathogenic Human Viruses" in July

24-26, 2011, Guangzhou, China.

Identification of the genetic molecular determinants that lead to EV71

adaptation to an increased temperature in vitro (Chapter 3). In-depth sequence

and structural analysis of the identified mutations, supported by the results of the

cell culture experiments with the generated EV71 mutants, determined the

contribution that different virus genes played in the virus adaptation. Results of

this work were presented at the Australian Society for Microbiology 2012

Annual Scientific Meeting in July 1-4, 2012, Brisbane, Australia.

Examination of the effect from SDM within the Vth and VIth domains of

the 5′UTR on the virus growth in vitro (Chapter 4). Analysis of the Sabin 3-like

EV71 revertants suggested the benefit that the stabilized RNA structure may

bring to the genetic stability of the attenuated phenotype in EV71. Results of this

work will be submitted for publication.

Identification of the molecular determinants of EV71 temperature

sensitivity within the VP1 structural protein (Chapter 5). Scanning charged-to-

alanine mutagenesis revealed the VP1 regions which may be potentially useful

for the virus attenuation in vivo. Combination of the newly identified ts

molecular determinants of the VP1 with the Sabin 3 attenuating mutation showed

further inhibition of the virus in cell culture experiments. This work was

presented at the 2nd World Congress on Virology “Innovations and Therapeutic


234

approaches in Virology” in August 20-22, 2012, Las Vegas, USA. Results of this

work will be submitted for publication.

6.2 Final conclusions of the experimental work

6.2.1 Construction of an infectious cDNA clone of EV71

Two strategies were employed to construct cDNA clones containing the

full-length genome of EV71: 1) the overlapping RT-PCR and 2) the long RT-

PCR of the virus genome. The successful production of cDNA clones by both

strategies, however, did not guarantee their in vitro RNA transcripts to be

biologically active in cell culture. Deleterious mutations introduced within the

EV71 genome during the overlapping PCR could probably account for the loss of

infectivity in viral clones from Strategy 1. There was no difference in viral titres

after transfection of cell culture with the infectious in vitro RNA transcripts

harbouring the poly(A) tail of 18 or 30 bases in length. The extra bases

downstream of the poly(A) tail did not reduce the infectivity of the RNA

transcripts. However, incorporation of the unique restriction enzyme recognition

site directly downstream of the poly(A) tail can be important if the vector does

not supply such a site or if it is located within a considerable distance from the

poly(A) tail. Polishing of the short 3′ end extension in dsDNA template did not

increase infectivity of the RNA transcripts and, therefore, can be eliminated in

order to simplify the procedure. Finally, cell culture status played an important

role in rescuing viral progeny from the infectious clones. The actively dividing

cells were able to establish viral infection at a higher level.


235

6.2.2 Temperature resistant molecular determinants of EV71 due to

natural selection in cell culture

Four passages of the ts EV71 at an elevated temperature (39˚C) in vitro

were sufficient to render the TR phenotype of the virus. Selection of the new

phenotype led to a decrease in the genetic diversity of the viral quasispecies.

Site-directed mutagenesis confirmed that only a single amino acid change

(Ser299Thr) within the 3Dpol was associated with the acquired temperature

resistance. The mutation was mapped within the core region of the protein,

constituting the catalytically active site. The amino acid changes within the

capsid proteins VP1 and VP3 had very little or no effect on temperature

resistance, and were most likely co-selected with the fittest EV71 genome.

Growth of the naturally selected TR variant was suppressed at temperatures

higher than that used for its selection.

