murine cytomegalovirus encodes proteins that regulate

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Murine Cytomegalovirus Encodes Proteins thatRegulate Viral Late TranscriptionTravis ChapaWashington University in St. Louis

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WASHINGTON UNIVERSITY IN ST. LOUIS

Division of Biology & Biomedical Sciences

Molecular Microbiology and Microbial Pathogenesis

Dissertation Examination Committee: Anthony French, Chair

Dong Yu, Co-Chair Jacco Boon

Michael Caparon Henry Huang Robyn Klein

Wayne Yokoyama

Murine Cytomegalovirus Encodes Proteins that Regulate Viral Late Transcription

By

Travis James Chapa

A dissertation presented to the Graduate School of Arts and Sciences

of Washington University in partial fulfillment of the

requirements for the degree of Doctor of Philosophy

August 2014

St. Louis, Missouri

TABLE OF CONTENTS

List of Figures ................................................................................................................................................ v

List of Tables ................................................................................................................................................ vii

List of Abbreviations .................................................................................................................................... viii

Acknowledgements ....................................................................................................................................... xi

Abstract of the Dissertation .......................................................................................................................... xii

Chapter I: Introduction ............................................................................................................................... 1

Herpesviruses ............................................................................................................................................... 2

Betaherpesviruses ........................................................................................................................................ 3

Cytomegalovirus ........................................................................................................................................... 4

Pathogenesis ................................................................................................................................................ 4

Virus structure ............................................................................................................................................... 6

Life cycle ..................................................................................................................................................... 10

Early gene expression ................................................................................................................................. 16

Late gene expression .................................................................................................................................. 19

Development of herpesvirus BAC system .................................................................................................. 24

Functional profiling in HCMV....................................................................................................................... 25

Aim and Scope of Thesis ............................................................................................................................ 25

References .................................................................................................................................................. 30

Tables .......................................................................................................................................................... 52

Figures and Legends .................................................................................................................................. 54

Chapter II: Murine Cytomegalovirus protein pM79 Is a Key Regulator of Viral Late Transcription 57

Preface ........................................................................................................................................................ 58

Summary ..................................................................................................................................................... 59

Introduction.................................................................................................................................................. 60

Results ........................................................................................................................................................ 63

ii

Discussion ................................................................................................................................................... 70

Acknowledgements ..................................................................................................................................... 72

Materials and Methods ................................................................................................................................ 73

References .................................................................................................................................................. 80

Tables .......................................................................................................................................................... 84

Figures and Legends .................................................................................................................................. 88

Chapter III: Murine Cytomegalovirus Protein pM92 Is a Conserved Regulator of Viral Late Gene

Expression ............................................................................................................................................... 100

Preface ...................................................................................................................................................... 101

Summary ................................................................................................................................................... 102

Introduction................................................................................................................................................ 103

Results ...................................................................................................................................................... 105

Discussion ................................................................................................................................................. 111

Acknowledgements ................................................................................................................................... 114

Materials and Methods .............................................................................................................................. 115

References ................................................................................................................................................ 121

Tables ........................................................................................................................................................ 126

Figures and Legends ................................................................................................................................ 127

Chapter IV: MCMV Late Transcription Regulator pM92 Interacts with pM79 and RNAP II During

Infection ................................................................................................................................................... 137

Preface ...................................................................................................................................................... 138

Introduction................................................................................................................................................ 139

Results ...................................................................................................................................................... 143

Future Directions ....................................................................................................................................... 148

Discussion ................................................................................................................................................. 150

Materials and Methods .............................................................................................................................. 152

iii

References ................................................................................................................................................ 155

Tables ........................................................................................................................................................ 160


Chapter V: Conclusions and Future Directions .................................................................................. 168

Conclusions ............................................................................................................................................... 169

Future Directions ....................................................................................................................................... 175

Concluding Remarks ................................................................................................................................. 182

References ................................................................................................................................................ 183


iv

List of Figures

Chapter I: Introduction

Figure 1. Human cytomegalovirus genome schematic ......................................................................... 54

Figure 2. A representative diagram depicting the strategy for creating recombinant MCMV BAC

Clones .................................................................................................................................... 55

Figure 3. Life cycle of CMV in a host cell .............................................................................................. 56

Chapter II: Murine Cytomegalovirus protein pM79 Is a Key Regulator of Viral Late Transcription

Figure 1. Gene M79 is essential for MCMV growth in fibroblasts ......................................................... 88

Figure 2. Expression of M79 gene products is markedly enhanced by viral DNA synthesis and protein

pM79 ...................................................................................................................................... 90

Figure 3. pM79 localizes to replication compartments during infection ................................................ 91

Figure 4. pM79 is not required for viral DNA synthesis......................................................................... 92

Figure 5. pM79 is not required for the maturation of viral replication compartments ............................ 94

Figure 6. pM79 is required for efficient expression of a representative viral late gene ........................ 95

Figure 7. Tiled array analysis of genome-wide transcription during MCMV infection ........................... 97

Figure 8. RT-qPCR analysis of representative PAA-sensitive transcripts in MCMV infection .............. 99


Expression

Figure 1. pM92 is essential for MCMV replication in fibroblasts .................................................... 127

Figure 2. pM92 accumulates abundantly at late times of infection and localizes to viral nuclear

replication compartments ................................................................................................ 129

Figure 3. pM92 is dispensable for viral DNA synthesis but is required for efficiently accumulation of

M55 late gene products during infection ......................................................................... 131

Figure 4. pM92 is required for efficient accumulation of a panel of late transcripts ....................... 133

Figure 5. CMV UL92/M92 proteins interact with UL79/M79 proteins during infection ................... 134

Figure 6. pM92 trans complements the growth of pUL92-deficient HCMV virus ........................... 135

v

Chapter IV: MCMV Late Transcription Regulator pM92 Interacts with pM79 and RNAP II During Infection

Figure 1. Proteomic analysis to identify binding partners of pM79 and pM92 ............................... 164

Figure 2. Immunoprecipitation analysis to confirm RNAP II interactions ....................................... 165

Figure 3. pM92 requires viral accessory proteins to facilitate interaction with pM79 ..................... 166

Figure 4. Amino acid alignment of the UL92 protein family ........................................................... 167

Chapter V: Conclusions and Future Directions

Figure 1. Amino acid alignment of the UL79 protein family ........................................................... 189

vi

List of Tables

Chapter I: Introduction

Table 1. HCMV gene families .............................................................................................................. 52

Table 2. CMV mutagenesis and phenotype characterization .............................................................. 53

Chapter II: Murine Cytomegalovirus protein pM79 Is a Key Regulator of Viral Late Transcription

Table 1. Primers used in quantitative PCR analysis .......................................................................... 84

Table 2. M79-dependent expression of MCMV ORFs ....................................................................... 85


Expression

Table 1. Primers used in PCR analysis ........................................................................................ 126

Chapter IV: MCMV Late Transcription Regulator pM92 Interacts with pM79 and RNAP II During Infection

Table 1. Mass spectrometry identities for sm79flag immunoprecipitation .................................... 160

Table 2. Mass spectrometry identities for smflag92 immunoprecipitation .................................... 161

Table 3. Mass spectrometry identities for viral proteins ................................................................ 162

Table 4. Primers used in QuickChange Mutagenesis ................................................................... 163

vii

List of abbreviations

AIDS Acquired Immunodeficiency Syndrome

ARF Anthony Roger Fehr

ARF1 Anthony R. French

ATP Adenosine Triphosphate

BAC Bacterial Artificial Chromosome

CA Christopher Affolter

CCMV Chimpanzee Cytomegalovirus

co-IP Co-Immunoprecipitation

CPE Cytopathic Effect

DNA Deoxyribonucleic Acid

DY Dong Yu

EBV Epstein-Barr Virus

FLP Flippase

FRT Flippase Recombination Target

GFP Green Fluorescent Protein

HCMV Human Cytomegalovirus

HDAC Histone Deacetylase

HFF Human Foreskin Fibroblasts

HHV-6 Human Herpes Virus 6

HHV-7 Human Herpes Virus 7

HIV Human Immunodeficiency Virus

HSV-1 Herpes Simplex Virus 1

HSV-2 Herpes Simplex Virus 2

IE Immediate Early

IRES Internal Ribosome Entry Site

KSHV Kaposi's Sarcoma-associated Virus

viii

LSJ L. Steve Johnson

mC-BP Minor Capsid Binding Protein

MCMV Murine Cytomegalovirus

MCP Major Capsid Protein

mCP Minor Capsid Protein

MCV Mark C. Valentine

MEF Murine Embryonic Fibroblasts

MHV68 Murine Herpesvirus 68

MIEP Major Immediate Early Promoter

MOI Multiplicity of Infection

ORF Open Reading Frame

PAA Phosphonoacetic Acid

PAGE Polyacrylamide Gel Electrophoresis

PBS Phosphate-Buffered Saline

PCNA proliferating Cell Nuclear Antigen

PCR Polymerase Chain Reaction

PFGE Pulse Field Gel Electrophoresis

PML Promyelocytic Leukemia Protein

RCMV Rat Cytomegalovirus

RNA Ribonucleic Acid

RNAP II RNA Polymerase II

RPA Replication Protein A

RRE RTA Responsive Element

RTA Replication and Transcription Activator

RT-qPCR Reverse Transcriptase-Quantitative PCR

SCP Smallest Capsid Protein

SDS Sodium Dodecyl Sulfate

ix

TAF TBP associated Factor

TBP TATA Binding Protein

TFII Transcription Factor II

TJC Travis James Chapa

TORCH Several Vertically Transmitted Infections Including Toxoplasmosis, Other Infections, Rubella, Cytomegalovirus, and Herpes Simplex Virus 2)

UL Unique Long

US Unique Short

UV Ultra Violet Light

VHS Virion Host Shut-off

VZV Varicella Zoster virus

WMY Wayne M. Yokoyama

YCP Yi-Chieh Perng

x

Acknowledgements

First and foremost I would like to thank my thesis committee. The many thoughtful suggestions they have

had for my project over the years have really shaped my research and challenged me to think about

controls and validations at an advanced level. Their guidance in this project has really made my thesis

what it is today. Particularly, I would like to thank former member Dr. Herbert “skip” Virgin for taking the

time to help me develop as both a scientist and a professional. He took the time to talk to me about the

importance of demeanor and communication in success. Without his guidance I could have sabotaged

my own career. I would also like to pay particular thanks to Dr. Henry Huang who spent many hours

outside of committee meetings thinking about my project. Henry was always available for a conversation,

and always had invaluable advice. I was lucky to have my committee, who all positively contributed to my

doctoral journey in innumerable ways. I am especially indebted to my former advisor, Dr. Dong Yu, for

seeing the potential in a very unpolished young graduate student like myself and allowing me to join his

research group. Dong set a high standard for quality, and devoted considerable time and energy to

graduate student training. Dong taught me everything I know about writing, public speaking, and critical

thinking. The successful completion of my thesis would not have been possible without his exceptional

mentorship throughout the years. I am also grateful to Dr. Anthony French who did not hesitate to

rescue my career in a time of turmoil. Without Anthony’s compassion and guidance, my doctoral journey

would have been prolonged and made difficult; with the very real possibility of a negative outcome. Thus,

I am especially thankful to Anthony and his research group for accommodating me for the passed year. I

am also appreciative for the Washington University community for providing an excellent environment in

which to do science and receive training. My thesis project would not have been possible without the

expertise of collaborators within the department, or the core facilities supported by the university. Finally I

would like to acknowledge the friends and family that have brought balance to my life outside of science.

Most important on that list is my fiancé Stephanie Yurash. Stephanie has been my biggest supporter for

the last seven years, and sometimes I think she values my dreams and goals more than her own. I am

truly grateful to everyone who has had a positive impact on my life and career.

xi

ABSTRACT OF THE DISSERTATION

Murine Cytomegalovirus Encodes Proteins that Regulate Viral Late Transcription

by

Travis James Chapa

Doctor of Philosophy in Biology and Biomedical Sciences

Molecular Microbiology and Microbial Pathogenesis

Washington University in St. Louis, 2014

Professor Anthony French, Chair

Professor Dong Yu, Co-Chair

Human Cytomegalovirus (HCMV) is a Beta-herpesvirus that causes severe disease in immuno-

compromised individuals (including AIDS patients), and is the leading viral cause of congenital birth

defects. Murine Cytomegalovirus (MCMV) is the primary surrogate model for HCMV, and resembles the

human virus with respect to virion structure, genome organization, gene expression, tissue tropism, and

clinical manifestations. In my graduate studies, I discovered the function of two novel MCMV proteins

during in vitro infection. I demonstrated that ORF M79 encoded protein pM79, and ORF M92 encoded

protein pM92. Both pM79 and pM92 have homologs in HCMV, and I showed that they regulate late viral

gene transcription. During infection, a mutant virus for either M79 or M92 accumulated representative

viral immediate early gene products, early gene products, and viral DNA sufficiently but had severe

reduction in the accumulation of late gene products, thus unable to produce infectious progeny. Analysis

of the viral transcriptome via tiled array and quantitative PCR analysis revealed that many late transcripts

sensitive to a viral DNA synthesis inhibitor (phosphonoacetic acid) were markedly reduced by pM79 or

pM92 mutation. Co-immunoprecipitation and mass spectrometry analysis revealed the interactions

between pM92 and pM79, and suggests that they are part of a larger virus transcription complex. A

colleague demonstrated that this function was conserved in the HCMV homolog pUL79, and I showed

that pM92 expression in trans could complement the growth defect of pUL92 deficient HCMV. My work

was the first evidence of this regulatory complex in MCMV. This complex represents a potential new

xii

target for therapeutic intervention in CMV disease, and a gateway into studying a largely uncharted viral

process that is critical to the viral life cycle.

xiii

Chapter I

Introduction

The final draft of this chapter was written and edited by TJC.

1

Herpesviruses

Herpesviruses are best known for causing lesions that form in the mouth and lips called “cold

sores”. Herpesviruses are also famous for causing chickenpox, and the sexually transmitted infection

Genital Herpes. These three annoying, but relatively mild diseases are caused by herpes simplex virus type

1 (HSV-1), varicella-zoster virus (VZV), and herpes simplex virus type 2 (HSV-2), respectively (57, 174).

The herpesvirus family consists of a group of over 100 viruses that are categorized by their large

double-stranded DNA genomes (~130-250 kbp) and the common architecture of their infectious particles.

Herpesviruses are further grouped into three subfamilies: alpha, beta, and gamma. These subfamilies are

based on biological properties such as growth characteristics, cell tropism, and gene conservation. Though

members of the herpesvirus family can infect a large variety of animal species including mammals, birds,

reptiles, fish, and oysters, there are nine herpesviruses that infect humans (242). These nine human viruses

span all three herpesvirus subfamilies. In the alpha subfamily the human viruses are the rapidly replicating,

neurotropic HSV-1, HSV-2, and VZV. In the beta subfamily the human viruses are human cytomegalovirus

(HCMV), human herpes virus 6a and b (HHV-6a and HHV-6b), and HHV-7, which are slow replicating

viruses with a broad cell tropism. In the gamma subfamily the human viruses are lymphotropic and include

Epstein-Barr virus (EBV) and Kaposi’s sarcoma-associated herpesvirus (KSHV), which are associated with

Burkitt’s lymphoma and Kaposi sarcoma, respectively (50, 241).

One of the hallmark characteristics of herpesviruses is their ability to establish latent infections (13,

106, 150, 151, 216, 260, 303, 324). After an initial infection, viral DNA is harbored in a state of latency with

minimal to no transcriptional activity. Each subfamily has a different location for latency;

alphaherpesviruses are latent in trigeminal ganglia, betaherpesviruses are latent in progenitor cells of the

bone marrow and monocytes of the blood, and gammaherpesviruses are latent in lymphocytes. Individuals

infected with latent virus can remain without symptoms for months or years, or even their entire life. This is

especially true for those persons that do not suffer from any long term immuno-suppression. However,

reactivation of latent virus, where the latent viral DNA becomes transcriptionally active, can lead to

recurrent disease (ex. recurring genital herpes or shingles). People who are immuno-compromised are at a

higher risk for suffering reactivation and more severe diseases (ex. retinitis or sarcoma) (64, 256). The

mechanisms by which herpesviruses establish latent infections and then reactivate are not fully understood,

2

but are under intense study (218).

The large number of herpesviruses and their complexity make it unwieldy to consider more than

one virus in any detail in this work. Though there will be references to other herpesviruses throughout the

remaining text, this work will mainly concentrate on a lineage of the betaherpesvirus subfamily,

cytomegaloviruses.

Betaherpesviruses

Viruses in the betaherpesvirus subfamily are distinct from those in the alpha or gamma subfamilies

because they replicate more slowly and are highly species specific for infection, not being able to cause

infection beyond one specific host species. There are two major lineages in the betaherpesvirus subfamily,

the cytomegaloviruses and the roseoloviruses. The best characterized members of these lineages are

human cytomegalovirus (HCMV) and human herpes virus 6A (HHV-6A), respectively. Though the genomes

of roseoloviruses are smaller (159-170 kbp) than those of cytomegaloviruses (248 kbp), the structure of the

genomes and the functions of many of the encoded genes are conserved between the two lineages. There

are 43 core genes that are thought to be inherited from the common herpesvirus ancestor that are encoded

by most herpesviruses. HCMV and HHV-6 contain 40 and 41, respectively, of the 43 core genes. HCMV

and HHV-6 lack homologues to two proteins that are well characterized in alphaherpesviruses, thymidine

kinase and ribonucleotide reductase. However, there are four genes that are common to HCMV and HHV-6

that have homologues in the gammaherpesvirus subfamily but not the alphaherpesvirus subfamily (HCMV

designation: UL49, UL79, UL87, and UL92) (16, 51, 52, 70, 135, 317, 321). This is not surprising as it has

been shown that beta- and gammaherpesviruses are more similar to one another than either subfamily is

similar to alphaherpesviruses, especially with regard to transcription (7, 52). In addition, there are 27 genes

that are specific to the betaherpesvirus subfamily; 22 of which are conserved by sequence between HCMV

and HHV-6. These include genes involved in tropism and inhibiting apoptosis, something that is important

for viruses with long replication cycles (206).

The thesis which is the subject of this work focuses on the cytomegalovirus lineage of the

betaherpesvirus subfamily. Thus, it is important to describe this genus in further detail.

3

Cytomegalovirus

Cytomegaloviruses are present in a wide range of mammalian species, including primates and

rodents (242). Though chimpanzee cytomegalovirus has the largest genome of all sequenced

herpesviruses, it is human cytomegalovirus (HCMV) that has the largest genome of human viruses. HCMV

is the best studied cytomegalovirus, and the prototype of the betaherpesvirus subfamily for studies of the

virus life cycle, especially gene expression and regulation. However, studies have been prolonged because

lab adapted strains of HCMV (including AD169) have been found to contain mutations that have important

functional consequences (6, 48, 53, 71, 80, 267). For example, the AD169 strain has mutations that limit the

cell tropism of the virus. Wild-type HCMV replicates in macrophages, dendritic cells, colonic and retinal

pigmented epithelial cells, endothelial cells, fibroblasts, smooth muscle cells, neuronal cells, glial cells,

hepatocytes, and trophoblasts (89, 90, 115, 124, 163, 182, 232, 254, 261, 262, 264, 265). In contrast, the

lab adapted strain AD169 preferentially infects and replicates in fibroblasts. Despite the differences

between wild-type and lab adapted strains of HCMV, a lot of progress has been made in understanding the

structure and viral life cycle of betaherpesviruses.

Pathogenesis

HCMV is a widespread pathogen that infects a majority of the world's population by early

adulthood. In fact, by the age of 40, between 50% and 85% of adults are infected by HCMV as indicated by

the presence of antibodies in much of the general population (82, 208, 257). Seroprevalence is

age-dependent, as 58.9% of individuals aged 6 and older are seropositive for HCMV while 90.8% of

individuals aged 80 and older are seropositive for HCMV (280). HCMV infection is typically asymptomatic in

healthy individuals but can cause life-threatening disease for the immuno-compromised, such as

HIV-infected persons, organ transplant recipients, or new born infants (36, 64, 82, 94, 96, 256). For

immuno-compromised individuals, symptoms consist of spiking fever, leucopenia (decrease in white blood

cells), malaise, hepatitis, pneumonia, gastrointestinal disease and/or retinitis (inflammation of the retina)

(270). HCMV is also responsible for approximately 8% of infectious mononucleosis cases (105).