6.2.3 Site-directed mutagenesis of EV71 within the 5′UTR

The Sabin 3 5′UTR attenuating determinant (472U) introduced into the

reciprocal position (nt 473) within the EV71 genome resulted in a reduced viral

growth at 39.5˚C in Vero cell culture. The Sabin 3 mutation was unstable within

EV71 and reverted to the wt sequence within five passages in vitro. The

reversion resulted in establishing the viral replication at the wt level. Genetic

stability of the Sabin 3 attenuating determinant was enhanced by the

complementary 538A substitution within the 5′UTR. When introduced together,

both the 473U and 538A mutations remained present within the EV71 genome

during the experimental period (up to 10 passages). Genetic stability of the

mutant viruses correlated with viral load accumulation in vitro. As a result, the

temperature sensitive phenotype of the 473U/538A EV71 mutant was more

pronounced during multiple passages when compared to the 473U mutant (Sabin

3-like EV71).


236

The replacement of the VIth domain of the 5′UTR of EV71 with the same

structural element of CAV8 and CAV16 did not affect viral growth in Vero cell

culture.

The human SK-N-SH cell line did not support replication of the parental

EV71 strain used in this study. Therefore, it was not possible to conclude if the

5′UTR mutations within the Vth and VIth domains lead to an attenuation of EV71

in neural cells.

6.2.4 Scanning the VP1 protein of EV71 for ts-determinants by charged-

to-alanine mutagenesis

In order to identify ts molecular determinants within the VP1 capsid

protein the charged-to-alanine mutagenesis was employed. Six clusters of

charged amino acids were identified within the VP1 sequence. Three of those

were mapped within the structural elements (α-helix and β-sheet), and the SDM

within those clusters did not produce infectious viruses. Another three clusters

were located within the VP1 loops, and alanine substitutions of those charged

amino acids resulted in eleven infectious mutant strains. Out of eleven, six EV71

mutants demonstrated growth kinetics impaired at the elevated temperature of

39.5˚C. Further inhibition of virus growth in vitro was demonstrated as a result of

a combination of the VP1 (164A and 213A) and IRES 5′UTR (473U/538A)

mutations.


237

6.3 Final comments and future directions

Although temperature sensitivity does not necessarily lead to virus

attenuation, the inhibitory effect observed from mutations in vitro can be a

potential marker of EV71 attenuation in vivo.

Revealing the EV71-specific molecular determinants of temperature

sensitivity and attenuation is an important part in the attenuated vaccine design

against this virus. Due to some differences in mechanisms of pathogenesis and

tissue tropisms, attenuating determinants derived from closely related species

might not have the same effect on the attenuation of EV71. Identification of ts

and attenuating determinants in different genomic regions of EV71, and their

combination in one strain seems to be essential in order to achieve both stronger

attenuation and increased genetic stability of the live-attenuated vaccine.

A combination of multiple mutations, each exhibiting a slight attenuated

but genetically stable phenotype, can be a more successful approach than a

combination of a few strong attenuating determinants, which revert to the wt

phenotype easily. Research efforts should be made towards the improvement of

the genetic stability of the vaccine strain. Stabilisation of every single attenuating

molecular determinant requires in-depth understanding of the protein functions,

its structure and those conformational changes which might be introduced by the

mutation.

Additionally, increasing the fidelity of the virus RNA synthesis, in

general, can be a promising approach. The single amino acid substitution, G64S,

within the 3Dpol was shown to lead to an increase in replication fidelity of the PV

(Pfeiffer & Kirkegaard 2003; Vignuzzi, Wendt & Andino 2008). Unfortunately,

the equivalent mutation introduced into the EV71 genome completely abolished

virus replication in this study (data not shown). Similar mutation introduced into

the FMDV 3Dpol was shown to impair RNA-binding, polymerization, and R

monophosphate incorporation activities (Ferrer-Orta et al. 2010).


238

Finally, revealing the molecular basis of EV71 tropism to neural cells can

enhance our understanding of the virus pathogenicity. The adaptive VP1, VP2

mutations selected by passaging the virus in the mouse brain have been shown to

increase EV71 neurovirulence in the murine model (Arita et al. 2008; Chua et al.