HCMV represents the most significant viral cause of congenital birth defects in industrialized countries, and

was estimated in the 1990’s to cost the United States health care system ~$2 billion annually (8, 17, 105).

4

The birth defects can range from deafness and mental retardation to fatal diseases that include

encephalitis, pneumonia, hepatitis, and hydrops fetalis (a condition in the fetus that leads to heart failure)

(147, 248). HCMV has been implicated in playing a role in inflammatory and proliferative diseases,

including atherosclerosis and cancers (184, 205, 259, 270). The severity of medical problems associated

with HCMV in these vulnerable populations underlies the necessity for developing safer and more effective

antiviral treatments and vaccine strategies to control its infection.

Antivirals. There are no vaccines for any of the betaherpesviruses, and the available antiviral therapies

have limited efficacy and high rates of adverse effects. The clinical use of drugs to treat viral diseases in

humans is difficult due to the fact that viruses replicate within the host cells, using many elements of the

host cell machinery. Therefore, there are fewer obvious targets for chemotherapy against viruses than for

bacterial or parasitic pathogens. A better understanding of betaherpesvirus replication and pathogenesis is

needed to delineate the functions of proteins involved in key viral processes. Ideally drugs would be

designed to target very early steps in infection such as attachment and entry, or very late steps in infection

like release. Inhibitors of early and late steps of infection do not have to enter the cell to exert their effect,

and therefore may be less toxic. Unfortunately, no drugs currently exist for such steps of the herpesvirus life

cycle.

The first antiviral drugs developed, and the most effective drugs for herpesviruses to date, are the

nucleoside analogues acyclovir and ganciclovir (225, 302). Nucleoside analogues mimic the natural

substrate of the viral DNA polymerase and act as competitive inhibitors to inhibit viral DNA synthesis.

Acyclovir is primarily used in alphaherpesvirus infections, while ganciclovir has been shown to inhibit CMV

replication, prevent CMV disease in AIDS patients and transplant recipients, and reduce the severity of

some CMV syndromes, such as retinitis and gastrointestinal disease (225, 302). Ganciclovir treatment is

made more effective when combined with immunoglobulin treatment, which reduces mortality for bone

marrow transplant patients with HCMV caused pneumonia; but long-term survival of these patients is still

very poor (56). Another drug approved for treating HCMV disease is foscarnet. Similar to ganciclovir,

foscarnet inhibits the HCMV DNA polymerase (22, 301). However, foscarnet’s toxic effects are primarily

renal and thought to be less severe than ganciclovir which can inhibit bone marrow function. The most

5

recently approved drug is the nucleotide analog cidofovir, which was approved for therapy of retinitis (22,

159). The side effects for cidofovir are nephrotoxic. Toxic effects aside, all herpesvirus drugs are proving

ineffective due to the emergence of resistant virus (22, 95, 201).

Viral life cycle steps such as genome replication are great targets for antivirals because the process

requires specific viral enzymes. Enzymes make good drug targets because they are usually at low

concentrations inside cells, they can be well-understood mechanistically, and they normally interact with

small-molecule substrates. Unfortunately, targeting one enzyme, like DNA polymerase, allows for escape

mutations that confer resistance to antivirals (as mentioned above). Thus, new drug targets are always

being sought after. One promising target is the cleavage of the viral genome and its packing into capsids,

which is mediated by the viral terminase. This virus-specific enzymatic process appears to be especially

vulnerable to antiviral inhibitors, and consequently, several substances have been described that interfere

with the function of the terminase (43, 123, 195, 295). The drug AIC246 is in phase 2 clinical trials (102,

170).

An effective vaccine for HCMV has still not been found. As a member of the TORCH complex

(several vertically transmitted infections including Toxoplasmosis, Other infections, Rubella,

Cytomegalovirus, and Herpes Simplex virus 2), cytomegalovirus is a priority for vaccine development by the

National Vaccine Program Office in the United States (17). The last vaccine candidate in 2009 had an

efficacy of 50%, thus the protection was limited and many subjects contracted HCMV infection despite the

vaccination (224). A better understanding of betaherpesvirus replication and pathogenesis is needed for

developing novel strategies to prevent disease to this important pathogen.

Virus structure

Like all herpesviruses, HCMV has a highly ordered icosahedral-shape nucleocapsid of about 130

nm in diameter, which encases the viral DNA genome. The capsid is enclosed in a polymorphic lipid bilayer

envelope containing multiple viral glycoproteins that are responsible for viral attachment and entry into host

cells. Unlike most enveloped viruses, herpesviruses have a large space between the capsid and the

envelope called the tegument. The tegument is a partially ordered proteinaceous layer that contains about

30 virus-coded proteins (138, 291). Tegument proteins are introduced into the cell upon viral entry and carry

6

out crucial functions during the early stages of HCMV infection. The following sections will describe each of

the viral structural elements in detail.

Envelope. The viral envelope is a lipid bilayer that contains both viral glycoproteins, and host membrane

proteins. Herpesvirus envelopment is believed to take place initially at the inner nuclear membrane, and

then further proceeds to acquire membranes from endosomes as well as the Golgi network (85, 100, 275).

Thus, the viral envelope contains lipid components that are associated with both the nuclear membrane and

the cytoplasmic membrane system. Whether these lipid components are important in maintaining the

integrity of the viral envelope has not been determined and their functional role in stabilizing virion structure

is unknown. A few examples of host proteins associated with the HCMV envelope include

β2-microglobumin, CD55 and CD59, and annexin II (107, 109, 194, 319). It’s thought that these molecules

may participate in the induction of host cellular responses and/or play a role in modulating virus attachment

(24, 59, 330).

In addition to host factors, viral envelopes contain virus-coded glycoproteins. Herpesviruses

encode a set of 20-80 glycoproteins, very few of which are conserved between all the Herpesviruses (203,

241, 242). HCMV encodes 57 putative glycoproteins, which is far more than other herpesviruses. The exact

organization of the glycoproteins in the envelope is not completely understood. It is known that some of the

glycoproteins are found to aggregate into three complexes: homodimers of glycoprotein B (gB; encoded by

UL55); heterodimer of glycoprotein M (gM; UL100) and glycoprotein N (gN; UL73); and a heterotrimer of

glycoprotein H (gH; UL75), glycoprotein L (gL; UL115), and glycoprotein O (gO; UL74) (121, 122, 180, 238).

gB is a type I integral membrane protein, which mediates a two step cell attachment starting with binding to

heparin sulfate proteoglycans, followed by a stabilizing interaction with another unknown receptor (32, 63).

Little is known about the function of gM (a type III membrane protein) or gN (a type I membrane protein), but

the gM/gN complex is the most abundant component of the viral envelope, and is essential for virus

replication (180, 181, 298). The gH/gL/gO heterotrimer is involved in mediating fusion of viral and host cell

membranes (121, 141, 274). The function of these glycoprotein complexes will be detailed in a later section

(the “Entry” subsection of the “Life Cycle” section).

7

Capsid. Cryo-microscopy and image reconstruction has been used to determine the structure of the HCMV

capsid (65, 66, 327). The architecture of the herpesvirus capsid consists of a hexamer-pentamer cluster

arranged in a icosadeltahedral lattice (44). The major structural subunit of the capsid is the caposomere, of

which there are two types in the CMV capsid. The penton caposomeres form the 12 vertices of the

icosadeltahedral, and the hexon caposomeres form the 150 subunits of the icosadeltahedral faces. The

CMV capsid consists of four structural proteins: the major capsid protein (MCP; encoded by UL86), the

minor capsid protein (mCP; UL85), the minor capsid protein-binding protein (mC-BP; UL46) and the

smallest capsid protein (SCP; UL48A) (99-101, 127, 255). The penton and hexon caposomeres are

composed of five or six copies of the MCP, respectively. The hexon caposomeres are further decorated

with six copies of SCP (326). The caposomeres are linked together by mCP and mC-BP triplexes that

stabilize the capsid structure (66, 215, 293).

Tegument. The tegument of herpesviruses occupies the space between the capsid and the envelope (138,

327). Since the capsid is ~125nm in diameter and the entire virion is ~220nm in diameter, the tegument

represents a significant part of the virion space; containing approximately 40% of the herpesvirus virion

protein mass (100). Many tegument proteins play roles in the early stages of virus infection, which is why

the virus has evolved to package them within the virion for infection of a new host (208). Mass spectrometry

analyses have been done to determine the protein content of the tegument. At least 30 virus-encoded

proteins have been found in the HCMV tegument (100, 203). The five most abundant proteins are: the lower

matrix protein pp65 (UL83); the basic phosphoprotein pp150 (encoded by UL32); the upper matrix protein

pp71 (UL82); the membrane-associated myristylated protein pp28 (UL99); the high molecular weight

tegument protein UL48, and its binding partner UL47 (19, 100, 203, 251).

pp65 is the most abundant tegument protein and accounts for more than 15% of the virion protein

mass (100). pp65 has been reported to inhibit presentation of viral proteins by the major histocompatibility

complex class I, and may inhibit the induction of host interferon responses (38). Furthermore, pp65 forms a

large portion of the protein mass of the noninfectious B capsids and dense bodies (45% and 90%,

respectively), which may be required to help the replicating virus evade immune surveillance (38, 100, 144).

pp150 and UL48 are both essential for viral replication, and have been proposed to interact intimately with

8

nucleocapsids (83, 200) . Blocking pp150 causes defects in tegumentation, and results in the accumulation

of nucleocapsids around the centrosome of host cells (200). pp71 is also believed to be involved in direct

interaction with the newly synthesized nucleocapsid, and is important for initiation of tegument assembly

(292). Moreover, pp71 protein is a transcriptional activator that helps to induce the expression of the

immediate-Early genes within the infected cells (172). pp28 has a wide array of functions, but is primarily

responsible for the cytoplasmic envelopment of tegument proteins and capsids in HCMV during the

assembly and egress process (138, 258). The function of UL47 is still unclear.

The requirement to incorporate specific proteins suggests that tegument morphogenesis may

involve specific interactions between individual components, leading to the formation of a structure that is at

least partially ordered. This hypothesis is supported by the fact that HCMV infections in cell culture give rise

to the production of many particles composed of enveloped tegument lacking a capsid (dense bodies),

suggesting that the tegument can spontaneously self-assemble (125). Cryo electron tomography has

shown that the HCMV tegument can be divided into two sub-compartments: an inner and an

outer tegument. The inner tegument consists of densely-packed proteins surrounding the capsid and the

outer tegument contains components that are loosely packed between the inner tegument and the

envelope (327). Moreover, immune-gold labeling experiments on thin sections of virus-infected cells can

reproducibly locate tegument proteins to the outer or inner regions of the tegument (160, 161).

Genome. All herpesviruses contain a double-stranded linear DNA genome, which is between 130 and 250

kbp in length depending on the virus species. The size of the HCMV genome is 230kb, which is 51% longer

than the HSV-1 genome, and as a result the HCMV capsid is 117% larger than HSV-1 (71, 193). Original

estimates of the HCMV genome coding capacity was 165 open reading frames (ORFs). However, it has

since been shown that HCMV may encode as many as 751 ORFs, making it one of the most complex

pathogens to infect humans (285).

HCMV, and all members of the betaherpesvirus subfamily, have a genome consisting of two unique

regions flanked by direct repeats. For cytomegaloviruses, the two unique regions are designated unique

long (UL) and unique short (US) (Fig. 1) (208). There are two sets of repeats in the HCMV genome; the

terminal direct repeats of approximately 300–600 bp, and internal repeats of the same length, which are a

9

duplication of the terminal repeats in an inverted orientation at the junction between the UL and Us

segments (272, 273, 286). Due to these repeat sequences, recombination takes place during viral DNA

replication to generate four isomers of the genome. Each isomer accounts for ¼ of the DNA packaged into

virions, and each isomer has a different relative orientation of the UL and US segments. Interestingly, DNA

of any one of the isomers is infectious, and limiting the ability to undergo recombination by removing the

repeat sequences also results in infectious virus (112, 253). Gene duplication has been employed widely by

large eukaryotic DNA viruses and their hosts as a means of generating diversity (236). There are 13 gene

families in HCMV that are presumed to have arisen by gene duplication (Table 1) (70).

The genomes of other cytomegaloviruses show variations on the theme of interspersed unique and

repeated DNA sequences. MCMV and RCMV have small terminal direct repeats of 30 and 504 bp,

respectively, but no internal repeat sequences (187, 299). Higher primates such as CCMV, have two unique

regions (UL and US) which are flanked by direct repeats (similar to HCMV) (71, 308). The biological reason

for why cytomegaloviruses maintain their genome structure remains unknown.

Life cycle

All viruses must deliver their genomes to host cells to initiate infection. For enveloped viruses like

HCMV, entry requires the use of virion envelope proteins to facilitate adherence to the cell surface and

fusion between the virus envelope and the cellular membrane. For HCMV, fusion occurs both at the cell

surface and in endosomes. Regardless of which entry location is used, fusion results in the deposition of

virion components into the cytoplasm that facilitate transport of the capsid to the nucleus. The viral genome

is then delivered to the nucleus through a nuclear pore in a process known as uncoating. The genome is

then used for viral transcription, which ultimately produces the proteins responsible for inhibiting the

immune response and high jacking the cellular machinery required for viral genome replication. At late

times of infection, proteins involved in capsid assembly and subsequent packaging of the viral genome are

expressed. Once assembled, the capsids travel from the nucleus to the Golgi—acquiring the viral tegument

and envelope in the process—and then are released from the cell (208). This entire process will be

described in detail in the following sub-sections (Fig. 2).

10

Tropism. To begin a discussion of the virus life cycle at the cellular level, one must first consider the basis

of cellular tropism since receptors involved in entry are expressed on permissive cells. In the human host,

HCMV causes systemic infection and exhibits a tropism for fibroblasts, endothelial cells, epithelial cells,

monocytes/macrophages, smooth muscle cells, stromal cells, neuronal cells, neutrophils, and hepatocytes

(213, 263). This exceptionally broad cellular tropism in the infected host is why HCMV disease manifests in

a variety of organs and tissue types in the host. However, in vitro, HCMV has a restricted cell tropism.

Though entry into many target cells is possible, productive HCMV infection in vitro is only supported by

primary fibroblast, endothelial cells, and certain differentiated myeloid cells (61, 124, 220). The exact

cellular receptors required for entry is unknown, and thus the reason for restricted tropism in vitro is still a

mystery.

Entry. HCMV initially binds to heparin sulfate proteoglycans at the host cell surface via gB to initiate entry

(61). HCMV engagement of heparin sulfate proteoglycans is thought to enhance binding of subsequent

receptors on the cell surface that ultimately lead to fusion in a multi-step cascade (45, 60, 139). Studies for

finding the exact HCMV receptor are complicated by the fact that HCMV has such a broad cell tropism and

that no group of cellular receptors have been shown to be necessary on all infectable cell types.

β-microglobulin, major histocompatibility complex class I molecules, annexin II, aminopeptidase N (CD13),

and epidermal growth factor have been suggested to be virus receptors, but all have since been shown to

be nonessential for virus infection and not present on all HCMV susceptible cells (20, 21, 40, 87, 107-109,

194, 220, 230, 233, 279, 290, 305, 319). Regardless of what receptor is used, receptor binding triggers

fusion of the viral envelope with the cellular membrane. This requires the conserved glycoprotein complex

gB/gH/gL. In this complex, gH appears to serve as the actual fusion glycoprotein (276, 297, 329).

Membrane fusion remains a poorly understood component of entry for any of the herpesviruses

because of the absence of a direct fusion assay. Any role that the gB/gH/gL complex has in fusion is only

inferred. To that end, entry of HCMV can be blocked by gB and gH dependent neutralizing antibodies at a

post-attachment stage of entry, which could be at the level of fusion (27, 35, 142, 294, 296). Integrins have

also been implicated in mediating HCMV fusion (88, 304). Integrins are a ubiquitous cell surface receptor

that can cause cytoskeletal reorganization. Analysis of the effects of various integrin blocking antibodies

11

show that integrins function at a post-attachment stage of infection, and are involved in delivering tegument

protein pp65 into infected cells (88). More work needs to be done to confirm that integrins are involved in

fusion. Development of a reliable fusion assay is required to confirm what factors are truly involved in

HCMV fusion.

After HCMV fusion with host cells the viral capsid is released into the cytoplasm. The tegument

proteins help to transport the capsid to nuclear pores, presumably by dynein motors along the microtubular

network, and then the DNA is released and enters the nucleus. Empty capsids remain at the cytoplasmic

side of the nuclear pores for several hours until they disintegrate (227). Viral DNA in the nuclease is

circularized, either by direct ligation of the ends or by recombination between the repeat sequences at the

terminal ends of the genome (208). The circularized DNA is localized near nuclear structures known as

nuclear domain 10 (ND10), where early transcription takes place (5, 93)

Transcription. Herpesvirus gene expression can be classified into at least four groups based on the time of

their expression during the viral replication cycle. The Immediate-Early (IE) or alpha (α) genes are

transcribed during the first several hours after infection; 0-2 hours in the case of HCMV. IE gene products

are required to combat innate immune defenses, and to make the cell suitable for viral genome replication.

Additionally, IE gene products function to set up a regulatory cascade that leads to the properly timed

transactivation of Early or beta (β) genes. Early genes are expressed between 4 and 8 hours post infection

(hpi) for HCMV. The Early gene products are mainly involved in viral genome replication. It’s only after

genome replication is occurring that the expression of Late or gamma (γ) genes occurs. The majority of viral

genes and all lytic genes are Late genes (~51% of the gene products in HCMV) (49, 51, 158, 185). There

are two classes of Late genes, designated γ1 and γ2. Genes in the γ1 group begin to be transcribed at low

levels before DNA replication, but their transcription is stimulated several fold after the onset of viral DNA

replication. For this reason, the genes in the γ1 class are also called Delayed Early or Early-Late genes.

Expression of γ2 genes begins only after DNA replication has initiated, and these genes are often called

True Late genes. Gene expression will be discussed in extensive detail in a later section (“Gene

Expression” section).

12

Genome replication. Genome replication of herpesviruses occurs in the nucleus of infected cells. The

process takes place in virally induced structures called replication compartments, where viral proteins

involved in DNA replication accumulate (314). These replication compartments form at the site of the

cellular ND10 nuclear bodies where the initial circular viral genome localizes after release from the

incoming capsid. Studies have shown that a cell can only support ~7 replication compartments during

infection (146). Since each replication compartment is derived from a single genome, the virus bottleneck

has been defined as seven entering particles which will have their genome expressed in an infected cell

(145). At late times of infection viral DNA levels can equal cellular DNA content, which implies that each

virus genome is replicated to high levels.

Herpesviruses initiate lytic DNA replication at defined sites on the viral genome called origin of

DNA/lytic replication (ori-lyt). Some herpesviruses have a single ori-lyt, such as in the betaherpesviruses

(including HCMV), while others have either two or three, as in the case of EBV or HSV-1, respectively (242).

The reason for multiple origins of lytic replication in the biology of these viruses remains unclear. After

initiation from the ori-lyt, herpesvirus DNA replication proceeds through either of two potential mechanisms:

1) genome circularization and theta form replication, which proceeds to a rolling circle (like bacteriophage

lambda), or 2) producing complicated branch structures from linear genomes that are then resolved by

homologous recombination and by viral cleavage machinery (25, 26, 132, 169, 219, 314). Either

mechanism of synthesis results in genome concatamers that are cleaved and packaged into newly

synthesized capsids.

HCMV encodes a virion-associated transcript that associates with the ori-lyt to form a

three-stranded structure, which results in targeted unwinding to enable the assembly of a replication fork

complex (235). HCMV encodes a core set of six conserved DNA synthesis enzymes that direct the

synthesis of viral DNA during lytic infection: DNA polymerase (encoded by UL54), the processivity factor

(UL44), a single stranded DNA binding protein (UL57), and a heterotrimeric helicase-primase complex

(UL70, UL102, UL105). Following ori-lyt engagement (which is still not fully understood), an interaction

involving UL57 leads to localized unwinding of the DNA and recruitment of other viral replication proteins.