2008; Huang et al. 2012). However, genetic determinants of the virus neuro-

tropism in humans are most likely to be different and remain to be determined.

The EV71 clone (GenBank accession #: JX025559) used in this study did not

replicate well in human neuroblastoma cells, although, the original 6F/AUS/6/99

virus was isolated from the cerebrospinal fluid of a patient with brainstem

encephalitis (McMinn et al. 2001a). Prior to cloning, the original virus was

propagated in RD and Vero cell cultures which resulted in a virus harbouring

additional mutations when compared to the original isolate. Further studies

would be required to determine which mutations in the viral clone led to the loss

of the neuro-tropic features of the virus. On the other hand, studying the low-

passage clinical isolates with no further adaptation to cell culture can provide

valuable information on genetic determinants of pathogenicity in circulating

viruses (Yeh et al. 2011).

239

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271

Appendices

APPENDICES

272

9 Appendices

Appendix I

6 x Crystal Violet Loading Dye:

30% Glycerol

20 mM EDTA

100 μg/ml Crystal Violet

To prepare 5 ml of 6 x Crystal Violet Loading Dye takes:

2.15 ml of 70% Glycerol

0.2 ml of 0.5M EDTA

50 µl of 1% Crystal Violet solution (w/v)

Add H2O up to 5 ml. Aliquot and store at room temperature.

Note:

Crystal Violet must not be heated. Therefore, it must be added once the agarose

is dissolved in TAE buffer.

APPENDICES

273

Appendix II Nucleotide and amino acid substitutions observed within the EV71 cDNA clones.

Region Mutations a Mutation frequencies (f) b

nt aa Strategy 1 Strategy 2 5′UTR T1 deletion n/a 1/2 0/2

A149G n/a 1/2 0/2 A282G n/a 1/2 0/2 A388G n/a 1/2 0/2 C640T n/a 1/2 0/2 T682C n/a 0/2 1/2

VP4 - - 0/2 0/2 VP2 - - 0/1 0/1 VP3 A680G K227R 1/1 0/1 VP1 A138G - 1/2 0/2

T209C L70P 1/2 0/2 A275G E92G 0/2 2/2 A292G K98E 0/2 1/2 A293C K98T 2/2 0/2 G314A G105E 1/2 0/2 T368C V123A 1/2 0/2 C658T L220F 0/2 2/2 A724C K242Q 0/2 1/2 T789C - 1/2 0/2 C848T S283F 1/2 0/2

2A A234G - 1/1 0/1 2B - - 0/1 0/1 2C A245G Y82C 1/1 0/1

G857A C286Y 1/1 0/1 3A - - 0/1 0/1 3B A35G K12R 1/2 0/1 3C A93G - 1/2 0/1 3D G6A - 1/2 0/2

T43G L15V 0/2 1/2 A342G - 1/2 0/2 T1119C - 2/2 2/2 G1125A - 1/2 0/2

3′UTR - n/a 0/2 0/2 a – Mutations are depicted according to their nt and aa positions in specified genes when compared to the 6F/AUS/6/99 reference strain (GenBank DQ381846). b – The mutation frequency (f) is presented as a number of clones, which contain the mutation, per total number of clones sequenced within the specified region.

APPENDICES

274

Appendix III

NZY+ broth (per liter)

10 g of casamino acids (DIFCO Laboratories)

5 g of yeast extract (DIFCO Laboratories)

5 g NaCl

1. Add deionized H2O to a final volume of 1 liter.

2. Adjust to pH 7.5 with 5M NaOH.

3. Autoclave.

4. Add the following filter-sterilized supplements prior to use:

12.5 ml of 1M MgCl2

12.5 ml of 1M MgSO4

10 ml of 2M glucose

APPENDICES

275

Appendix IV

Standard deviation of growth kinetic curves presented in Figure 4-3:

In vitro replication of the parental and mutant (473U, 473U/538A) EV71 viruses

at 37.0˚C and 39.5˚C in Vero (A) and SK-N-SH (B) cells.

A. 37.0˚C

0 6 12 24 48 96 Parental 0.66 0.80 0.52 0.43 0.38 0.52 473U 0.90 0.54 0.29 0.52 0.29 0.88 473U/538A 0.23 0.14 0.25 0.29 0.25 0.25

39.5˚C

0 6 12 24 48 96 Parental 0.50 0.54 0.50 0.14 0.29 0.29 473U 0.86 0.75 0.48 0.66 0.58 0.74 473U/538A 0.00 0.18 0.00 0.12 0.00 0.00

B. 37.0˚C

0 6 12 24 48 96 Parental 0.82 0.35 0.18 0.07 0.95 0.71 473U 0.28 0.18 0.00 0.37 0.25 1.06 473U/538A 0.07 0.18 0.53 0.28 0.60 0.88

39.5˚C

0 6 12 24 48 96 Parental 0.00 0.42 0.00 0.00 0.21 0.00 473U 0.59 0.42 0.00 0.11 0.00 0.00 473U/538A 0.71 0.11 0.21 0.11 0.00 0.00

APPENDICES

276

Appendix V


In vitro replication of the parental and mutant (SL6-CAV8, SL6-CAV16) EV71

viruses at 37.0˚C and 39.5˚C in Vero (A) and SK-N-SH (B) cells.

A. 37.0˚C

0 6 12 24 48 96 Parental 0.66 0.80 0.52 0.43 0.38 0.52 SL6-CAV8 1.09 0.79 0.14 0.43 0.58 0.46 SL6-CAV16 0.58 0.69 0.25 0.50 0.52 0.29

39.5˚C


B. 37.0˚C


39.5˚C


APPENDICES

277

Appendix VI

Nucleotide and amino acid changes observed within the TR EV71 clone used in

this study, when compared to the 6F/AUS/6/99 EV71 strain.

Genome region nt mutations a aa mutations b

5′UTR - n/a VP4 - - VP2 G1284A - VP3 A1947G -

G2030A G429E VP1 A2732C K663T

C3287T S848F 2A - - 2B - - 2C - - 3A - - 3B - - 3C - - 3D T6832A S2030T

T7056C - 3′UTR - n/a

a – nucleotide (nt) changes are numbered in accordance with the position within

the EV71 genome; b – amino acid (aa) changes are numbered in accordance with the position within

the EV71 polyprotein.

APPENDICES

278

Appendix VII


In vitro replication of the parental and VP1 mutant viruses at 37.0˚C and 39.5˚C

in Vero cells.

The standard deviation values of EV71 mutants within the cluster 1, 4 and 5

presented by the table A, B and C, respectively.

A. 37.0˚C

0 6 12 24 48 96 Parental 0.66 0.80 0.52 0.43 0.38 0.52 38A 0.14 0.68 0.43 0.29 0.63 0.52 40A 0.88 0.51 0.58 0.50 0.38 0.80 43A 0.72 0.39 0.58 0.43 0.14 0.29

39.5˚C

0 6 12 24 48 96 Parental 0.50 0.54 0.50 0.14 0.29 0.29 38A 0.43 0.17 0.52 0.87 0.25 0.58 40A 0.95 0.65 0.75 1.01 0.66 0.76 43A 0.52 0.61 0.00 0.66 0.58 0.88

B. 37.0˚C

0 6 12 24 48 96 Parental 0.66 0.80 0.52 0.43 0.38 0.52 162A 1.01 0.80 0.75 0.52 0.00 0.00 164A 0.20 0.16 0.38 0.50 0.00 1.01 167A 0.66 1.03 0.63 0.66 0.66 0.88

39.5˚C

0 6 12 24 48 96 Parental 0.50 0.54 0.50 0.14 0.29 0.29 162A 0.58 0.14 0.38 0.58 0.43 0.20 164A 0.26 0.15 0.27 0.18 0.00 0.33 167A 1.06 0.73 0.76 0.52 0.58 1.00