The helicase-primase complex has DNA dependent ATPase, helicase, and primase activities. Thus, this

complex unwinds DNA and produces RNA oligonucleotides to aid polymerase elongation. The polymerase

13

enzyme consists of UL54 and processivity factor UL44. UL54 has the main enzymatic activity of

polymerizing, but also contains a 3’-5’ exonuclease activity for proofreading. The UL44 protein increases

the processivity of the polymerase which is essential for DNA replication (26, 313, 314). Cellular enzymes

such as topoisomerases are highly likely to be required for replication; however, a complete understanding

of this process will require a defined cell-free assay.

Capsid assembly. The assembly pathway of the HCMV particle is highly similar across all herpesviruses

species (100, 242). The assembly of the HCMV capsid occurs in the nucleus of infected cells starting with

the assembly of the procapsid, which consists of the capsid shell and the internal scaffolding structure.

Second, the procapsid is filled with viral DNA genome while the scaffolding proteins are simultaneously

removed (227). This results in a major conformation change of the capsid shell, converting the procapsid

into a mature nucleocapsid (214, 326, 328).

In order to package its genome, HCMV requires the terminase complex, which is comprised of viral

proteins UL51, UL56 and UL89 (31). The terminase interacts with both the viral DNA and the portal protein

(UL104) to initiate genome encapsidation (76-78, 149). The terminase recognizes specific sequences

termed packaging signals on the viral genomes, docks at the portal vertex of the capsid, and by ATP

hydrolysis provides the energy necessary for genome insertion, followed by cutting of the genome

concatamer after exactly one genome length is packaged. However, the precise mechanism for how

concatameric HCMV DNA is resolved into unit-length genomes that undergo packaging is not completely

understood.

Maturation and envelopment. The HCMV envelope is acquired in the cytoplasm from Golgi membranes

and secretory vesicles. However, the HCMV nucleocapsids is assembled in the nucleus, and must first

escape this compartment before acquire its envelope. Since the nuclear pores are thought to be too small to

allow escape of the HCMV capsid, it is generally believed that HCMV uses the nuclear membrane in an

envelope/de-envelope mechanism to escape from the nucleus (126, 127, 227). In this model, HCMV buds

through the inner nuclear membrane into the lumen between the inner and outer nuclear membranes,

acquiring an envelope and a layer of tegument proteins. The nucleocapsid then loses the nuclear envelope

14

by fusion with the outer nuclear membrane, which releases the nucleocapsids into the cytoplasm with the

acquired tegument proteins. These nucleocapsids subsequently reacquire an envelope by budding into

Golgi membranes located in the viral assembly compartment (9). The viral assembly compartment results

from extensive reorganization of the cellular secretory apparatus (67-69). As a result, the viral assembly

compartment contains many cellular and viral components (including early endosomes, Golgi, viral

structural proteins, and viral tegument proteins) that are necessary for virion maturation. The final step in

egress is fusion of an exocytic vesicle with the plasma membrane, a process that is likely to follow cellular

vesicle trafficking pathways (208).

Latency. A critical component for HCMV persistence in the non-immune compromised host is the ability of

the virus to establish cellular sites of latency. Viral latency can be operationally defined as the maintenance

of the viral genome in the absence of production of infectious virions but with the ability of the viral genome

to reactivate under certain conditions. During latency, the HCMV genome is maintained within the cell with

limited viral gene expression and little to no genome replication. The virus can be reactivated upon cellular

stimulation, including radiation, UV, and other stress responses. Such reactivation from latency is thought

to occur routinely in healthy virus carriers, but is limited by the host immune response. On the other hand,

reactivation of HCMV in an immuno-compromised or immuno-suppressed host background is a

well-established cause of morbidity and mortality (81, 244). Cytomegaloviruses latency occurs in CD34+

progenitor myeloid cells, but stay with the differentiated monocyte-macrophage lineage (152, 266). It is

unclear why other lineages which arise from CD34+ progenitors, such as T and B cells, do not carry viral

genomes or why even some cells of the myeloid lineage itself, such as polymorphonuclear cells, do not

carry viral genomes (288, 289). Interestingly, the number of cells carrying viral genomes during latency is

very small, estimated to be 1 in 104–105 peripheral blood mononuclear cells or bone marrow cells (268).

Within these carrier cells, the viral genomes are maintained as episomes (29). How the episome is

maintained, and why lytic infection does not occur is not completely understood. What is known is that

myeloid cell differentiation is crucial for virus reactivation and that reactivation is mediated by the

expression of IE genes. Analysis of the major IE enhancer promoter (MIEP) suggests that DNA sequences

within the MIEP not only interact with cellular transcription factors which activate the IE promoter but also

15

with cellular transcriptional repressors like chromatin (18, 98, 120, 178, 197, 247, 318). It is now well

established that the regulation of promoter activity of many cellular genes involves regulation at the level of

their chromatin structure, and that the chromatin profile during cell differentiation is such that there are

more active promoters (84, 157, 179, 306). The same correlation between chromatin structure of the MIEP

and virus reactivation has also been observed in models of experimental latency (240). Changes in

chromatin structure of IE gene promoters likely control reactivation as seen with other herpesviruses (11,

15, 134, 156). Though it is clear that the absence of the IE protein is required for latency, however, both

spliced and unspliced RNAs, termed cytomegalovirus latency-specific transcripts (CLTs) (153). Though

some of them are increased or decreased during monocyte differentiation, when latent virus reactivates,

no role for them has been confirmed in latency (103, 310). Understanding the relationships between

HCMV and myeloid cells will be important for a full understanding of how this virus persists in the host and

the complexities of its interaction with the host immune system. A thorough understanding of the molecular

biology and immunology of HCMV persistence in vivo can only help to generate specific strategies that

more effectively control virus reactivation.

Early gene expression

A viral process that was briefly described earlier is gene expression. Since this topic is the focus of

this dissertation, much more elaboration is required. Below you will find details of Immediate Early and Early

gene regulation, but the section will extensively focus on Late gene expression and regulation.

Immediate Early genes. A major function of the IE genes is to set up a regulatory cascade that leads to

properly timed expression of other viral genes. The IE genes are the first viral genes transcribed after

infection, and their transcription does not require de novo viral protein synthesis. These gene products

optimize the cell for viral gene expression and replication. Downstream of the MIEP are two IE genes

designated IE1 and IE2 in HCMV. In MCMV these genes are designated ie1 and ie3, respectively. Multiple

proteins are encoded by these two IE genes through differential mRNA splicing throughout infection (217,

282-284). The resulting viral proteins are designated according to their apparent molecular weight; IE1 =

IE72, IE2 = IE86. The minor products are designated IE38, IE55, and IE18, and have received less study

16

and attention than IE72 and IE86. During the two hours after HCMV infection, the mRNA for the IE genes

are expressed abundantly, but as the protein products are made, the IE72 and IE86 proteins negatively

regulate the MIEP and decrease transcription of IE genes (14, 54, 162, 231, 278). After synthesis in the

cytoplasm, the IE proteins are transported to the nucleus and targeted to ND10 nuclear bodies (mentioned

earlier as the place where viral replication compartments eventually form). IE72 is involved in alleviating the

repressive effects of ND10 on the viral genome presumably by binding to its associated proteins, such as

PML, SP1 00, and hDaxx (3, 4, 166, 168, 315). Once the ND10 repressive effects are alleviated, the basal

transcription initiation complex is activated and viral Early gene transcription can take place (128, 209, 287).

It is unclear how the IE72 protein activates promoters, but an association of IE72 with TATA box

-associated factors (TAFs) and transcription factors (Sp-1 , E2F-1 , CTF-1 ) has been proposed (176, 186).

The mechanism by which the IE72 protein activates promoters may also be related to inhibition of HDAC-2

activity (222, 287). Interestingly, the IE72 gene is only required for efficient HCMV replication at low

multiplicity of Infection (MOI). Deletion of the IE72 encoding gene, IE1, results in reduced virus replication

due to insufficient levels of Early gene products, which results in low efficiency genome replication (97, 104,

204). However, at high MOI viral replication levels of IE1 mutant virus are similar to wild-type virus. This is

likely due to virion-associated proteins present in infectious and non-infectious particles that compensate

for the absence of functional IE72. The same phenotype is true for mutants of the homologue ie1 in MCMV

infections.

On the other hand, the IE2 gene product, IE86, and its functional homologue in MCMV (ie3) are

absolutely essential for virus replication (12, 183). Recombinant viruses with deletion of the HCMV IE2

gene or the MCMV ie3 gene are unable to activate Early viral gene expression, which abrogates viral DNA

synthesis and Late gene expression. IE86 protein is considered a master regulator of productive virus

infection. It regulates activation of transcription from viral and cellular promoters, negatively auto-regulates

the MIEP, and induces cell cycle progression (118, 208, 309). Though IE86 localizes adjacent to ND10

where the viral DNA is located, IE86 does not interact with ND10 (128). Instead IE86 promotes gene

expression by directly activating promoters. IE86 interacts with a wide variety of cellular transcription factors

(TBP, TFIIB, TAF4, and histone acetyl-transferase) to link various transcription regulators to the viral

transcription complex (42, 47, 93, 111, 176, 177). IE86 also regulates gene expression by interacting with

17

regulators of cell cycle progression. Replication of HCMV appears to be best in a cell that progresses to the

G/S transition point, but is prevented from entering the S phase (46, 92, 110, 212, 271, 311, 312).

Though not well studied, minor Immediate Early proteins also have functions important to HCMV

replication. Immediate Early proteins TRS1 and IRS1 were initially recognized as regulatory proteins

working in conjunction with IE1 and IE2 in activation of delayed Early and Late gene expression (129, 281).

The mechanism of their action is thought to be related to the interferon response (23, 55). HCMV genes

UL36 and UL37 are also IE genes that encode cell death suppressors (188-190). These proteins inhibit

apoptosis in infected cells, but are both dispensable for replication.

Early genes. Early gene products constitute a significant proportion of the essential genes for HCMV

replication. Mutagenesis analysis has shown that 23 to 25 essential and augmenting genes expressed with

Early or Early–Late kinetics (49). Most of the viral Early genes function either by participating in genome

replication, or by creating an environment that is conducive for genome replication.

An experiment in which fragments of the HCMV genome were tested for their ability to support

ori-lyt dependent DNA replication identified 11 loci required for viral genome replication (223). Of the genes

encoded in these regions, six were the core set of conserved herpesvirus genes involved in DNA replication

(UL44, UL54, UL57, UL70, UL102, and UL105, mentioned earlier in the “Genome replication” sub-section

of the “Life Cycle” section). Each of these genes is expressed with Early kinetics (49, 269). These loci also

encoded UL112–113 protein products which are important for the formation of viral replication

compartments, and may play a role in the recruitment of additional factors to these sites (5, 131, 226). The

IE86 proteins were also encoded by these loci, along with UL84. The product of the UL84 gene is essential

for viral DNA synthesis and productive infection (83, 252, 322, 325). The UL84 protein interacts with IE86 in

viral replication compartments. Though, the importance of this interaction is not known (171, 277). It is also

thought that UL84 is involved in the formation of replication compartments (322). A protein that is important

for viral DNA replication, but was not identified in the loci complementation experiment, is UL114. The

UL114-encoded uracil-DNA glycosylase is not strictly required for growth in fibroblasts, but a mutant lacking

this gene is delayed in the initiation of DNA replication (62, 234).

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How Early gene expression makes host cells suitable for DNA replication is less clear. Early gene

expression during infection is associated with the stimulation of host cell genes that encode proteins

involved in host DNA synthesis and cell proliferation (ex. thymidine kinase and ornithine decarboxylase)

(28, 39, 58, 86, 116). Along those lines, tumor suppressor protein p53 and host DNA synthesis proteins

PCNA and RPA are sequestered to viral replication compartments (79, 92, 133). Early gene expression is

also required to induce elevated levels of cyclin E and cyclin B and their associated kinase activities (133,

192, 246, 250). In contrast to the activation of cyclins E and B, the expression of cyclin A and its associated

kinase activity is inhibited by Early proteins during infection (133). In this way the virus is able to both

positively and negatively regulate host cell DNA synthesis and cell division (33, 34, 79, 133, 175). The

challenge remains to determine which viral genes are involved and elucidate the mechanisms governing

their activity. Given the large number of Early genes, most of which have not yet been studied, the task is

not trivial.

Aside from their roles in DNA replication, some Early genes play roles at a later stage. Following

synthesis, viral DNA is cleaved into genome-length segments and packaged into preformed capsids. Early

genes UL89 and UL56 are involved in DNA cleavage (43, 155, 295). Four Early proteins, UL51, UL52,

UL77, and UL104, are predicted to be involved in packaging cleaved DNA into progeny capsids. Recently,

it has been shown that the TRS1 protein also may be involved in packaging at a step that occurs after the

cleavage of the DNA (2).

Late gene expression

Our understanding of Late gene regulation in the betaherpesviruses lags far behind that of Early

gene regulation. Late genes code for viral structural proteins, proteins involved in the assembly of capsids

and packaging of viral DNA, and may other proteins whose functions are only beginning to be discovered. It

is known that DNA replication is required for the transcription of Late genes. The γ1 genes can be

transcribed at very low levels in the absence of viral DNA replication, but their expression is dramatically

increased after viral DNA synthesis. In contrast, the transcription of γ2 genes is totally dependent on viral

DNA synthesis, and occurs exclusively after genome replication has begun. The mechanism that links Late

gene activation to DNA replication has not yet been determined. However, some of the theories include:

19

restriction on expression related to DNA structure; DNA modifications that promote expression; the

availability of certain viral or cellular proteins that inhibit repressors or recruit transcription activators; or

inefficient Late gene promoter that requires DNA abundance for efficient transcript accumulation. To that

end, only a handful of HCMV Late gene promoters have been analyzed in detail. One of the primary

reasons for this is that Late gene promoters are utilized with Early gene kinetics when put into an ectopic

plasmid (74, 245). What DNA elements are required to recapitulate the appropriate kinetic class has been a

mystery. However, it has been shown for gammaherpesviruses that Late gene regulation is only restored in

ectopic plasmids if the plasmid contains the Ori-lyt element in cis (10, 73). Recently, a plasmid that contains

the HCMV ori-lyt in cis with the Late promoter has been developed, and transient transfection of this

plasmid does indeed express the reporter gene in the appropriate kinetic class (207). Thus, studies in the

next few years are likely to provide great insight into the importance of the Late promoter sequence

elements in regulation of Late gene expression.

Recent research has also identified a series of cellular and viral factors that are specifically involved

in Late gene regulation during herpesvirus infection. Though the fields of alpha and gammaherpesviruses

are more advanced than that of betaherpesvirus, research, including that done in this dissertation, has

made progress in understanding the protein requirements for Late gene expression (16, 51, 52, 137, 317,

321). To give us a better understanding about how HCMV may be regulating Late gene expression, it is

important to take into consideration what is already known.

Promoter requirements. Typically, Late promoters seem to require a TATA element but no further

upstream sequences for transcriptional activation (75, 148, 196, 316). Similar findings have been observed

for Late gene promoters of herpes simplex virus, where the TATA box and downstream elements are

sufficient to direct Late gene activation (91, 119, 307). This is in contrast to the betaherpesviruses Early

promoters, where specific upstream sequence elements capable of binding to cellular transcription factors

are critical for promoter activation.

The most extensive studies on Late gene regulation have examined the promoter of the UL99 gene

encoding the virion tegument protein pp28 (74, 143, 148). UL99 is expressed as a γ2 gene, and requires

only sequences from -40 relative to the cap site to restrict expression to late times (148). Thus, it was

20

thought that Late gene promoters are relatively simple, consisting primarily of a TATA element and

downstream sequences and lacking a requirement for multiple transcriptional regulatory elements.

However, studies of other HCMV Late gene promoters suggest that regulation can be more complex (75,

196). Analysis of the UL75 promoter, which promotes expression of glycoprotein gH, sequences from −38

to +15 relative to the cap site were sufficient to activate this promoter (196). Closer examination of those

sequences revealed that sequences downstream of the UL75 cap site appeared to function as a dominant

regulatory element, because in the absence of this region, both activation and repressor elements were

identified in the upstream sequences (196). The same type of dominant regulatory element was found

downstream of the UL94 promoter cap site (316). In the absence of this downstream element, deletion of

the upstream sequences enhanced promoter activity, suggesting the presence of an upstream negative

regulatory element. Further analysis of the UL94 promoter identified two p53 binding sites that were

important, but not sufficient for the repressive effects (316). These studies suggest that the regulation of

Late gene expression may actually be more complex than previously estimated. However, it remains to be

determined if these elements truly play a role in Late gene regulation in the context of a normal viral

infection, or if these are artifacts resulting from the transient transfection systems used.

The regulation of Late gene promoters is even more interesting if you consider that a number of

Late promoters are located in regions of IE and Early gene expression (164, 165, 237). In fact, some Late

transcripts with alternate TATA elements and start sites have been identified within HCMV Early promoters

(136, 164). Thus it might be expected that the association of repressors and activators may be critical for

restricting viral Late gene expression within the promoter context. A system for studying Late promoters in

context of their natural genomic location will be important for elucidating the mechanism behind their

regulation. Intriguingly, a more global analysis of the putative promoter regions of HCMV Late genes

revealed a palindromic GC-rich sequence (CCGCGGGCGCGG) in the promoters of 17% of viral Late

genes (49). The significance of this sequence element in Late gene regulation remains to be determined.

Regulatory cellular proteins. Some efforts have been made to identify cellular regulatory proteins that are

involved in the activation of HCMV Late promoters. Analysis of the UL75 promoter indicated an activation

role for a cellular PEA3-related protein (196). Analysis of the UL94 promoter indicated a role for p53 in

21

transcriptional repression (316). Studies also suggest that the NF-κB transcription factor may also be

involved in activating viral Late gene expression (72). As mentioned earlier, there are sequence elements

that have been identified as having activating or repressive effects on Late gene promoters, but the specific

proteins that are required for that domain are unknown (74, 75). In addition to cellular proteins, there is

evidence for the role of viral factors in the activation of Late promoters.

The role of IE genes. It has been shown that the HCMV immediate–Early proteins can activate some Late

promoters, although to minimal extents (74, 75, 243, 249, 281, 309, 320). Deletion of specific sequences of

the IE2 gene results in reduced accumulation of the Late proteins pp65, pp28, and UL83, despite DNA

replication being minimally affected (249, 309). Even more interesting is that certain sequence deletions in

IE2 had a completely different effect. Certain deletions resulted in an IE2 protein that did not activate Early

gene expression, but expressed certain Late genes at early times after infection (309). Sequence analysis

identified a potential IE2-binding site within one of these Late gene promoters, which suggests that IE2 may

be involved in repression of Late gene transcriptional (309). Thus, IE2 appears to play both a positive and

negative regulator of Late gene expression during HCMV infection. The mystery still remains how it is that a

gene expressed at Immediate Early times of infection can be involved in Late gene regulation without

inducing Late gene expression at earlier times post infection. Though many possibilities exist, the front

running theories are that the IE2 protein is modified at late times of infection (phosphorylation and/or

sumoylation), or that Late promoters are epigenetically regulated in such a way that they are not accessible

until late times of infection (113, 114, 117, 167).

Late gene transactivators. Recent studies have identified multiple HCMV genes that appear to

specifically act as Late gene transactivators. The first genes of this nature were discovered in

gammaherpesviruses. ORF18, ORF24, ORF30, ORF31, and ORF34 of gammaherpesvirus MHV68

encode proteins that are required for Late gene expression, but have no impact on DNA replication (16,

135, 317, 321). Homologues for these genes exist in HCMV and MCMV. To this end, UL79 (homologue of

ORF18) and UL92 (homologue of ORF31) have been shown to be required for HCMV Late gene

expression, but dispensable for viral DNA replication (16, 130, 135, 221, 229). However, the mechanism for

22

how these proteins regulate Late gene expression during HCMV replication is unknown. Identifying

additional viral regulatory factors in Late gene expression will be critical to understand how these proteins

function. The MCMV homologues M79 and M92, which are the focus of this thesis, remained

uncharacterized.

Direct role for DNA replication. There has only been one study addressing the direct role of DNA

replication in stimulating Late gene expression. A vector containing both the Late gene TRL7 and the ori-lyt

in cis were transfected into mammalian cells and infected with HCMV (300). The ori-lyt prevented the

plasmid from replicating outside of infection. What was observed is that the ability of the plasmid to replicate

during HCMV infection increased the TRL7 promoter-directed transcript to levels higher than could be

explained by plasmid replication alone. Furthermore, this increase in transcription was not dependent on

methylation or specific upstream promoter elements. Therefore it is possible that factors required for DNA

replication may directly be involved in the transcription of some Late genes as well, with replication

providing a mechanism for bringing such factors into proximity of the promoter.