APPENDICES

279

C. 37.0˚C

0 6 12 24 48 96 Parental 0.66 0.80 0.52 0.43 0.38 0.52 213A 0.14 0.53 0.14 0.14 0.38 0.38 215A 0.92 0.74 0.43 0.25 0.52 0.76 217A 0.25 0.32 0.58 0.50 0.25 0.00 218A 0.58 0.16 0.00 0.52 0.52 0.38 219A 0.58 0.38 0.52 0.29 0.29 0.00

39.5˚C

0 6 12 24 48 96 Parental 0.50 0.54 0.50 0.14 0.29 0.29 213A 0.69 0.33 0.23 0.00 0.23 0.38 215A 1.01 1.00 0.50 0.72 0.52 0.25 217A 0.38 0.43 0.00 0.25 0.14 0.31 218A 0.87 0.15 0.58 0.72 0.14 0.38 219A 0.78 0.14 0.29 0.52 0.43 0.38

APPENDICES

280

Appendix VIII

The efficiency of plating (EOP) was calculated with the TCID50 titres obtained at

12 h and 96 h p.i. The data is the mean ± standard error mean (SEM) of three

experiments. P-values were calculated in the “unpaired” version of the Student’s

t-test, with the unequal variance and one-tailed distribution.

Tables A, B and C represent results for EV71 VP1 mutants within the cluster 1, 4

and 5, respectively, in comparison to the parental strain.

A. 12 h 96 h

Parental EOP 0.65 0.45 ±SEM ±0.05 ±0.03 P n/a n/a

38A EOP 0.46 0.37 ±SEM ±0.13 ±0.07 P 0.14 0.19

40A EOP 0.67 0.33 ±SEM ±0.12 ±0.08 P 0.45 0.15

43A EOP 0.54 0.33 ±SEM ±0.06 ±0.12 P 0.11 0.22

B.

12 h 96 h Parental EOP 0.65 0.45

±SEM ±0.05 ±0.03 P n/a n/a

162A EOP 0.32 0.09 ±SEM ±0.03 ±0.03 P 0.003 0.0003

164A EOP 0.29 0.15 ±SEM ±0.09 ±0.06 P 0.02 0.01

APPENDICES

281

167A EOP 0.62 0.31 ±SEM ±0.10 ±0.12 P 0.42 0.18

C.

12 h 96 h Parental EOP 0.65 0.45

±SEM ±0.05 ±0.03 P n/a n/a

213A EOP 0.31 0.10 ±SEM ±0.08 ±0.05 P 0.01 0.01

215A EOP 0.65 0.35 ±SEM ±0.16 ±0.04 P 0.50 0.08

217A EOP 0.54 0.11 ±SEM ±0.13 ±0.05 P 0.24 0.01

218A EOP 0.48 0.25 ±SEM ±0.19 ±0.05 P 0.23 0.02

219A EOP 0.75 0.19 ±SEM ±0.18 ±0.05 P 0.32 0.01

APPENDICES

282

Appendix IX

The COBALT (multiple BLAST) alignment of the Picornaviridae VP1 containing a highly conserved motif PxGzP. PxGzP* *