Post-transcriptional regulation. In addition to transcriptional regulation, viral genes are regulated at the

post-transcriptional level as well. Analysis of the UL99 5’ untranslated region revealed that these

sequences influenced translational regulation, via a putative stem–loop structure (143). While the exact

mechanism of this regulation has not been determined, it is thought that IE2 is involved (309).

Post-transcriptional regulation for HCMV is in line with what has been observed for the other

betaherpesviruses. Studies in HHV-6 and HHV-7 show that certain splice variants are only expressed at

late times of infection (198, 202). Thus, post-transcriptional Late gene regulation is likely to be a conserved

mechanism in the betaherpesviruses. Furthermore, post-transcriptional mechanisms also play an important

role in the regulation of Late gene expression. During HSV1 infection it has been shown that reduction in

gene expression can occur despite unaltered transcript levels, suggesting that post-transcriptional

regulation may be conserved throughout the herpesvirus family (191, 228).

23

Development of herpesvirus BAC system

The large genome and complicated reverse genetics system for HCMV have made the creation of

mutant viruses difficult. The only way to make mutant viruses was to use homologous recombination in

mammalian cells, which was particularly difficult for HCMV due to the large genome size and

slow-replication kinetics. Recently, the herpesvirus field has seen a revolution in the genetic system for

large DNA viruses. A system was created whereby herpesvirus genomes are maintained as infectious

bacterial artificial chromosomes (BACs) within Escherichia coli (30). This system allows researchers to do

genetic manipulations of herpesviruses by using the well established bacterial genetic techniques. The

creation of BACs required that virus genomes were inserted into F-plasmids, flanked by loxP sites. The

F-plasmid maintained the genome in host cells, and allowed for reconstitution of infectious viral particles.

Since the F-plasmid also encoded an intron-containing Cre recombinase, during viral reconstitution in

mammalian cells, the F-plasmid was excised from the viral genome to prevent expression or packaging

problems downstream. Due to the presence of an intron, the Cre is not expressed in E. coli cells, preventing

any problems that Cre might cause during genetic modification of the BAC. This development has greatly

increased the rate of making individual ORFs mutations in herpesvirus genomes. This method created a

system where viral recombinants could be created in the absence of viral growth, thus one can create a viral

mutant independently of its viral fitness. Furthermore, the viral genome could be characterized before

reconstitution of viral progeny, reducing the likelihood of spurious point mutations or deletions occurring in

the process of producing recombinant virus.

In HCMV, several strains have been cloned as BACs including the laboratory strains Towne and

AD169, and several clinical isolates including TR, TB40, and Merlin. The ability to create recombinant

clinical BACs will be critical to the future study of HCMV, as the laboratory strains contain large deletions

and many point mutations and thus does not behave precisely like virus acquired directly from patients. It

should also be noted that a BAC has been created for the MCMV smith strain. This will be relevant to the

studies of this dissertation.

24

Functional profiling in HCMV

Not long after the advent of the BAC, several groups set out to create a complete library of

ORF/gene mutations in HCMV and screen these mutants for their growth in fibroblasts. The functions of

many of the 166 HCMV ORFs remains unknown, and thus these mutant libraries represented a significant

step in the ability to understand the biology of this virus at the molecular level. Complete mutant libraries

were created in both the Towne and AD169 strains (17, 143). These studies identified whether each gene of

these HCMV strains was essential (no growth of mutant virus), nonessential (mutant virus grows like

wild-type virus), or augmenting (mutant virus has >10 fold growth defect) for growth on fibroblasts. The

conclusions of these two studies largely agreed with each other; and occasional differences have been

attributed to the location of the mutation (i.e. UL35) (173). In libraries of HCMV BACs constructed to disrupt

each unique ORF 41–45 of the ORFs examined appear essential for replication in fibroblasts, 117 are not

required for viral replication in fibroblasts (83, 325). Of the non-essential genes, deletion in 88 of them had

growth kinetics identical to wild type, and thus were truly non-essential, and 27 when deleted, give rise to a

severe growth defect, and were designated augmenting. Interestingly, some of these dispensable ORFs

(UL24, UL64, and US29) are required for viral growth in cell types other than fibroblasts, such as endothelial

or epithelial cells (83, 325). In fact some of these nonessential genes have turned out to be tropism factors

for these cell types. In addition, four of the mutants with non-essential genes deleted (UL10, UL16, US16,

and US19) grow significantly better than the wild type in cell types other than fibroblasts. Furthermore,

although many of the HCMV genes are dispensable for viral growth in cell culture, studies with MCMV or

RhCMV suggest that many dispensable genes are important for modulating the virus–host interaction.

Aim and scope of thesis

Developing effective anti-HCMV therapeutics requires a better understanding of the molecular

mechanisms by which the virus replicates and causes disease within the host. Late gene expression and

regulation is a vital part of the HCMV life cycle. Proteins involved in this process would make for great drug

targets. Due to the lack of a robust genetics system, comprehensive analysis of much of the HCMV genome

has not been achieved. As mentioned above, the advent of the BAC system has allowed functional profiling

of the entire HCMV genome. However, due to species restrictions, the attenuation of lab strains, and

25

difficulty of complementing mutant viruses, understanding the function of novel genes during HCMV

pathogenesis has been difficult. Murine CMV (MCMV) is a useful surrogate model to bridge the knowledge

gap between aspects of HCMV infection and the functions of undefined genes.

MCMV as a surrogate model. MCMV shares 45% sequence identity with HCMV, and is the most

commonly used animal model for the study of CMV induced disease (140). MCMV allows researchers to

investigate tissue tropism, virulence, latency, and reactivation of MCMV in mice with hopes of gaining

insight into HCMV infection in humans. Remarkably, the effects of MCMV infection in mice resemble those

of HCMV in humans with respect to pathogenesis during acute infection, persistent infection and

reactivation from latency after immune-suppression. In addition, the major cell types and organs infected,

the course of infection, and the type of pathology seen are identical for both viruses. There is the notable

exception that MCMV does not cross the placenta to cause congenital defects in newborn pups (154).

Nonetheless, the genetic and pathobiological similarities between these two viruses make the use of

MCMV a sensible model system to study HCMV infection.

Functional profiling. Analysis of the complete nucleotide sequence of MCMV has revealed that the

MCMV genome is collinear with HCMV over the central 180kb and that 78 open reading frames have

significant sequence homology to those of HCMV (53, 71, 210, 211, 239). Characterization of these

conserved genes should provide insight into the functions of their HCMV counterparts in viral infections in

humans.

One of the most powerful approaches to identify the function of virus-encoded genes is to create and

analyze viral mutants. The construction of herpesvirus mutants via site-directed homologous recombination

and transposon-mediated insertional mutagenesis has been reported (41, 199). MCMV mutants can be

generated from the BAC-based viral genome by both of the aforementioned strategies. The BAC-based

mutagenesis approach provides a powerful and convenient strategy to generate viral mutants, facilitating

studies of the functions of viral genes in tissue culture and in animals. Our experimental plan to identify

MCMV protein function is based on the published sequence and ORF predictions of the Smith Strain of

MCMV. There are 170 ORFs predicted for the MCMV genome assuming a minimum 100aa protein size and

26

<60% gene overlap (239). However, most of the MCMV genome coding capacity has not been confirmed

(37).

We set out to mutate a subset of the 78 MCMV genes that have homologues in HCMV, and to

characterize those mutants for their ability to replicate in tissue cultured fibroblasts (239). This would both

expand our capacity to study the functions of the chosen homologous genes, and refine the use of MCMV

as a model for studying HCMV biology and pathogenesis.

Create insertion/deletion mutations in MCMV. With genome wide mutagenesis outside the scope of my

studies, we chose to focus on those genes in MCMV that have homologues in HCMV. A reasonable list of

candidate genes was selected from the 78 conserved genes. For this study, we have decided not to include

the conserved genes that either have been well studied in MCMV, such as M27, or have well-studied

homologues in other human herpesviruses (ex. HCMV or HSV-1), such as core viral proteins like DNA

polymerase or glycoprotein B (1). This process of elimination left us with 28 genes of interest to target

(Table 2).

To inactivate a MCMV gene by insertion/deletion mutation, a kanamycin cassette flanked by FRT

sites was recombined into the gene of interest using the MCMV BAC and linear homologous recombination.

The insertion site was chosen such that it was as N-terminal as possible within the ORF but would not

disrupt an overlapping ORF; often placed 200bp from the terminus of the neighboring ORF. The

homologous recombination in the BAC was achieved by designing primers that had 20bp of homology to

the Kan-containing vector on the 3’ end and 50bp homology to the MCMV gene target sequence on the 5’

end. After electroporation of the PCR product into BAC containing E.coli cells, Kan resistant bacteria were

treated with arabinose. The arabinose treatment induced flippase expression from the bacteria which

caused site specific recombination of the FRT sites. This removed the Kan-cassette and left behind an 88bp

sequence including the FRT site. The remaining sequence caused a frame shift of the ORF and theoretical

disruption of its expression downstream of the insertion (Fig. 3). The success of the insertion was confirmed

by restriction digest, PCR analysis, and sequencing.

27

Analyze the growth of mutant MCMV. After the creation of the mutant MCMV BACs, it was necessary to

determine the effect of the mutation on virus replication. Mutant BACs were transfected into mouse

fibroblasts, and monitored via microscopy for seven days post transfection. Wild-type MCMV BAC and

mutants that did not disrupt viral replication produced virus and lysed the cell monolayer by seven days post

transfection. Mutations that were located in regions that were important for virus replication took longer than

seven days to lyse the monolayer, or in some cases never lysed the monolayer at all. The mutants that did

not lyse the monolayer even out to 15 days post transfection were categorized as “essential” genes.

The genes that were able to lyse the monolayer by 15 days post transfection were further analyzed in a

controlled growth curve analysis. Following reconstitution of the virus and titering of the stock, each virus

was subjected to a multistep growth analysis on cultured fibroblasts to differentiate mutations as

“non-essential” or “augmenting” genes. Each stock of virus was used to infect murine fibroblasts at a

multiplicity of infection (MOI) of 0.01. Growth analysis was carried out over a course of 12 days post

infection. Supernatants were collected every two days and tittered by plaque assay. Mutant viruses that

grow to viral titers comparable to wild-type virus were classified as “non-essential” genes. Mutant viruses

that produced progeny at a reduced level when compared to wild-type virus were categorized as

“augmenting” genes.

Though most viruses were reconstituted to wild-type titer levels during the analysis, seven viruses

emerged with a marked growth defect (Table 2). Mutant viruses with mutations in genes M49, M79, M92,

M94 or M96 were able to express the SV40 driven GFP marker and cause cellular swelling as is

characteristic of MCMV infection, but failed to spread to surrounding cells. Thus these genes were

classified as “essential” for viral growth; which is consistent with the classification of their respective HCMV

homologues. Mutants of M47 and M88 spread from cell to cell, but growth curve analysis showed that the

virus titers were three and two logs lower than wild-type virus, respectively. Thus, M47 and M88 are

augmenting genes, which is also consistent for functional profiling done in HCMV (325). The remaining 15

viruses, for which mutants were created, have demonstrated no defect during transfection or in subsequent

infections into murine fibroblasts (data not shown). Six of the 28 mutant viruses that we set out to create

proved to be difficult to obtain despite multiple attempts. As a result, those genes were excluded from the

goal of the thesis.

28

Complement mutant viruses. In order to study the function of the essential viral proteins in MCMV

infection it is necessary to produce a high titer stock of the mutant virus lacking the targeted gene. The

complementation strategy that was used was to create cells over-expressing the viral gene in trans in order

to complement the growth of a corresponding mutant MCMV BAC. This is a routine approach that is used in

our laboratory with the pRetro-EBNA retroviral vector (323). A pRetro-EBNA retroviral vector containing our

gene of interest was constructed and transfected into the Phoenix packaging cell line to produce retrovirus.

Phoenix cells are derived from 293T cells but express the retroviral gag-pol and envelope proteins for

generating amphotropic retrovirus. The resulting retrovirus was transduced into 10.1 fibroblasts to produce

cells that express the gene of interest. The pRetro-EBNA vector contained a dsRED cassette, which

allowed visual screening by fluorescent microscopy. Cells that expressed the gene of interest would show

up as red under the fluorescent scope. Clonal cell lines could be created by limiting dilution and clonal

expansion of dsRED expressing cells. The over-expressing cell lines were then used to reconstitute the

corresponding mutant viruses from BAC-MCMV transfections.

Two of the three essential genes could be complemented by this system; M79 and M92. Defining

the role of M79 and M92 will set the stage for using the MCMV model to elucidate the mechanism of action

for this CMV gene family, and explore novel antiviral strategies targeting this viral factor. Determining the

molecular mechanism of even one conserved CMV gene would greatly enhance our knowledge of CMV

biology and may also provide fundamental discoveries in cell biology as many of these genes are likely to

modulate critical virus-host cell interactions.

29

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Table 1. HCMV gene families

Family name Genes in the family Details

RL11 RL5A, RL6, RL11, RL12, RL13, UL1, UL4, UL5, UL6, UL7, UL8, UL9, UL10, UL11

Membrane glycoproteins that share a common immunoglobulin domain

UL14 UL14, UL141 Membrane glycoprotein containing an immunoglobulin domain, putatively involved in NK cell evasion.

UL18 UL18, UL142 MHC-1-realted membrane glycoproteins involved in immune evasion

US22 UL23, UL24, UL26, UL28, UL29, UL36, UL43, US22, US23, US24, US26, IRS1, TRS1

Tegument proteins involved in modulating the cellular response

UL25 UL25, UL35 Tegument proteins

GPCR UL33, UL78, US27, US28 Chemokine receptors

DURP UL31, UL72, UL82, UL83, UL84 Tegument proteins derived from dUTPase that have multiple roles in modulating the cellular response

UL120 UL120, UL121, UL119 Membrane glycoproteins

UL146 UL146, UL147 CXC chemokines

US1 US1, US31, US32 potentially regulating both innate and adaptive immunity

US2 US2, US3 Membrane glycoproteins involved in immune evasion

US6 US6, US7, US8, US9, US10, US11 Membrane glycoproteins involved in immune evasion

US12 US12, US13, US14, US15, US16, US17, US18, US19, US20, US21 Transmembrane proteins

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TABLE 2. CMV mutagenesis and phenotype characterization MCMV HCMV Homolog Mutant MCMV Growth

ORF Mutant Created ORF Class Fibroblasts

M024 Yes UL024 NE Nonessential


M026 Yes UL026 A Nonessential





M071 Yes UL071 E Nonessential







M121 Yes UL121-P NE Nonessential

M049 Yes UL049 E Essential M079 Yes UL079 E Essential M092 Yes UL092 E Essential M094 Yes UL094 E Essential M096 Yes UL096 E Essential M047 Yes UL047 A Augmenting

M088 Yes UL088 A Augmenting

M023 No UL023-P NE M034 No UL034 E M083 No UL083 NE M95 No UL95 E M103 No UL103 A M114 No UL114 A

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Figure 1. Human cytomegalovirus genome schematic. Schematic map of the AD169 HCMV genome.

The CMV genome is organized as two regions of unique sequences, unique long (UL) and unique short

(US) (designated by the light shaded boxes), flanked by two sets of inverted repeats (TRL/IRL) (designated

by black boxes) and (IRS/TRS) (designated by white boxes). Arrows indicate the relative orientations of the

repeated and unique genome blocks.

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Figure 2. A representative diagram depicting the strategy for creating recombinant MCMV BAC

clones. An insertion mutation was created by two step recA-mediated homologous recombination. A site

downstream of the translation initiation codon was chosen for insertion of a kanamycin cassette (indicated

by red bar) flanked by two FRT sites. After the induction of flp recombinase, the BAC clone carried an 88-bp

insert (indicated by black bar) downstream of the M92 translation initiation codon, resulting in a frame-shift

mutation.

55

Figure 3. Life cycle of CMV in a host cell. CMV enters host cells (1) either through direct fusion or

through the endocytic pathway. The virus attaches to the cell via interactions between viral glycoproteins

and an unknown cell surface receptor(s), followed by the fusion of the envelope with the cellular membrane

to release the capsid into the cytoplasm. The capsid is then translocated into the nucleus, where viral DNA

is released. This initiates the expression of the IE genes (2). The IE genes make the environment suitable

for viral DNA replication, and then transactivate the Early genes. Early genes facilitate viral DNA replication

(3). Once viral DNA replication is occurring, the Late gene transcription program is initiated (4). Viral Late

genes encode proteins involved in DNA packaging and capsid assembly. Once the nucleocapsid is

assembled, it leaves the nucleus and acquires the virion envelope in the cytoplasm (5). Secondary

envelopment occurs in the cytoplasm at the endoplasmic reticulum (ER)-Golgi intermediate compartment.

This is followed by a complex two-stage final envelopment and egress process that leads to virion release

by exocytosis at the plasma membrane.

56

Chapter II

Murine Cytomegalovirus Protein pM79 Is a Key Regulator of Viral Late Transcription

57

Preface

TJC designed and performed the majority of experiments, analyzed the data, generated the figures and

wrote the first draft of the manuscript. CA assisted with the preparation of the RNA samples for the tiled

array analysis, LSJ and MCV performed the computational analysis of the tiled array data, WMY provided

key reagents and insights for the tiled array analysis, ARF and DY supervised the studies and greatly

contributed to the design and analysis of the experiments, TJC, DY, and WMY greatly contributed to the

writing and editing of the manuscript.

This chapter has been previously published:

T. J. Chapa, L. S. Johnson, C. Affolter, M. C. Valentine, A. R. Fehr, W. M. Yokoyama, and D. Yu. Murine

cytomegalovirus protein pM79 is a key regulator for viral late transcription. J Virol 87:9135-47.

58

Summary

Herpesvirus genes are temporally expressed during permissive infections, but how their

expression is regulated at late times is poorly understood. Previous studies indicate that the human

cytomegalovirus (CMV) gene, UL79, is required for late gene expression. However, the mechanism

remains to be fully elucidated and UL79 homologues in other CMVs have not been studied. Here we

characterized the role of the conserved murine CMV (MCMV) gene M79. We showed that M79 encoded a

protein (pM79) which was expressed with early-late kinetics and localized to nuclear viral replication

compartments. M79 transcription was significantly decreased in the absence of viral DNA synthesis but

markedly stimulated by pM79. To investigate its role, we created the recombinant virus SMin79, in which

pM79 expression was disrupted. While marker-rescued virus grew efficiently in fibroblasts, SMin79 failed

to produce infectious progeny but was rescued by pM79 expression in trans. During SMin79 infection,

representative viral immediate early and early gene products as well as viral DNA accumulated

sufficiently. Formation of viral replication compartments also appeared normal. Pulsed field gel

electrophoresis analysis indicated that the overall structure of replicating viral DNA was indistinguishable

between wild-type and SMin79 infection. Viral tiled array and quantitative PCR analysis revealed that

many late transcripts sensitive to a viral DNA synthesis inhibitor (phosphonoacetic acid) were markedly

reduced by pM79 mutation. This study indicates that cytomegaloviruses use a conserved mechanism to

promote transcription at late stages of infection, and that pM79 is a critical regulator for at least a subset

of viral DNA synthesis-dependent transcripts.

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Introduction

Cytomegalovirus (CMV) is the prototypical member of the β-herpesvirus subfamily. Human CMV

(HCMV) is a ubiquitous human pathogen, which causes asymptomatic infection in healthy adults.

However, in immuno-compromised hosts, such as neonates, transplant recipients, persons with

advanced AIDS, and cancer patients, HCMV is a common cause of severe and even life-threatening

disease (5, 10, 12, 38). The severity of medical problems associated with HCMV in these vulnerable

populations underlies the necessity for developing safer and more effective antiviral treatments and

vaccine strategies to control its infection. HCMV infection is limited to human hosts, as the members of

the CMV family are species specific. Murine CMV (MCMV) infection provides a tractable small animal

model to study CMV biology. MCMV shares conservation with HCMV in regard to its collinear genome,

gene expression program, tissue tropism, and pathology (35, 37). Over 40% of MCMV genes have

sequence or functional homologues in HCMV (34). This conservation provides us with excellent

opportunities to explore MCMV as a tool to dissect the mechanistic basis of shared features of viral

replication and pathogenesis. Furthermore, revealing the function of homologous viral genes in their

respective hosts will allow antiviral therapeutics or vaccine candidates targeting these conserved genes to

be tested in the mouse model.

Gene expression during herpesvirus lytic infection is highly coordinated and sequentially ordered

such that viral genes are traditionally divided into three kinetic classes: immediate early (IE), early, and

late. IE genes are transcribed following viral DNA translocation to the nucleus and require only incoming

virion-associated proteins and cellular factors for their expression. Products of IE genes transactivate

early genes and remodel the host cell to be permissive for virus replication. Early gene transcriptions are

initiated prior to viral DNA synthesis but some persist at late times of infection, even after the onset of

DNA synthesis. Early gene products are required for both viral DNA synthesis and formation of replication

compartments, which are virus-induced subnuclear structures where viral DNA synthesis occurs.

Transcription of late viral genes occurs after the onset of viral DNA synthesis, and peaks at late times of

infection. Many late genes encode structural proteins required for virion assembly, maturation, and

release. Largely consistent with this temporal regulation, transcription of many IE and early genes is

resistant to viral DNA synthesis inhibitors, such as phosphonoacetic acid (PAA). Late transcripts, defined

60

by their abundant accumulation at late times of infection and dependency on viral DNA synthesis, are

largely derived from late genes. In addition, some genes have both early and late properties, as their

expression initiates prior to DNA synthesis but the transcripts continue to accumulate to high levels at late

times in a DNA synthesis-dependent manner.

While IE and early transcription has been extensively studied in herpesviruses, little is known

about how viral late transcription is regulated. Viral DNA synthesis is required in cis for viral late promoter

activity but the precise mechanism remains elusive (17, 26, 28). Herpes simplex virus 1 (HSV-1), the

prototypical α-herpesvirus, is perhaps the best studied example. Multiple HSV-1 proteins (ICP0, ICP4,

ICP22, and ICP27) have been shown to regulate late expression (8, 32, 36, 41, 48, 49). For some late

genes, the TATA box as well as DNA sequences downstream of the transcription start site are also main

determinants of transcription (11, 16, 27). However, a majority of these HSV-1 genes lack homologues in

β-herpesviruses (1), and evidence suggests that the requirement for these sequence elements is not

universal (11, 14, 20, 27, 39).

Understanding how CMV regulates late gene expression is important to understand its biology

and identify novel targets for antiviral therapeutics. The HCMV UL79 family is a viral gene family

conserved between β- and γ-herpesviruses (2). We and others have recently shown that UL79 is required

for HCMV late gene expression (15, 31). However, the MCMV homologue of UL79, M79, remains

uncharacterized. Defining the role of M79 will set the stage for using the MCMV model to elucidate the

mechanism of action for this CMV gene family, and explore novel antiviral strategies targeting this viral

factor.

In this study, we characterized M79 during MCMV infection. We show that pM79, the protein

product of M79, acts downstream of viral DNA synthesis to facilitate viral late transcription. Importantly,

viral oligonucleotide tiled array analysis reveals at least two subsets of late transcripts. Both require viral

DNA synthesis for their expression, but they have different degrees of dependence on pM79 for

expression. As a result, abrogation of pM79 results in a complete failure in virus growth. These results,

along with studies of HCMV UL79 and murine gammaherpesvirus 68 (MHV68) ORF18 (2, 31), suggest

that divergent herpesviruses use similar mechanisms to promote late gene expression. Furthermore, our

study provides evidence to support the model that CMV late transcription is tightly regulated beyond its

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dependency on viral DNA synthesis, and that pM79 is a key regulator for at least a subset of MCMV late

transcription, highlighting the complex regulatory mechanisms governing CMV late transcription.

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Results

M79 is essential for MCMV replication. The homolog of M79 in HCMV, UL79, is essential for virus

replication (15, 31), but the role of M79 has not been characterized. To investigate this, we created a

mutant BAC clone of MCMV Smith strain, pSMin79, by BAC recombineering. In this mutant clone, an 88-

nt insertion was introduced at 403-nt downstream of the start codon of the M79 coding sequence,

resulting in a frame-shift mutation (Fig. 1A). We anticipated that this site of insertion made it unlikely to

interfere with expression of neighboring genes, particularly M80, an essential gene that overlaps with M79

at its N-terminus (3, 23, 47).

To reconstitute recombinant virus, both pSMin79 and its wild-type parental clone, pSMgfp, were

electroporated into 10.1 mouse embryonic fibroblasts (MEF10.1). Cells transfected with pSMgfp readily

initiated virus production and spread. By 5 days post transfection, the monolayer of transfected cells

demonstrated complete cytopathic effect (CPE) and full virus spread, indicated by virus-driven GFP.

However, even though pSMin79 transfection was efficient, evidenced by the presence of individual GFP-

positive cells upon initial inspection at day 2, it repeatedly failed to produce any CPE or GFP spread even

at two weeks post transfection (Fig. 1B). Transfection of multiple independently isolated pSMin79 clones

yielded the same result, suggesting that M79 is essential for MCMV replication.

To provide a means to propagate BAC-derived M79 mutant virus, we created multiple clonal

MEF10.1 cell lines (10.1-M79flag) that stably expressed C-terminally FLAG-tagged M79 by retroviral

transduction. Transfection of pSMin79 into 10.1-M79flag cells supported efficient virus reconstitution,

producing complete CPE and full spread of virus-driven GFP expression on the monolayer (Fig. 1B).

Importantly, transfection of pSMin79 in 10.1-M79flag cells produced virus with titers similar to that of

reconstituted wild-type virus, SMgfp (Fig. 1B).

Finally, to provide definitive evidence for the essential role of M79 in MCMV infection, we

performed growth curve analysis of SMin79 in MEF10.1 cells. For this experiment, we also created

SMrev79, a marker-rescued virus of SMin79. The SMin79 virus failed to produce cell-free or cell-

associated progeny virus in both multistep (data not shown) and single step growth analysis (Fig. 1C). On

the other hand, SMrev79 replicated indistinguishably from wild-type virus (Fig. 1D). Together, our results

63

indicate that the defect of SMin79 is the direct result of M79 ablation, and that M79 is essential for MCMV

replication at steps prior to virus release.

Expression of M79 gene products is markedly enhanced by viral DNA synthesis and protein pM79.

To better understand the function of the M79 gene, we first characterized its potential protein product

(pM79). M79 is predicted to encode a protein of 258 amino acids (aa) with a molecular weight of 29 kDa.

As no specific antibody to the M79 protein was available, we created a recombinant virus, SM79flag, in

which the M79 coding sequence was tagged with 3x FLAG at the C-terminus (Fig. 1A). SM79flag was

reconstituted efficiently from BAC transfection and grew indistinguishably from wild-type virus (Fig. 1D),

indicating that the 3x FLAG tag did not interfere with M79 function. To determine expression of pM79

during infection, we infected MEF10.1 cells with SM79flag and analyzed the accumulation of FLAG-

tagged pM79 over the course of a single viral replication cycle by immunoblotting (Fig. 2A). FLAG-tagged

pM79 migrated at the apparent molecular weight of 31 kDa, consistent with the predicted size, and was

detected at 24-36 hours post infection (hpi) (Fig. 2A). We then profiled M79 transcription by reverse

transcription-coupled quantitative PCR analysis (RT-qPCR) to more precisely characterize M79

expression (Fig. 2B). When viral DNA synthesis was inhibited by PAA, low levels of M79 transcription

persisted and increased modestly from 10 hpi to 30 hpi, suggesting that a small amount of M79

transcripts were produced independent of viral DNA synthesis. Importantly however, the majority of M79

transcription was inhibited by PAA, and in the absence of PAA, M79 transcript levels accumulated to high

abundance at late times (20-30 hpi). Together, our results indicate that low levels of M79 expression

occurs independent of viral DNA synthesis but that the majority of its expression requires viral DNA

synthesis, similar to the previously described viral gene expression pattern of early-late kinetics (28).

It was interesting to note that during SMin79 infection, M79 transcript levels were drastically

reduced relative to that of SMgfp virus infection (Fig. 2B). As the small insertion mutation in SMin79 was

designed to abolish only the M79 protein but not its transcript, we interpreted this to suggest that pM79

enhances its own transcription, particularly at late times of infection, correlating with the previous report of

its homolog UL79 during HCMV infection (31).

64

M79 protein localizes to viral nuclear replication compartments. We next examined the intracellular

localization of pM79 during infection of SM79flag using a rabbit anti-FLAG antibody. At 24 hpi, FLAG

staining was found exclusively in the nucleus, closely co-localized with the viral polymerase processivity

factor pM44 in the nuclei of infected cells (Fig. 3A). As pM44 is a widely used marker of viral replication

compartments, this result indicates that the nuclear pM79 localizes within replication compartments. The

rabbit anti-FLAG antibody also produced a diffuse, weak cytoplasmic staining, which was likely

nonspecific as it was also present in SMgfp infected control cells (Fig. 3A). To test this, a separate set of

infected cells were stained with a mouse anti-FLAG antibody, even though this strategy precluded us

from co-staining cells with the mouse anti-pM44 antibody to mark replication compartments (Fig. 3B).

Nonetheless, in these cells there was only specific staining in the nuclei resembling replication

compartments, and there was no cytoplasmic background staining observed. Collectively, we conclude

that pM79 is a nuclear protein that localizes to replication compartments during MCMV infection.

M79 is not required for viral DNA synthesis or development of nuclear replication compartments.

As pM79 localized to viral replication compartments during infection, we next determined if this protein

was involved in viral DNA replication processes, particularly the ability of the virus to synthesize its

genome and form nuclear replication compartments. To test if pM79 was required for viral DNA synthesis,

we first examined the kinetics of viral DNA accumulation during SMin79 infection by quantitative PCR

(qPCR) analysis. Viral DNA accumulation over the course of SMin79 infection was comparable to that in

SMgfp infection, indicating that pM79 is not required for the virus to synthesize its DNA (Fig. 4A).

To probe if its overall structure was altered in the absence of pM79, viral DNA from infected cells

was analyzed by a pulsed field gel electrophoresis (PFGE) (Fig. 4B). SMgfp infected cells produced both

concatemeric replicating viral DNA, which was retained in the well, and cleaved 232-kb monomeric viral

genome, which migrated into the gel (lane 2). In addition, a minor population of viral DNA with the

apparent molecular weight greater than 232 kb also migrated into the gel, likely representing polymeric

DNA intermediates. In contrast, SMin79 infected cells produced only concatemeric viral DNA and

polymeric DNA intermediates without any monomeric viral genomes (lane 3). This suggests either a

failure of the mutant virus to cleave replicating viral DNA into mature viral genomes, or altered viral DNA

65

structures that prevent monomeric viral genomes from migrating into the gel. To differentiate these

possibilities, intracellular viral DNA was digested with Pac I, a restriction enzyme that cuts the MCMV

genome four times. As expected, Pac I cut the 232-kb monomeric virion genome (lane 7) into linear

fragments of 92.8 kb, 90.3 kb (which co-migrated together ), 43.0 kb, 1.3 kb and 4.2 kb (the last two run

off the gel) (lane 8). Furthermore, Pac I digested intracellular DNA from SMgfp infected cells produced an

additional 135.8-kb linear fragment that resulted from joint ends within concatemeric viral DNA (lane 5).

Pac I digestion of SMinM79 infected cells released the same fragments of 90.3-kb and 135.8-kb from

concatemeric DNA , but did not produce the monomer-derived fragments (lane 6). Therefore, the overall

structure of replicating viral DNA produced by pM79-deficient virus was not appreciably different from that

of wild-type virus. Together, our data indicate that loss of M79 does not have a deleterious effect on viral

genomic amplification.

We also tested if pM79 was involved in the development of replication compartments, virus-

induced nuclear structures critical to successful viral DNA replication (30). We infected cells with wild-type

or M79-deficient virus, and monitored the formation and maturation of replication compartments marked

by pM44 staining. pM44 staining became evident at 12 hpi, forming multiple small nuclear foci (i.e. pre-

replicative compartments), and these foci coalesced into larger and fewer mature replication

compartments at 24 hpi (Fig. 5). Importantly, no appreciable difference was observed in this temporal

progression of replication compartments between infections of SMgfp and SMin79 virus (Fig. 5).

Therefore, pM79 does not appear to play a role in the development of replication compartments.

These data taken together suggest that the defect seen in SMin79 infection occurs downstream of

viral DNA synthesis. Moreover, the failure of mutant virus to process replicating viral DNA into monomeric

genomes suggests the defect occurs at or prior to genome cleavage and packaging, such as the step of

late gene expression or capsid assembly.

M79-deficient virus is defective in the efficient accumulation of representative viral Late gene

products. To continue to define the stage of the viral replication cycle where pM79 acts, we analyzed the

accumulation of representative viral proteins from each kinetic class: immediate early protein IE1, early

protein E1 (M112/113), and late protein gB (M55). M79-deficient virus demonstrated two defects in viral

66

protein accumulation (Fig. 6A). One was a modest delay in IE1 and E1 protein accumulation, whereas the

other was the complete loss of gB. As this modest delay in IE1 and E1 had no deleterious consequence

on viral DNA synthesis (Fig. 4), it was unlikely responsible for defects in events beyond viral DNA

synthesis, such as late protein accumulation and ultimately virus growth. The delay in IE1 and E1

expression might be due to the characteristics of the mutant viral stocks, such as tegumentation or virion

composition, which could be slightly different from wild type stocks because of the efficiency of

complementation. To rule out any effect of IE1 and E1 expression, we infected cells with SMin79 at a MOI

of 5 and SMgfp at a MOI of 1 to compensate for IE1 and E1 expression levels. Despite elevated IE1 and

E1 accumulation in SMin79 infection under this condition, gB remained absent even after 48 hpi (Fig. 6B).

We interpreted this result to indicate that the major defect of SMin79 was the inability to express viral

gene products at late times of infection.

To test if this defect was at the transcriptional level, we analyzed the accumulation of E1 and M55

transcripts during wild-type and M79 mutant virus infection. E1 transcript accumulated at comparable

levels during SMin79 and SMgfp infection, particularly at early times (10 hpi) (Fig. 6C). In contrast,

accumulation of M55 transcript in SMin79 infected cells was effectively reduced to levels comparable to

those under PAA treatment. All transcripts detected were specific and were not the result of genomic

DNA contamination as mock cells and reactions done in the absence of reverse transcriptase failed to

produce any products (data not shown). This result supports the hypothesis that both pM79 and viral DNA

synthesis are required for efficient viral late transcription.

pM79 regulates the accumulation of a subset of viral late transcripts. The failure of SMin79 to

express a representative late transcript could be due to a global down-regulation of MCMV late

transcription, or it could be due to a down-regulation of only a subset of late transcripts. To differentiate

these two possibilities, we profiled the entire MCMV transcriptome with or without pM79. We designed a

high density oligonucleotide tiled array with probes to both the forward and reverse strand of the MCMV

genome, allowing us to measure transcription across the entire viral genome. We first determined viral

regions where transcription was dependent on viral DNA synthesis. To test this, MEF10.1 cells were

infected with MCMV in the presence or absence of PAA. At 20 hpi, RNA was harvested, converted to

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fluorescently labeled cDNA, and hybridized to the MCMV DNA array. The normalized mean intensity of

probes across the genome was plotted in Fig. 7. The sensitivity of viral transcription to PAA was variable

across the genome, ranging from no change to greater than 100-fold reduction. Although most viral

regions exhibited some sensitivity to PAA, many regions that were highly regulated did not correspond to

annotated ORFs. To minimize false positives, we considered a region of transcription to be dependent on

viral DNA synthesis only if the intensity of the PAA-treated sample was at least 3-fold lower than that of

the untreated sample in this analysis. By this criterion, among viral regions corresponding to the 172

annotated ORFs examined, efficient transcription of 115 ORFs was dependent on viral DNA synthesis

(Table 2). Among them, 80 ORFs have been previously analyzed for their temporal expression, and 61

have been found to be expressed at elevated levels at late stages of MCMV infection (i.e. late genes) (19,

25). Therefore, transcription of a large set (76%) of annotated ORFs sensitive to PAA are also known to

be highly expressed at late times, consistent with the notion that most viral DNA synthesis-dependent

transcripts are derived from late genes.

We then examined the transcriptome profile in SMin79 infection. Overall, the effect of pM79

mutation on viral transcription was less pronounced than that of PAA (Fig. 7). Compared to PAA

treatment, fewer regions of transcription were reduced by greater than 3-fold in the absence of pM79 in

array analysis. Interestingly, many un-annotated regions of RNA expression were affected by PAA but not

by pM79 mutation. Transcription from regions corresponding to 43 ORFs was reduced by greater than 3-

fold in the absence of pM79 by array analysis, and 41 of them (95%) were also reduced by PAA

treatment (Table 2). Transcription from the regions corresponding to the remaining 74 PAA-sensitive

ORFs was less affected by pM79 mutation based on the criterion used in our analysis. These results

suggest that at least a subset of DNA synthesis-dependent viral transcripts also have a high dependence

on pM79 for their expression.

This differentiated dependency of late transcripts on pM79 was validated by RT-qPCR analysis.

Transcription of M74 and M116, which showed a greater dependency on pM79 in array analysis (Table

2), was reduced by 9.8 and 37.7 -fold by pM79 mutation in qPCR analysis (Fig. 8A). Conversely,

transcription of M25 and M121 was reduced by only 5.5 and 4.5 -fold, respectively, in pM79 mutant virus

68

infection (Fig. 8B), thus consistent with the result of array analysis (Table 2) and showing relatively less

dependency on pM79.

Together, our results show that M79 is critical for the accumulation of at least a subset of DNA

synthesis-dependent viral late products (Fig. 8). These results, along with the observations of UL79 in

HCMV and ORF18 in MHV68 (2, 15, 31), underscore a conserved function of the UL79 family of genes in

β- and γ- herpesvirus replication cycles.

69

Discussion

MCMV is the commonly used model virus for HCMV, so revealing HCMV genes that are

functionally conserved in MCMV will allow the use of the robust mouse genetic system to elucidate their

role and test novel antivirals targeting these products. Previously, we have found that the HCMV gene

UL79 regulates viral late gene expression (31). Here, we characterized the function of its MCMV

sequence homologue, M79, by analyzing mutant MCMV virus in which pM79 expression was disrupted.

pM79 accumulated with early-late kinetics in nuclear viral replication compartments, and if disrupted,

abrogated the ability of MCMV to replicate. In particular, we showed that pM79 was critical for MCMV to

promote expression of a set of late transcripts. Moreover, MCMV DNA was not only synthesized

efficiently in the absence of pM79, PFGE analysis shows that the overall structure of replicating viral DNA

was unimpaired. The development of viral replication compartments during mutant virus infection was

indistinguishable from those during wild-type virus infection. This body of evidence further excludes the

involvement of pM79 in viral DNA synthesis or other events preceding viral late transcription.

Furthermore, the failure of pM79-deficient virus to cleave viral concatameric replicating DNA is consistent

with its defect in late viral transcription. For example, genes required for genomic cleavage (e.g. M56 and

M104) or capsid assembly (e.g. M80, M85, and M86) are down-regulated during SMinM79 infection

(Table 2). Our work, together with the reported role of its homologues, including pUL79 in HCMV and

ORF18 in MHV68 (2, 15, 31), suggests a common mechanism governing late transcription among β and

γ-herpesviruses. How viral late transcription is regulated remains largely unknown and viral/host factors

involved are poorly defined. The pM79/pUL79 protein family represents an invaluable tool to gain insight

into this key viral process.

Our oligonucleotide tiled array analysis has identified a set of annotated genes whose

transcription is substantially reduced at late stages of virus infection when pM79 is abrogated (Fig. 7).

Comparative analysis of viral transcriptomes among MCMV infections with PAA, without PAA, or in the

absence of pM79 leads to two interesting observations. First, while viral DNA synthesis is required for

transcription from genomic regions containing many previously reported late genes, it also facilitates the

continued transcription from genomic regions containing several previously reported early genes at late

times of infection. Second, viral transcripts that are dependent on viral DNA synthesis also have a

70

dependency on pM79 for their accumulation. There seems to be a striation in dependence, such that

without pM79, some transcripts are markedly reduced, whereas others are reduced to a much less

extent. Thus, pM79 is a key viral regulator of late transcription in MCMV infection. In future studies, similar

transcriptome analysis should be applied to other viral genes known or predicted to be regulators of late

gene expression. Examples are HCMV UL79 and MHV68 ORF18, as well as MCMV M87 and M95

(homologues of HCMV UL87 and UL95). Such analysis will reveal whether these viral regulators control

an overlapping or distinct set of viral late gene expression.