Query(TRc EV71)150 --LLQYMFVPPGAPKPDSRESLVWQTATNPSVFVKLSDPPAQVSVPFMSPASAYQWFYDGYPTFGEHKQEKDL----EYG 223

3VBF_A (EV71) 150 --LLQYMFVPPGAPKPDSRESLAWQTATNPSVFVKLSDPPAQVSVPFMSPASAYQWFYDGYPTFGEHKQEKDL----EYG 223

1BEV_1 (BEV) 140 --TYQVMYVPPGAPVPSNQDSFQWQSGCNPSVFADTDGPPAQFSVPFMSSANAYSTVYDGYARFMDTDP--DR-----YG 210

1EV1_1 (ECV1) 139 VLTHQIMYVPPGGPIPVSVDDYSWQTSTNPSIFWTEGNAPARMSIPFISIGNAYSNFYDGWSHFSQAG---------VYG 209

1M11_1 (ECV7) 139 PLTHQIMYIPPGGPVPNSVTDFAWQTSTNPSIFWTEGNAPPRMSIPFISIGNAYSNFYDGWSHFSQNG---------VYG 209

1H8T_A (ECV11) 139 PLTHQIMYIPPGGPIPKSVTDYTWQTSTNPSIFWTEGNAPPRMSIPFISIGNAYSNFYDGWSHFSQNG---------VYG 209

2C8I_A (ECV12) 139 PLTHQIMYIPPGGPIPKSVTDYTWQTSTNPSIFWTEGNAPPRMSIPFISIGNAYSNFYDGWSHFSQNG---------VYG 209

1D4M_1 (CAV9) 139 VLTHQIMYVPPGGPIPAKVDDYAWQTSTNPSIFWTEGNAPARMSIPFISIGNAYSNFYDGWSNFDQRG---------SYG 209

1Z7S_1 (CAV21) 147 NQVYQIMYIPPGAPRPSSWDDYTWQSSSNPSIFYMYGNAPPRMSIPYVGIANAYSHFYDGFARVPLEGENTDA-GDTFYG 225

1COV_1 (CBV3) 136 ILTHQIMYVPPGGPVPDKVDSYVWQTSTNPSVFWTEGNAPPRMSVPFLSIGNAYSNFYDGWSEFSRNG---------VYG 206

1FPN_1 (HRV2) 138 HITMQYMYVPPGAPVPNSRDDYAWQSGTNASVFWQHGQAYPRFSLPFLSVASAYYMFYDGYDEQD--QN---------YG 206

1RHI_1 (HRV3) 146 -LTVQAMYVPPGAPNPKEWDDYTWQSASNPSVFFKVGE-TSRFSVPFVGIASAYNCFYDGYSHDDPDTP---------YG 214

1RUF_1 (HRV14) 146 -LVVQAMYVPPGAPNPKEWDDYTWQSASNPSVFFKVGD-TSRFSVPYVGLASAYNCFYDGYSHDDAETQ---------YG 214

1AYN_1 (HRV16) 137 HIVMQYMYVPPGAPIPTTRDDYAWQSGTNASVFWQHGQPFPRFSLPFLSIASAYYMFYDGYDGDTYKSR---------YG 207

1HXS_1 (PV1) 152 NQVYQIMYVPPGAPVPEKWDDYTWQTSSNPSIFYTYGTAPARISVPYVGISNAYSHFYDGFSKVPLKDQSAAL-GDSLYG 230

3EPF_1 (PV2) 129 NQVYQIMYIPPGAPIPGKWNDYTWQTSSNPSVFYTYGAPPARISVPYVGIANAYSHFYDGFAKVPLAGQASTE-GDSLYG 207

3EPD_1 (PV3) 129 NQVYQIMYIPPGAPTPKSWDDYTWQTSSNPSIFYTYGAAPARISVPYVGLANAYSHFYDGFAKVPLKTDANDQIGDSLYS 208

1OOP_A (SVDV) 138 VLTHQIMYVPPGGPVPTKVNSYSWQTSTNPSVFWTEGSAPPRMSVPFIGIGNAYSMFYDGWARFDKQG---------TYG 208

The Picornaviridae VP1 sequences aligned to the EV71 VP1 fragment between 150 and 223 amino acids are shown. The PxGzP motif is highlighted in grey. The 162 and 164 amino acids targeted in SDM with alanine and which resulted in ts phenotypes are indicated with stars (*). The sequence name consists of the PDB accession code and virus name (in parentheses).

site-directed in vitro mutagenesis of enterovirus ev71

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