How does pM79 regulate expression of late transcripts? A late transcript regulator could be

involved in epigenetic regulation of replicating viral DNA. For instance, viral DNA-associated histones are

modified when herpesviruses replicate their genomes, and this may render late promoters accessible for

transcription (21, 22, 40). However, the pM79 coding sequence does not resemble those of histone

modifying enzymes, such as histone acetyltransferase or histone deacetylase (data not shown). If pM79

had a role in epigenetic regulation of gene expression, it would likely act indirectly, for instance, by

recruiting modification enzymes to histones associated with late viral promoters. Late regulators may also

act as transcription factors. However, pM79 does not contain any identifiable putative DNA binding

domains (data not shown). Therefore, in this capacity pM79 would have to act as a modulator of cellular

and viral transcriptional regulators or RNA polymerase to facilitate viral late gene transcription. Finally, it is

tempting to speculate that regulation of late gene expression may function as a switch to decide a lytic or

latent viral infection. Cellular or viral repressors may associate with viral late promoters by default to keep

them silent, but viral regulators such as pM79 could displace these repressors to favor a productive, lytic

infection. Work is underway to identify viral and cellular binding partners of pM79 in order to reveal the

mechanism of its activity.

The identification of pM79 as an essential protein for MCMV late transcription indicates that it is a

functional homologue of HCMV pUL79. MCMV infection in mice is an important model for preclinical

evaluation of antiviral compounds, most of which have been directed at highly conserved viral DNA

replication proteins. The pM79/pUL79 protein family presents an attractive, alternative target for

therapeutic intervention in CMV disease. The functional conservation between pM79 and pUL79 justifies

MCMV as a credible model to test this novel antiviral strategy in vivo.

71

Acknowledgements

We thank Herbert Virgin and the members of his laboratory for helpful discussion and invaluable

advice; Dr. Ulrich Koszinowski (Max von Pettenkofer-Institute, Ludwig Maximilians-University, Germany),

Dr. Martin Messerle (Hannover Medical School, Hannover, Germany), and Dr. Wolfram Brune (Heinrich

Pette Institute, Leibniz Institute for Experimental Virology, Germany) for the MCMV BAC clone pSM3fr;

Dr. Anthony Scalzo (University of Western Australia) for M44 and gB antibodies; Dr. Stipan Jonjic

(University of Rijeka, Croatia) for IE1 and E1 antibodies; Jian Gao (Washington University) for assistance

in array analysis, and members of the Yu lab for critical reading of the manuscript.

We thank the Genome Technology Access Center in the Department of Genetics at Washington

University School of Medicine for help with genomic analysis. The Center is partially supported by NCI

Cancer Center Support Grant #P30 CA91842 to the Siteman Cancer Center and by ICTS/CTSA Grant#

UL1RR024992 from the National Center for Research Resources (NCRR), a component of the National

Institutes of Health (NIH), and NIH Roadmap for Medical Research. This publication is solely the

responsibility of the authors and does not necessarily represent the official view of NCRR or NIH.

This study was supported by Public Health Service grants (RO1CA120768 and RO1AI51345).

D.Y. holds an Investigators in the Pathogenesis of Infectious Disease award from the Burroughs

Wellcome Fund and W.M.Y. is an investigator of the Howard Hughes Medical Institute.

72

Material and methods

Plasmids, antibodies, and chemicals. pYD-C245 and pYD-C571 were retroviral expression vectors

derived from pRetro-EBNA (18). pYD-C245 expressed the red fluorescent protein (DsRed) (4) from an

internal ribosome entry site (IRES). pYD-C571 was derived from pYD-C245. It carried the coding

sequence of C-terminally 1x FLAG tagged M79 that was expressed together with DsRed as a bicistronic

transcript. pYD-C191 carried a kanamycin selection cassette bracketed by two Flp recognition target

(FRT) sites. pYD-C630 was derived from pGalK (44) and carried a FRT-bracketed GalK/kanamycin dual

selection cassette (29). pYD-C746 was derived from pYD-C630, where a 3x FLAG sequence preceded

the FRT-bracketed selection cassette.

The primary antibodies used in this study included: anti-actin (clone AC15; Abcam); anti-FLAG

polyclonal rabbit antibody (F7425) and monoclonal mouse antibody (F1804) (Sigma); anti-MCMV IE1

(CROMA101) and E1 (CROMA103) (generous gifts from Dr. Stipan Jonjic, University of Rijeka, Croatia);

anti-MCMV M44 (3B9.22A) and gB (2E8.21A) (generous gifts from Dr. Anthony Scalzo, University of

Western Australia). The secondary antibody used for immunoblotting was horseradish peroxidase (HRP)-

conjugated goat anti-mouse IgG (Jackson Laboratory). The secondary antibodies used for immuno-

fluorescence were Alexa Fluor 594-conjugated goat anti-mouse IgG and Alexa Fluor 488-conjugated goat

anti-rabbit IgG (Invitrogen-Molecular Probes).

Other chemicals used in this study include phosphonoacetic acid (PAA) (284270-10G; Sigma

Aldrich); L-(+)-Arabinose (A3256-25G; Sigma Aldrich); and TO-PRO3 iodide (T3605; Invitrogen).

Cells and viruses. Mouse embryonic fibroblast 10.1 cells (MEF10.1) (13) were propagated in Dulbecco

modified Eagle medium supplemented with 10% fetal bovine serum, nonessential amino acids, and 1 mM

sodium pyruvate. Cells were maintained at 37oC and 5% CO2 in a humidified atmosphere. To create cell

lines stably expressing FLAG-tagged M79 (10.1-M79flag), MEF10.1 cells were transduced three times

with retrovirus reconstituted from pYD-C571 and allowed to recover for 48 hours. Clonal cells expressing

DsRed were isolated by limiting dilution and expanded to produce stocks of cell lines. Individual clonal

cell lines were tested by transfecting them with the recombinant MCMV BAC clone pSMin79 (see below)

and determining the titer of reconstituted virus at 5 days post transfection. The cell line that yielded the

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highest titer was used in this study. Recombinant MCMV viruses SMgfp, SMrev79, and SM79flag (see

below) were reconstituted from electroporation of corresponding BAC clones into MEF10.1 cells.

Recombinant virus SMin79 (see below) was reconstituted from electroporation of the BAC clone pSMin79

into 10.1-M79flag cells.

BAC recombineering. Recombinant MCMV BAC clones used in this study were derived from the self-

excisable parental MCMV BAC clone, pSM3fr, which carried a full length genome of the MCMV Smith

strain (43). All recombinant MCMV BAC clones in this study were created using the linear recombination-

based BAC recombineering protocol that we have previously established (29). Recombination was

carried out in E. coli strain SW105 that harbored an MCMV BAC clone and expressed an arabinose

inducible Flippase gene for transient expression of Flp recombinase (44). We inserted the green

fluorescent protein (GFP) expression cassette at the C-terminus of the IE2 loci within pSM3fr to produce

the BAC clone pSMgfp. This clone was used to produce wild-type virus SMgfp as IE2 has been shown to

be dispensable for MCMV infection in vivo and in vitro (6, 7, 24). We independently confirmed that the

insertion of the GFP cassette at this locus had no deleterious consequence on virus growth in our

infection system (data not shown). The BAC clone pSMin79 carried a frame-shift mutation in the viral

gene M79 (Fig. 1A). To construct pSMin79, the FRT-bracketed GalK/kanamycin cassette was PCR

amplified from pYD-C630 and recombined into pSMgfp at 403 nucleotide (nt) downstream of the start

codon of the M79 coding sequence. Transformants were selected by kanamycin resistance. The selection

cassette was removed by arabinose induction of Flp recombinase and subsequent Flp-FRT

recombination (29), leaving an 88-nt insert within the M79 coding sequence and creating a frame-shift

mutation. The BAC clone pSM79flag contained a C-terminally 3x FLAG-tagged M79 (Fig. 1A). To

construct pSM79flag, a DNA fragment that contained the FRT-bracketed GalK/kanamycin selection

cassette preceded by a 3x FLAG sequence was PCR amplified from pYD-C746 and recombined into the

C-terminus of the M79 coding sequence. The selection cassette was subsequently removed by Flp-FRT

recombination, resulting in the 3x FLAG fused in frame with the M79 coding sequence. The BAC clone

pSMrev79 was derived from pSMin79 and contained the repaired M79 coding sequence. To create

pSMrev79, a patched PCR fragment containing the wild-type M79 coding sequence followed by a FRT-

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bracketed kanamycin selection cassette was recombined into pSMin79 to replace the M79 frame-shift

mutation. The selection cassette was subsequently removed by Flp-FRT recombination, leaving an 81-bp

sequence insert after the stop codon of the M79 coding sequence, which had no deleterious effect on

virus replication (Fig. 1D). All the final BAC clones were validated by restriction digestion, PCR analysis,

and direct sequencing as previously described (46).

To reconstitute recombinant viruses that did not require complementation to grow, confluent

MEF10.1 cells were electroporated with 5 µg of MCMV BAC DNA and plated on a 10 cm plate. Culture

medium was changed 24 hours post transfection, and virus was harvested by collecting cell free culture

medium after the entire monolayer of cells was lysed. Alternatively, virus stocks were produced by

collecting cell free supernatant from infected culture at a multiplicity of infection (MOI) of 0.001. Virus titers

were determined in duplicate by a tissue culture infectious dose 50 (TCID50) assay in MEF10.1 cells. To

reconstitute, propagate, and titer SMin79 virus, 10.1-M79flag cells were used as described above. In

experiments where comparative analysis was performed between SMin79 and other recombinant viruses,

titers of all viruses were determined in 10.1-M79flag cells.

Viral growth analysis. MEF10.1 cells were seeded in 12-well plates overnight to produce a confluent

monolayer. Cells were inoculated with recombinant MCMV viruses for 1 hour at a MOI of 2 for single step

or 0.01 for multistep growth analysis. The inoculum was removed, the infected monolayer was rinsed with

phosphate-buffered saline (PBS), and fresh medium was replenished. At various times post infection,

cell-free virus was collected in duplicate by harvesting medium from infected cultures. Cell-associated

virus was collected by rinsing infected cells once with PBS and scraping cells into fresh medium. Cells

were lysed by one freeze-thaw cycle followed by sonication. Lysates were cleared of cell debris by low

speed centrifugation and supernatants were saved as cell-associated virus. Virus titers were determined

by TCID50 assay.

DNA and RNA analysis. Intracellular DNA was measured by quantitative PCR (qPCR) as previously

described (46). Briefly, MCMV-infected cells were collected in a lysis buffer (200 mM NaCl, 20 mM Tris

[pH 8.0], 20 mM EDTA, 0.2 mg/ml proteinase K, 0.4% sodium dodecyl sulfate [SDS]), and lysed by

75

incubation at 55°C overnight. DNA was extracted with phenol-chloroform and treated with RNase A (100

µg/ml) at 37oC for 1 hour. Samples were extracted again with phenol-chloroform, precipitated with

ethanol, and resuspended in nuclease-free water (Ambion). Viral DNA was quantified by qPCR using

SYBR Advantage qPCR Premix (Clonetech) and a primer pair specific for the MCMV IE1 or M55 gene

(Table 1). Cellular DNA was quantified using a primer pair specific for the mouse actin gene (Table 1)

(45). A standard curve was generated using serially diluted pSMgfp BAC DNA or DNA from infected cells,

and used to calculate relative amounts of viral or cellular DNA in a sample. The amount of viral DNA was

normalized by dividing IE1 or M55 equivalents over actin equivalents. The normalized amount of viral

DNA in SMgfp infected cells at 2 hour post infection (hpi) was set at 1.

Intracellular RNA was determined by reverse transcription-coupled qPCR (RT-qPCR) as

previously described (46). Total RNA was extracted by Trizol reagent (Invitrogen) and treated with

TURBO DNA-free reagents (Ambion) to remove contaminating DNA. First strand cDNA synthesis was

performed with the High Capacity cDNA Reverse Transcription Kit using random hexamer primered total

RNA (Applied Biosystems). Each sample also included a control without the addition of reverse

transcriptase to determine the level of residual contaminating DNA. cDNA was quantified using SYBR

Advantage qPCR Premix (Clonetech) and primer pairs specific for viral genes or the mouse actin gene

(Table 1). A standard curve was generated for each gene using serially diluted cDNA from infected cells

and used to calculate the relative amount of a transcript in each sample. The amounts of viral transcript

were normalized by dividing viral transcript equivalents over actin equivalents. The normalized amount of

transcript during SMgfp infection at 10 hpi in the absence of PAA was set to 1.

Tiled array design, experimental procedure, and analysis. The array was designed using Agilent’s

eArray package. The array consisted of 103,347 60-mer oligonucleotide probes for each strand of the

MCMV genome, and each probe advanced 4-5nt. In addition, the array also contained probes

complementary to Agilent’s RNA Spike-Ins, and 400 negative control probes against Arabidopsis with no

homology to mouse or MCMV genomes.

To prepare viral RNA, total RNA was harvested from infected MEF10.1 cells at 20hpi using Trizol

(Invitrogen) and mRNA was purified using RNAeasy columns (Qiagen) according to the manufacturer’s

76

instructions. cDNA probes were synthesized, fluorescently labeled by random hexamer-primed

polymerization using the SuperScript Plus Indirect cDNA Labeling module (Invitrogen), and hybridized to

the array chip at Genome Technology Access Center of Washington University School of Medicine

(GTAC). Agilent’s RNA Spike-In Kit was used to monitor the linearity, sensitivity, and accuracy of the

array.

The total signal intensity for each probe was log2 transformed, and the mock signal was

subtracted from the experimental signal after they were normalized to each other using the spike-in RNA

signals. To enable comparisons among samples, raw data from each MCMV infected sample was

normalized using spike-in controls. This normalization was further refined and validated using qRT-PCR

data for several viral probes. Normalized intensities of experimental probes were mapped back to the

MCMV genome, and mean fluorescence of each nucleotide was calculated from all overlapping probes.

Changes in fluorescence intensity greater than 3-fold between compared samples were used for data

interpretation. MCMV open reading frames (ORFs) were annotated based on the studies by Rawlinson

and coworkers (34) and by Cheng and coworkers (9), and updated with details from additional

publications whenever possible. Data were converted to gff3 file format and visualized using gBrowse

(42). The data are in the process of being deposited into the Gene expression Omnibus (GEO) database.

Pulsed-field gel electrophoresis (PFGE) and southern blot analysis. PFGE was performed on a Bio-

Rad CHEF Mapper XA Pulsed Field Electrophoresis System. To prepare DNA from infected cells,

MEF10.1 cells were seeded onto a 60 mm dish at a density of 1.4 x 106 cells per dish and infected at a

MOI of 2. At 36 hpi, cells were scraped off the dish, collected by centrifugation at 200 xg for 5 minutes,

and resuspended in 180 μl of 55oC 1% low-melting-point agarose (NuSieve GTG Agarose, Lonza) in

PBS. 90µl of cell suspension was casted into a disposable casting mold (Bio-Rad) and solidified at 4oC for

15 minutes. To prepare DNA from cell-free virions, liquid viral stock equivalent to 106 PFU was casted in

one low-melting-point agarose block. Agarose blocks were transferred into lysis buffer (20 mM Tris.Cl

pH8.0, 200 mM NaCl, 400 mM EDTA, 1% SDS, 1 mg/mL proteinase K) to lyse imbedded samples by

incubation at 37oC overnight. Blocks were then rinsed five times with TE buffer (10mM Tris-HCl, pH8.0,

0.1mM EDTA) at 50oC for 15 minutes each and stored in TE Buffer at 4oC.

77

Restriction enzyme digestions were carried out by incubating one-half of a block (~45uL) in 200µl

digestion buffer containing 50 units of Pac I at 4oC overnight, and then at 37oC for 6 hours. Digested

blocks were loaded into wells of a 1% megabase agarose gel (Biorad Pulse Field Certified Agarose) in

0.5 x TBE (0.045 M Tris pH 8.0, 0.045 M boric acid, 0.001 M EDTA pH 8.0) and sealed with 1% low

melting-point agarose in 0.5 x TBE. PFGE was performed at 6 V/cm at 14oC for 16 hours, with a linear

pulse increase from 0.26 to 20.01 seconds over the course of the run.

Resolved viral fragments were analyzed by Southern blot analysis as previously described (33).

Briefly, the gel was transferred to a Nytran SuPerCharge Membrane as described in the Turboblotter

transfer system protocol (VWR), and the membrane was air dried and cross-linked by UV at 125mJ. The

32P-labeled probe was generated by random hexamer-priming of pSMgfp BAC DNA using the Prime-It II

Labeling Kit (Stratagene), purified with ProbeQuant G-50 micro columns (Amersham), and denatured in

10mM EDTA at 90oC for 10 minutes before use. The membrane was pre-hybridized with 10 ml

ULTRAhyb solution (Ambion) at 42oC for 1 hour, and hybridized with 107 cpm of denatured probes at

42oC for 3 hours. The membrane was washed once with buffer I (2x SCC and 0.1% SDS) for 10 minutes

followed by two washes with buffer II (0.1x SSC and 0.1% SDS) for 15 minutes before exposure on a

Kodak film.

Protein analysis. Protein accumulation was analyzed by immunoblot. Cells were washed and lysates

were collected in sodium dodecyl sulfate (SDS)-containing sample buffer. Proteins were resolved by

SDS-containing polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene

difluoride (PVDF) membrane. Proteins of interest were detected by hybridizing the membrane with

specific primary antibodies followed by HRP-coupled secondary antibodies, and visualized by using

SuperSignal West Pico enhanced chemiluminescent (ECL) substrate (Thermo Scientific).

Intracellular localization of proteins of interest was analyzed by immunofluorescence assay. Cells

were seeded onto cover-slips 24 hours prior to infection. At various times, cells were washed with PBS,

fixed and permeabilized with methanol (-20oC) for 10 minutes, and blocked with 5% FBS in PBS at room

temperature for 1 hour. Cells were incubated with primary antibodies for 30 minutes at room temperature

and subsequently labeled with secondary antibodies coupled to Alexa Fluor 488 or Alexa Fluor 594

78

(Invitrogen-Molecular Probes). Cells were counterstained with TO-PRO3 and mounted on slides with

Prolong Gold antifade reagent (Invitrogen-Molecular Probes). Confocal microscopic images were

captured by a Zeiss LSM510 Meta confocal laser scanning microscope.

79

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TABLE 1. Primers used in quantitative PCR analysis

Primer Name Primer Sequence

MCMV IE1 forward 5'-CAGGGTGGATCATGAAGCCT-3'

MCMV IE1 reverse 5'-AGCGCATCGAAAGACAACG-3'

MCMV M25 forward 5'-AAGACATGTCACGCGACGGA-3'

MCMV M25 reverse 5'-CTATTGCCCATCATCGCCCG-3'

MCMV gB (M55) forward 5'-GCGATGTCCGAGTGTGTCAAG-3'

MCMV gB (M55) reverse 5'-CGACCAGCGGTCTCGAATAAC-3'

MCMV M74 forward 5'-AGGAGGCTGTGACTTTGAAA-3'

MCMV M74 reverse 5'-CTCATCAGCCGTTACTCGAG-3'

MCMV M79 forward 5'-CTACCTGATCGCCTGGAAAAAG-3'

MCMV M79 reverse 5'-TAGTCCTGGATCAGGAAGGAAAAG-3'

MCMV M112/113 (E1) forward 5'-GAATCCGAGGAGGAAGACGAT-3'

MCMV M112/113 (E1) reverse 5'-GGTGAACGTTTGCTCGATCTC-3'

MCMV M116 forward 5'-TCCTTGGTGGTGATGGCGGT-3'

MCMV M116 reverse 5'-GCATCCCGTACCTGACCACA-3'

MCMV M121 forward 5'-CCCGTTCGCTTCTGAAACTG-3'

MCMV M121 reverse 5'-GCTTCTCGAGGCAGCAGCAA-3'

Mouse actin forward 5'-GCTGTATTCCCCTCCATCGTG-3'

Mouse actin reverse 5'-CACGGTTGGCCTTAGGGTTCA-3'

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TABLE 2. M79-dependent expression of MCMV ORFs

ORF Strand a PAA b M79 c Kinetics d ORF Strand PAA M79 Kinetics

m011 Y Y L m025.3 C Y N ND

m012 Y Y L m025.4 C Y N ND

m014 Y Y L M026 C Y N M032 C Y Y L M027 C Y N M035 Y Y L m029 Y N m039 C Y Y L m030 Y N m040 C Y Y ND M031 Y N M046 C Y Y M031b Y N ND

M053 Y Y L M033 Y N ND

M055 C Y Y L M044.1 Y N ND

M056 C Y Y L M044.3 Y N ND

M072 C Y Y L m045.2 C Y N ND

M073 Y Y L M050 C Y N L

M074 C Y Y L M052 Y N L

M075 C Y Y L m069.1 Y N ND

M076 Y Y ND M071 Y N L

M077 Y Y L M073x2 Y N ND

M080 Y Y L M082 C Y N L

M084 C Y Y ND M083 C Y N L

M085 C Y Y L M087 Y N M086 C Y Y L M088 Y N M096 Y Y L M089x1 C Y N L

M100 C Y Y L m090 C Y N L

M104 C Y Y L M091 Y N M114 C Y Y L M092 Y N M115 C Y Y L M093 Y N L

M116 C Y Y L M094 Y N L

M118 C Y Y L M095 Y N L

85

m119.1 C Y Y L M097 Y N m131 C Y Y L M098 Y N m155 C Y Y L M099 Y N L

m156 C Y Y L M103 C Y N L

m157 C Y Y ND m106 C Y N L

m158 C Y Y ND m106.1 C Y N ND

m159 C Y Y L m106.3 C Y N ND

m160 C Y Y L m107 Y N L

m161 C Y Y L m108 C Y N L

m162 C Y Y L m117 C Y N ND

m163 C Y Y m117.1 Y N ND

m165 C Y Y m119.5 Y N L

m168 Y Y L M121 C Y N L

m164 C N Y m123x2 C Y N ND

m166 C N Y m124 Y N L

m001 C Y N ND m124.1 C Y N ND

m002 Y N ND m125 Y N ND

m003 Y N m126 Y N ND

m007 Y N L m127 C Y N ND

m015 Y N L m128x3 Y N m016 Y N L m129 C Y N L

m018 C Y N m130 Y N ND

m019 Y N ND m134 Y N ND

m021 Y N ND m136 C Y N ND

m022 Y N ND m137 C Y N M023 C Y N m144 C Y N ND

m023.1 Y N ND m148 Y N L

M024 C Y N ND m149 Y N ND

M025 Y N L m167 C Y N L

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m025.1 C Y N L m170 C Y N L

m025.2 C Y N L

a "C", complementary strand.

b "Y", PAA treatment reduces the mean intensity of a transcriptional region by at least 3-fold in array

analysis.

c "Y", M79 mutation reduces the mean intensity of a transcriptional region by at least 3-fold in array

analysis.

d Transcription of an ORF is considered to be late kinetics ("L") when it is elevated by at least 50% from

12 to 24 hpi as described by Marcinowski and coworkers (25), or absent in 6.5 hpi but becomes

detectable at 24 hpi as described by Lacaze and coworkers (19). "ND", kinetics class has not been

previously described.

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Figure 1. Gene M79 is essential for MCMV growth in fibroblasts. (A) Diagram depicting recombinant

MCMV BAC clones used in this study. The BAC clone pSM79flag carried a 3xFLAG tag that was fused in

frame at the C-terminus of the M79 coding sequence (indicated by shaded region). The BAC clone

pSMin79 carried an 88-bp insert (indicated by black region) at 403-nt downstream of the start codon of

the M79 coding sequence, resulting in a frame-shift mutation. See Materials and Methods for details. (B)

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Growth of SMin79 virus on MEF10.1 cells expressing FLAG-tagged M79 (10.1-M79flag). Left panel; virus-

driven GFP expression in normal MEF10.1 cells or 10.1-M79flag cells 7 days post transfection with

pSMin79. Right panel; titers of wild-type virus (SMgfp) and M79 mutant virus (SMin79) produced at 72

hour post infection (hpi) in 10.1-M79flag cells that were infected at a multiplicity of infection (MOI) of 2.

Shown is a representative from at least two reproducible, independent experiments. (C-D) Growth kinetic

analysis of M79 recombinant viruses used in this study. Normal MEF10.1 cells were infected at a MOI of

2, cell free and cell associated viruses were collected at indicated times, and viral titers were determined

by tissue culture infectious dose 50 (TCID50) assay in 10.1-M79flag cells. The detection limit of TCID50

assay is indicated by a dashed line. Shown is a representative from two reproducible, independent

experiments.

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Figure 2. Expression of M79 gene products is markedly enhanced by viral DNA synthesis and

protein pM79. (A) Accumulation of the M79 protein product during MCMV infection. MEF10.1 cells were

infected with SM79flag virus at a MOI of 2, total cell lysates were collected at indicated times and

analyzed by immunoblotting. The M79 protein was detected with anti-FLAG antibody. Actin was used as

a loading control, and viral proteins IE1, E1 and gB were used as representative immediate early, early,

and late proteins, respectively. Shown is a representative from three reproducible, independent

experiments. (B) Accumulation of the M79 transcript during MCMV infection. MEF10.1 cells were infected

with SMgfp in the presence or absence of viral DNA synthesis inhibitor phosphonoacetic acid (PAA) (200

µg/ml) or with SMin79 at a MOI of 2. Total RNA was isolated at indicated times, and the amount of M79

transcript was measured by reverse transcription-coupled quantitative PCR (RT-qPCR) analysis with the

primers listed in Table 1. The values were normalized to that of actin, and the normalized amount of M79

transcript during SMgfp infection at 10 hpi in the absence of PAA was set to 1. Shown is a representative

from three reproducible, independent experiments.

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Figure 3. pM79 localizes to replication compartments during infection. MEF10.1 cells were mock

infected or infected with SMgfp or SM79flag at a MOI of 2. At 24 hpi, cells were fixed with methanol

(which quenches GFP fluorescence), and stained with either rabbit polyclonal (A) or mouse monoclonal

(B) anti-FLAG antibody for detection of the tagged M79 protein (green). In panel A, cells were co-stained

with antibody to pM44 to mark replication compartments (red). The last row of images in panel A

represent the magnified view of an infected nucleus where the M79 protein (pM79) localized. Cells were

counterstained with TO-PRO3 to visualize the nuclei (blue). Scale bars are equivalent to 20 µm. Shown is

a representative from four reproducible, independent experiments.

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Figure 4. pM79 is not required for viral DNA synthesis. (A) Accumulation of viral DNA during pM79-

deficient virus infection. MEF10.1 cells were infected with SMgfp or SMin79 at a MOI of 2 and total DNA

was harvested from infected cells at indicated times. Viral DNA accumulation was analyzed by qPCR

using primers specific for viral genes IE1 (left panel) or M55 (right panel) (see Table 1 for primer

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sequences), and the values were normalized to that of actin. The normalized values of viral DNA during

SMgfp infection at 2 hpi were set to 1. Shown is a representative from five reproducible, independent

experiments. (B) Pulsed field gel electrophoresis (PFGE) analysis of intracellular viral DNA during M79-

deficient virus infection. MEF10.1 cells were infected with SMgfp or SMin79 at a MOI of 2, and collected

at 36 hpi. Cell-free virions or infected cells were suspended in a low melting agarose block, lysed,

digested with Pac I, and subjected to pulsed field gel electrophoresis. The gel was then transferred to a

membrane, hybridized with a 32P-labeled probe specific to the entire SMgfp BAC sequence. The positions

of wells, high-molecular-weight viral DNA (HMW DNA), 232-kb monomer viral DNA, and prominent

digested viral DNA fragments are indicated. Shown is a representative from two reproducible,

independent experiments. The top panel shows the schematic diagram of monomer and concatamer viral

DNA with Pac I sites indicated.

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Figure 5. pM79 is not required for the maturation of viral replication compartments. MEF10.1 cells

were infected with SMgfp or SMin79 virus at a MOI of 2. At 12 and 24 hpi, cells were fixed with methanol

and stained with antibody to pM44 to mark replication compartments (red). Cells were also

counterstained with TO-PRO3 to visualize the nuclei (blue). Scale bars are equivalent to 20 µm. Shown is

a representative from three reproducible, independent experiments.

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Figure 6. pM79 is required for efficient expression of a representative viral late gene. (A)

Accumulation of representative viral proteins during pM79-deficient virus infection. MEF10.1 cells were

infected with SMgfp or SMin79 at a MOI of 2, and the accumulation of viral proteins IE1, E1, and gB at

indicated times was analyzed by immunoblotting. Shown is a representative from five reproducible,

independent experiments. (B) Failure of gB accumulation during pM79-deficient virus infection cannot be

rescued by elevating expression of viral early genes. Cells were infected with SMgfp or SMin79 at a MOI

of 1 or 5, respectively, so SMin79 infected cells expressed E1 protein at levels comparable to that in

SMgfp infected cells. Cell lysates were collected at indicated times and analyzed by immunoblotting.

Shown is a representative from three reproducible, independent experiments. (C) Accumulation of viral

transcripts during pM79-deficient virus infection. Cells were infected with SMgfp in the presence or

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absence of 200 μg/ml PAA, or with SMin79 as described in (A). Total RNA was isolated at indicated

times, and amounts of viral E1 and M55 transcript were measured by RT-qPCR with the primers listed in

Table 1. Shown is a representative from at least three reproducible, independent experiments. The values

were normalized to that of actin, and normalized values of viral transcript during SMgfp infection at 10 hpi

in the absence of PAA were set to 1.

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Figure 7. Tiled array analysis of genome-wide transcription during MCMV infection. MEF10.1 cells

were infected with SMgfp in the presence or absence of PAA, or with SMin79 at a MOI of 2. Total RNA

was isolated at 20 hpi, reverse transcribed, and labeled cDNAs were hybridized to an oligonucleotide tiled

array of the MCMV genome. The mean fluorescence of probes overlapping each nucleotide position is

plotted on a log2 scale underneath the annotated viral genomic sequence. Blue or red arrows represent

annotated open reading frames on the positive or negative strand of the viral genome, respectively. The

colored lines represent the transcriptional signals from SMgfp infection (black), SMgfp infection with PAA

(red), and SMin79 infection (blue). These probe intensities were compared on a nucleotide by nucleotide

basis between SMgfp infections with or without PAA treatment, or between infections of SMgfp and

SMin79. Regions in which the fluorescent intensity was reduced by greater than 3-fold by PAA treatment

or mutation of M79 are plotted on a linear scale below the transcriptional intensity plots, and labeled as

"PAA" or "in79", respectively; scales for these plots reflect fold-reduction. (A) Genomic sequence 1-80 kb.

(B) Genomic sequence 80-160 kb. (C) Genomic sequence 160-230 kb. Shown is a representative from

two reproducible, independent experiments.

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Figure 8. RT-qPCR analysis of representative PAA-sensitive transcripts in MCMV infection.

MEF10.1 cells were infected with SMgfp in the presence or absence of 200 μg/ml PAA, or with SMin79 at

a MOI of 2. Total RNA was isolated at indicated times, and amounts of indicated late transcripts were

measured by RT-qPCR with the primers listed in Table 1. The values were normalized to that of actin,

and normalized values of viral transcript during SMgfp infection at 10 hpi in the absence of PAA were set

to 1.

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Chapter III

Murine Cytomegalovirus Protein pM92 is a Conserved Regulator of Viral Late Gene Expression

100

Preface

TJC designed and performed the majority of experiments, analyzed the data, generated the figures and

wrote the first draft of the manuscript. YCP developed the UL92 and M92 MRC5 complementing cells

and created the UL92 mutant HCMV, ARF1 provided reagents, advice, and support for completion of the

study, DY supervised the studies and greatly contributed to the design and analysis of the experiments,

TJC and DY greatly contributed to the writing and editing of the manuscript.

This chapter has been previously published:

T. J. Chapa, Y. C. Perng, A. R. French, and D. Yu. Murine Cytomegalovirus Protein pM92 Is a

Conserved Regulator of Viral Late Gene Expression. J Virol 88:131-42.

101

Summary

In this study, we report that murine cytomegalovirus (MCMV) protein pM92 regulates viral late

gene expression during virus infection. Previously, we have shown that MCMV protein pM79 and its

human cytomegalovirus (HCMV) homologue pUL79 are required for late viral gene transcription.

Identification of additional factors involved is critical to dissecting the mechanism of this regulation. Here

we showed that pM92 accumulated abundantly at late times of infection in a DNA synthesis-dependent

manner and localized to nuclear viral replication compartments. To investigate the role of pM92, we

constructed a recombinant virus SMin92, in which pM92 expression was disrupted by an

insertional/frame-shift mutation. During infection, SMin92 accumulated representative viral immediate

early gene products, early gene products, and viral DNA sufficiently but had severe reduction in the

accumulation of late gene products, thus unable to produce infectious progeny. Co-immunoprecipitation

and mass spectrometry analysis revealed the interactions between pM92 and pM79 as well as between

their HCMV homologues pUL92 and pUL79. Importantly, we showed that the growth defect of pUL92-

deficient HCMV could be rescued in trans by pM92. This study indicates that pM92 is an additional viral

regulator of late gene expression, that these regulators (represented by pM92 and pM79) may need to

complex with each other for their activity, and that pM92 and pUL92 share a conserved function in CMV

infection. pM92 represents a potential new target for therapeutic intervention in CMV disease, and a

gateway into studying a largely uncharted viral process that is critical to the viral life cycle.

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Introduction

Human cytomegalovirus (HCMV), the prototypical member of the betaherpesvirus subfamily, is a

ubiquitous pathogen limited to the human host (37). After the resolution of acute infection, HCMV

establishes a persistent, life-long infection characterized by alternate stages of virus production and

latency (37). In immuno-competent hosts, the infection is typically asymptomatic. However, in immuno-

compromised hosts, lytic infection, during both primary infection and reactivation from latency, can cause

significant morbidity and mortality (37, 48). HCMV is the leading viral cause of birth defects, such as

deafness and mental retardation, in perinatally infected infants (1, 20). It is a major cause of retinitis and

blindness in AIDS patients (48). It is a common source of infectious complications in transplant recipients

and cancer patients (50). Emerging evidence also provides possible association of HCMV infection with

cardiovascular disease and proliferative diseases such as cancer (47). Currently there is no vaccine to

this virus, and antiviral therapies are limited by poor toxicity scores, low availability, and emergence of

resistant viruses (6). Understanding the role of viral genes in lytic infection is paramount and will yield

novel targets for antiviral therapies.

Murine CMV (MCMV) is the homologue of HCMV and model of choice to study CMV biology and

pathogenesis. It shares conserved features with HCMV with regard to virion structure, genome

organization, gene expression, tissue tropism, and clinical manifestations (18, 42-44). Many genes of

MCMV are conserved in HCMV and its ability to infect mice provides a tractable small animal model to

investigate virus infection in vivo. The use of MCMV to explore conserved viral genes will shed light on

the roles of their counterparts in replication and pathogenesis of HCMV.

The lytic replication cycles of herpesviruses are characterized by highly ordered cascades of

gene expression, which can be sequentially divided into immediate early (IE), early (E), and late (L)

phases (37). Expression of IE genes only requires cellular factors and viral proteins associated with

incoming virions. IE proteins transactivate expression of early genes that are required for viral DNA

synthesis. Many of the early proteins localize to viral nuclear replication compartments, where viral DNA

synthesis, late gene transcription, and viral genome encapsidation take place (37). Following viral DNA

synthesis, late genes, many of which encode structural proteins, are expressed to allow virion assembly,

maturation, and egress. It is also worth noting that some genes have both early and late properties; their

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transcriptions start prior to viral DNA synthesis, but the accumulation of their transcripts is enhanced

considerably by DNA synthesis.

Though the regulation of IE and E gene expression has been studied extensively, less is known regarding

the regulation of late gene expression during CMV infection. Previously we have shown that MCMV

protein pM79 is dispensable for viral DNA synthesis but is a key regulator of late gene transcription during

MCMV infection (10) . pM79 is conserved in both beta- and gamma-herpesviruses, and its homologues in

HCMV (pUL79) and MHV68 (ORF18) have been shown to play similar roles during virus infection (3, 27,

41). Identifying additional viral regulatory factors in late gene expression will be critical to understand this

process. In this study, we report that pM92 is another key regulator of MCMV late gene transcription. Like

pM79, pM92 is also conserved in both beta- and gamma-herpesviruses. Its homologues include pUL92 of

HCMV, ORF31 of MHV68, and U63 of HHV-6. However, the role of pM92 during MCMV infection has not

been defined, even though genome-wide mutagenesis analyses have previously shown that both MHV68

ORF31 and HCMV pUL92 are essential for lytic virus replication. Here we create a pM92-deficient MCMV

mutant virus, and show that in the absence of pM92, MCMV is capable of synthesizing its DNA at wild-

type levels but unable to efficiently produce late gene products. The M92 gene products abundantly

accumulate at 20 hours post infection (hpi) and localize to nuclear replication compartments. We also

provide evidence that pM92 interacts with pM79 during virus infection, and that their HCMV homologues

pUL92 and pUL79 interact as well. These results support the role of pM92 as a key regulator of viral late

transcription, and suggest that pM92 and pM79 are part of multi-component regulatory complex

controlling late transcription. Finally, we demonstrate the functional conservation between MCMV pM92

and HCMV pUL92 by rescuing the growth defect of pUL92-deficient HCMV virus with pM92 expression in

trans. pM79 and pM92 offer attractive targets for novel antivirals, and MCMV provides a powerful system

to dissect the regulatory mechanism of CMV late gene transcription as well as to test antivirals targeting

steps other than viral DNA synthesis.

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Results

pM92 is essential for MCMV replication in fibroblasts. The M92 open reading frame (ORF) is

predicted to encode a gene product of 231 amino acids (aa), and is a sequence homologue to the HCMV

ORF UL92 (202aa). UL92 is essential for virus replication during HCMV infection (61), but the importance

of M92 in MCMV infection has not yet been established. To investigate this, a frame-shift mutation was

introduced at nt 358 of the predicted M92 ORF by BAC recombineering (10, 40), producing mutant BAC

pSMin92 (Fig. 1A). This insertion is not expected to interfere with the expression of neighboring genes,

particularly the 5’-terminally overlapping M93. Transfection of wild-type pSMgfp BAC in MEF10.1 cells

produced virus (termed SMgfp), resulting in complete CPE of the monolayer, and full spread of the virus-

driven GFP expression at 5 days post transfection; whereas pM92-deficient virus (termed SMin92) failed

to show any sign of CPE even at two weeks post transfection (Fig. 1B). However, pSMin92 was rapidly

reconstituted to wild-type levels from pSMin92 upon transfection into MEF 10.1 cell that stably expressed

N-terminally 1×FLAG-tagged pM92 (10.1-flagM92). Thus, the defect of SMin92 is the direct result of

pM92 ablation.

To more precisely define the growth defect of SMin92, we performed growth curve analyses to

quantify the defect of the recombinant virus and validate the essentiality of pM92 (Fig. 1C). SMin92 failed

to produce detectable levels of cell-free or cell-associated progeny through the entire course of analysis,

indicating that pM92 is essential for MCMV replication at steps prior to virus release.

pM92 is a 25kDa protein that accumulated at high levels at late times of infection. A thorough

search of available nucleotide and amino acid sequence databases failed to identify any significant

homology of pM92 to proteins with known function. To acquire basic information and gain insights into the

role of pM92, we first characterized potential protein and transcript products from this gene. As no

antibody was available for detecting the M92 protein product, we created a recombinant MCMV BAC,

pSMflag92, in which the M92 coding sequence was tagged with 3×FLAG at the N-terminus (Fig. 1A).

Transfection of pSMflag92 BAC in MEF 10.1 cells rapidly reconstituted recombinant virus, termed

SMflag92. Both single-step and multi-step growth curve analyses indicated that SMflag92 grew similarly

to SMgfp (Fig. 1D), suggesting that the 3×FLAG tag did not interfere with the function of M92 or

105

neighboring gene M91 (Fig. 1A). 3×FLAG-tagged pM92 (pflagM92) migrated at the expected size of 25

kDa, became detectable at 24 hpi, and accumulated at more abundant levels at 48 hpi during SMflag92

infection (Fig. 2A). To profile its transcription, we determined the accumulation of M92 transcript by

reverse transcription-coupled quantitative PCR analysis (RT-qPCR). In agreement with its protein

accumulation profile, M92 transcript levels were low at 10 hpi but increased 8-fold at 20 hpi (Fig. 2B).

Importantly, M92 transcription was dramatically reduced when viral DNA synthesis was inhibited by

phosphonoacetic acid (PAA) at 20 hpi (Fig. 2B). Therefore, M92 gene products accumulate abundantly in

a viral DNA synthesis-dependent manner at late times of infection.

pM92 localizes to viral nuclear replication compartments during infection. To further characterize

pM92, we next examined the intracellular localization of 3×FLAG-tagged pM92 during infection of

SMflag92. Infected cells were fixed and permeablized with methanol, which also quenched GFP

fluorescence, thus allowing visualization of pM92 localization using Alexa Fluor 488 conjugated mouse

anti-flag antibody by indirect immunofluorescence. pM92 localized to the nucleus of infected cells, and in

particular, it localized to subnuclear structures resembling those of nuclear replication compartments (Fig.

2C). This led us to hypothesize that pM92 localized to viral replication compartments during infection. To

test this, we compared intracellular localization of 3x FLAG-tagged pM92 to that of viral protein pM44 (i.e.,

the viral polymerase processivity factor and commonly used marker for replication compartments) during

infection of SMflag92. As the anti-pM44 antibody is of mouse origin, we used a rabbit anti-FLAG antibody

to co-stain pM92 in this experiment. At 24hpi, FLAG staining strongly co-localized with pM44 in the

nucleus of SMflag92-infected cells (Fig. 2D), indicating that the majority of pM92 localizes within

replication compartments. The rabbit anti-FLAG antibody has been previously shown to have high

background staining (10). In this experiment, it also produced a diffuse, weak cytoplasmic staining, which

likely represented nonspecific background as it was also present in SMgfp infected control cells.

Collectively, we conclude that pM92 is a nuclear protein that localizes to replication compartments during

MCMV infection.

106

pM92 is dispensable for viral DNA synthesis but required for efficient Late gene expression. To

define the function of pM92, we first determined where it acted in the viral lifecycle, hypothesizing that

pM92 might be required for viral DNA synthesis as it localized to replication compartments (Fig. 2).

However, quantitative PCR analysis showed that viral DNA accumulation over the course of SMin92

infection was comparable to that during SMgfp infection (Fig. 3C). This result indicates that pM92 is not

required for viral DNA synthesis.

To test if pM92 is required for late gene expression, a viral event immediately downstream of viral

DNA synthesis, we next examined the accumulation of immediate early protein IE1, early protein E1

(M112/113), and late protein gB (M55) during SMin92 infection by immunoblot analysis. Compared to

wild-type control SMgfp, SMin92 appeared to have two defects during infection. The first was a modest

decrease in the accumulation of E1 protein at early times of infection. The second, and more striking

defect, was that accumulation of late protein gB was reduced to undetectable levels in SMin92 infection

(Fig. 3A). Early genes primarily function prior to viral DNA synthesis. Since no measurable defect was

observed in DNA synthesis during SMin92 infection (Fig. 3C), the modest decrease in E1 accumulation

was unlikely the main cause for the growth defect of SMin92. It is unclear why SMin92 has this minor

defect in early gene expression. It may be a result of sub-optimal complementation by the 10.1-flagM92

cells used to produce the mutant virus. It is also reminiscent of a similar observation reported for pM79-

deficient virus (10). We therefore hypothesized that the inability of SMin92 to replicate was likely due to

the failure to efficiently produce late proteins during infection.

To determine if the defect was at the transcriptional level, we measured transcript accumulation

of representative immediate early (IE1), early (E1), and late (M55) genes in the presence or absence of

pM92 during virus infection by RT-qPCR. The expression kinetics of these genes was validated by

treatment with the DNA synthesis inhibitor phosphonoacetic acid (PAA). As expected, both IE and E1

gene expression was resistant to PAA, whereas late gene M55 expression was markedly sensitive to

PAA (Fig. 3B). Importantly, in the absence of pM92, IE1 and E1 transcripts accumulated at wild-type

levels, but the accumulation of late transcript M55 was significantly reduced (Fig. 3B). Therefore, pM92 is

required for efficient transcript accumulation of late gene M55 during MCMV infection.

107

This result led us to hypothesize that pM92 is a new member of the viral late transcription

regulators, which includes recently reported pM79 (10). To test if pM92 had a global regulatory role in

viral late transcription, we examined transcript accumulation of multiple early genes (M34, M37, M45,

M102) and late genes (M46, M74, M85, M96, M116) during SMin92 infection. Transcription of early genes

was independent of viral DNA synthesis, and consequently only modestly affected by PAA treatment (< 2-

fold), consistent with previous reports (Fig. 4A) (10). Importantly, the effect of pM92 mutation on early

gene transcription was as modest as PAA treatment, indicating that pM92 is not required for efficient early

gene expression. In stark contrast, late gene transcription was dependent on viral DNA synthesis, and

therefore significantly sensitive to PAA (Fig. 4B). Importantly, transcription of these genes was also

significantly reduced during SMin92 infection. It was also noted that transcription of individual genes

showed various dependency on pM92 relative to that on viral DNA synthesis. Reduction in M116 and

M46 transcriptions in the absence of pM92 was comparable to that with PAA treatment, whereas the

reduction in M96 and M74 transcription in the absence of pM92 was less pronounced. This is reminiscent

of the previous report that different late gene transcriptions have different dependency on the viral late

transcription regulator pM79 (10). Our results indicate that pM92 plays an important role in regulating late

gene transcription during MCMV infection.

CMV UL92/M92 proteins interact with UL79/M79 proteins during infection. We have previously found

that MCMV protein pM79 regulates viral late transcription (10). As we showed here that pM92 also played

a critical role in late gene expression, we hypothesized that these two proteins might interact and form a

functional complex to exert this regulatory activity. To test this, we created a retroviral vector expressing

the C-terminally HA-tagged M79 ORF (pM79ha) or MCMV M38 ORF as a control (pM38ha), and

transfected it into MEF10.1 cells to generate expression cells. Transfected cells were subsequently

infected with SMflag92 and cell lysates were collected at 48 hpi. FLAG-tagged pM92 complexes were

immunoprecipitated using the mouse anti-FLAG antibody and analyzed by immunoblotting (Fig. 5).

pM79ha co-immunoprecipitated with pflagM92, indicated by its relative abundance in the eluted sample

as compared to that in the flow-through sample. The interaction was specific, as the control pM38ha, a

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protein not thought to be involved in late gene regulation, was only detected in the flow-through wash but

not in the eluted sample. These results suggest that pM79 and pM92 interact during MCMV infection.

As MCMV M79 and HCMV UL79 play a similar role in late gene expression during infection (10,

41), we wanted to determine whether pUL79 also interacted with pUL92, the HCMV homologue of pM92,

during infection. To test this, we infected human foreskin fibroblasts (HFFs) cells with recombinant HCMV

expressing C-terminally 3×FLAG-tagged pUL79 (AD flag UL79), or wild-type HCMV expressing GFP

(ADgfp), collected lysates at 72 hpi, and isolated pUL79-containing complex by co-immunoprecipitation

using anti-FLAG antibody. Immunoprecipitants were resolved by SDS-PAGE gel followed by silverstain

analysis. Protein bands present in AD flagUL 79-infected samples but absent in ADgfp control samples

were extracted, and their identity was determined by mass spectrometry analysis. pUL92 was among the

pUL79-associated viral proteins identified by this analysis.

Taken together, our results suggest that two viral regulators of late gene expression, pM79 and

pM92, interact during MCMV infection, and this interaction is conserved between MCMV and HCMV.

pM92 trans complements the growth of pUL92-deficient HCMV virus. MCMV pM92 and HCMV

pUL92 share 50% identity and 71% similarity, and notably pM92 has an additional 30aa at the N-terminus

(Fig. 6A). As pM92 and pUL92 share significant sequence homology and a similar interaction partner (i.e.,

pM79 and pUL79, respectively), we hypothesized that pM92 and pUL92 were functional homologues. To

test this, we first constructed a pUL92-deficient HCMV recombinant BAC clone, pADinUL92, by FLP/FRT-

mediated BAC recombineering (Fig. 6B). pADinUL92 carried an 88-nt insertion at nt 124 of the UL92 ORF

to replace a 282-nt segment of the coding sequence (Fig. 6A). The location of the mutation was expected

not to interfere with expression of neighboring genes, namely the overlapping 3’-terminus of UL91 or 5’-

terminus of UL93. Transfection of pADinUL92 BAC into MRC5 cells failed to produce any infectious virus

even after 4 weeks of incubation, whereas cells transfected with the wild-type BAC pADgfp readily

developed complete CPE and full spread of virus-driven GFP expression. This was in accordance with

previous reports that UL92 is essential for HCMV viral replication in fibroblasts (61). To reconstitute

pUL92-deficient virus, we constructed a lentivirus carrying the C-terminally 3×FLAG tagged UL92 ORF,

and subsequently generated pUL92 expressing cells by lentiviral transduction (MRC5-UL92flag).

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Transfection of pADinUL92 into MRC5-UL92flag cells could now reconstitute infectious progeny virus,

ADinUL92, with wild-type titers. Therefore, pUL92 is essential for HCMV replication, and the growth

defect of the HCMV recombinant virus ADinUL92 was due to the disruption of pUL92 expression.

To determine if pM92 is the functional homologue of pUL92, we tested if pM92 expression could

trans complement the growth of pUL92-deficient virus. We created a lentivirus containing the C-terminally

3×FLAG-tagged M92 ORF, and subsequently generated pM92-expressing MRC5 cells by lentiviral

transduction (MRC5-M92flag). We then infected MRC5 cells expressing pUL92 (MRC5-UL92flag), pM92

(MRC5-M92flag), or empty vector (MRC5-vector) with ADinUL92 at an MOI of 0.01, and determined the

titer of the cell free virus produced at 14 dpi. Both infected MRC5-UL92flag and MRC5-M92flag showed

spread of the pUL92-deficient virus at 14 dpi whereas MRC5-vector showed little sign of CPE (Fig. 6C).

Analysis of the final titers of infected culture supernatants indicated that pM92 could complement

ADinUL92 to titers similar to that by pUL92 (Fig. 6D). PCR analysis of genomic DNA from MRC5-

UL92flag and pM92 MRC5-M92flag confirmed that there was no cross-contamination of these two cell

types (Fig. 6E). Therefore, pM92 and pUL92 have a conserved function during CMV infection.

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Discussion

The expression of late genes is an essential step for CMV to complete its lytic infection cycle. Key

viral factors required in this process could be attractive targets for antiviral strategies to prevent CMV

infection and disease. In this study we have determined the role of MCMV protein pM92 during virus

infection. pM92 accumulated at late times, and localized to nuclear replication compartments during

infection (Fig. 2). When pM92 was abolished, the accumulation of early gene products and viral DNA was

minimally affected, but the accumulation of late gene products was markedly reduced (Figs. 3-4). As a

result, the mutant virus failed to complete the infection cycle to produce progeny virus (Fig. 1). Therefore,

pM92 is a novel regulator of viral late gene expression and thus plays an essential role in the MCMV lytic

infection cycle.

Our study provides additional evidence that the regulatory mechanism of viral late gene

expression is conserved between MCMV and HCMV. We have previously shown that MCMV protein

pM79 and its HCMV homologue pUL79 regulate viral late gene expression (10, 41). In this study, we

demonstrated that pM92 interacted with pM79 during MCMV infection; likewise pUL92 could interact with

pUL79 during HCMV infection (Fig. 5). This suggests that during betaherpesvirus infection, a complex

containing similar components of virus-encoded factors forms to promote late gene expression. How this

complex functions and what additional protein components are in this complex remain important

questions. Furthermore, we demonstrated that viral protein pUL92, the HCMV homologue of pM92, was

also essential for virus infection (Fig. 6). Importantly, pM92 could trans complement the growth of pUL92-

deficient HCMV recombinant virus (Fig. 6). These experiments do not specify whether the compensation

occurs at the transcriptional or translational level during HCMV infection, and further work is required to

define the exact mechanism at play. Regardless, this work suggests a conserved function for pM92

homologues among betaherpesviruses.

Our study also provides additional evidence that viral DNA synthesis is necessary but not

sufficient to drive late viral gene expression during herpesvirus infection. Inhibition of the viral polymerase

by PAA abolishes the accumulation of late transcripts (23, 25, 26, 51). This dependence on viral DNA

synthesis has been linked to the origin of lytic replication (oriLyt), as the oriLyt sequence is required in cis

for proper expression of late transcripts in many herpes viruses (2, 11, 14, 29, 55). However, our previous

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data and the data presented here demonstrate that viral gene expression at late times of infection

depends not only on viral DNA in cis, but also on viral factors such as pM92 and pM79 in trans (10). In

the absence of these viral factors, DNA synthesis kinetics was indistinguishable from wild-type virus

despite a defect in late transcript accumulation. Therefore, pM92 does not function as a viral DNA

synthesis protein; rather it specifically acts on gene expression at late times of infection.

What is the mechanism of pM92 activity? It has been established that herpesviral genomes

associate with histones during infection and require epigenetic regulation for gene expression (7, 12, 21,

30, 34, 38, 39). One possible mechanism is that the pM92/pM79 complex may activate late gene

transcription by remodeling the chromatin structure of the viral genome. This could be accomplished by

recruiting chromatin-remodeling complexes to the late gene loci, and rendering their promoters accessible

for transcription. Such activity has been observed for HSV-1 late gene trans factors (13, 24). Alternatively,

pM92/pM79 could play a more direct role in transcription. Herpesvirus genes are transcribed by the

cellular RNA polymerase II (RNAPII) (5, 33, 53, 58), a 12-subunit multi-protein enzyme that requires a

host of accessory scaffold and regulatory proteins for its activity. The pM92/pM79 complex could play an

essential role in the recruitment or assembly of these components on viral late gene promoters. Such a

mechanism has been suggested for regulation of late gene expression in both alpha and

gammaherpesviruses (30, 59). Finally, the activity of pM92/pM79 complex could be required at the stage

of post transcriptional modification. It is clear that accumulation of late transcripts was defective during

mutant virus infection, but it remains to be determined if this defect results from a failure in transcription or

an alteration in mRNA stability. HSV-1 endoribonuclease VHS-RNase is tightly regulated by at least four

other viral proteins in order to prevent it from degrading viral mRNAs (32, 45, 46, 52). Precedent for viral

regulation of viral mRNA accumulation also exists in HCMV, as viral protein IE2 can inhibit its own

transcription by binding to the MIEP promoter (35, 49). Thus we cannot rule out the possibility that during

pM92-deficient mutant virus infection, a defect in RNA trafficking, stability, or processing results in higher

RNA turnover rates.

Our efforts are currently underway for a better mechanistic understanding of the role of pM92 in

late gene regulation. Identification of cellular and viral factors that interact with pM92 is anticipated to

provide important insights into its function. Though pM79 is one interaction partner of pM92, it is almost

112

certain that many additional partners exist. Furthermore, genetic and protein analysis to identify functional

domains and structural elements of pM92 will be invaluable to understand the mechanistic basis for its

activity and to determine additional functions that pM92 may have. Finally, it is tempting to speculate that

late gene expression regulators such as pM92 and pM79 could play a role in the establishment of latency.

As both proteins are essential for the lytic viral life cycle, regulation of their activity and/or expression may

be a deciding factor for viral latency and reactivation.

In summary, we have identified pM92 as a novel late gene regulator in MCMV lytic infection,

shown its interaction with another late gene regulator pM79, and demonstrated its conserved function

with its HCMV homolog, pUL92. pM92 represents a potential new target for therapeutic intervention in

CMV disease, and a gateway into studying a largely uncharted viral process that is critical to the viral life

cycle.

113

Acknowledgements

We thank Herbert Virgin and the members of his laboratory for helpful discussion and invaluable

advice; Dr. Ulrich Koszinowski (Max von Pettenkofer-Institute, Ludwig Maximilians-University, Germany),

Dr. Martin Messerle (Hannover Medical School, Hannover, Germany), and Dr. Wolfram Brune (Heinrich

Pette Institute, Leibniz Institute for Experimental Virology, Germany) for the MCMV BAC clone pSM3fr;

Dr. Anthony Scalzo (University of Western Australia) for M44 and gB antibodies; Dr. Stipan Jonjic

(University of Rijeka, Croatia) for IE1 and E1 antibodies; and members of the Yu lab for critical reading of

the manuscript.

This study was supported by Public Health Service grants RO1CA120768. D.Y. holds an

Investigators in the Pathogenesis of Infectious Disease award from the Burroughs Wellcome Fund.

114


Plasmids, antibodies, and chemicals. pYD-C433, pYD-C569, pYD-C245, and pYD-C618 were

retroviral vectors derived from pRetro-EBNA (31). pYD-C433 and pYD-C569 contained the C-terminally

HA tagged M38 and M79 coding sequences, respectively. pYD-C245 expressed the red fluorescent

protein (DsRed) (4) from an internal ribosome entry site (IRES). pYD-C618 was derived from pYD-C245,

and carried the N-terminally 1×FLAG tagged M92 coding sequence that was expressed together with

DsRed as a bicistronic transcript. pYD-C755 (gift from Roger Everett, University of Glasgow Center for

Viral Research), pYD-C678, and pYD-C780 were pLKO.1-based lentiviral expression vectors that carried

a puromycin resistance marker (15, 17). Both pYD-C780 and pYD-C678 were derived from pYD-C755,

and they carried the C-terminally 3×FLAG tagged M92 and UL92 coding sequences, respectively. pYD-

C191 carried a kanamycin selection cassette bracketed by two Flp recognition target (FRT) sites. pYD-

C630 was derived from pGalK (57) and carried a FRT-bracketed GalK/kanamycin dual selection cassette

(40). pYD-C746 was derived from pYD-C630, where a 3×FLAG sequence preceded the FRT-

bracketed selection cassette.

The primary antibodies included: anti-actin (clone AC15, Abcam); anti-FLAG polyclonal rabbit

antibody (F7425) and monoclonal mouse antibody (F1804) (Sigma); rat anti-HA (11867423001, Roche);

anti-MCMV IE1 (CROMA101) and E1 (CROMA103) (generous gifts from Dr. Stipan Jonjic, University of

Rijeka, Croatia); anti-MCMV M44 (3B9.22A) and gB (2E8.21A) (generous gifts from Dr. Anthony Scalzo,

University of Western Australia). The secondary antibodies used for immunoblotting were horseradish

peroxidase (HRP)-conjugated goat anti-mouse IgG, goat anti-rabbit IgG, and goat anti-rat IgG (Jackson

Laboratory). The secondary antibodies used for immunofluorescence were Alexa Fluor 594-conjugated

goat anti-mouse IgG and Alexa Fluor 488-conjugated goat anti-rabbit IgG (Invitrogen-Molecular Probes).


Aldrich); L-(+)-Arabinose (A3256-25G; Sigma Aldrich); TO-PRO3 iodide (T3605; Invitrogen); Dyanbeads

(Novex, Life technologies); Benzonase (Novagen, Fisher Scientific).

Cells and viruses. Mouse embryonic fibroblast 10.1 cells (MEF10.1) (22), human embryonic lung

fibroblasts (MRC5) (19, 28), and human foreskin fibroblasts (HFF) were propagated in Dulbecco modified

115

Eagle medium supplemented with 10% fetal bovine serum, nonessential amino acids, 1 mM sodium

pyruvate and 100 U/mL penicillin-streptomycin. Cells were maintained at 37oC and 5% CO2 in a

humidified atmosphere. To create cell lines stably expressing N-terminally 1×FLAG tagged pM92 (10.1-

flagM92), MEF10.1 cells were transduced three times with pYD-C618-derived retrovirus and allowed to

recover for 48 hours. DsRed-positive cells were cloned by limiting dilution. Clonal cell lines were tested for

their ability to produce virus upon transfection with the mutant MCMV BAC pSMin92 (see below). The cell

line yielding the highest titer at 5 days post transfection was used in this study. To create cells expressing

C-terminally 3×FLAG-tagged pM92 (MRC5-M92flag) or pUL92 (MRC5-UL92flag), MRC5 cells were

transduced with lentivirus reconstituted from pYD-C780 or C678, respectively, and allowed to recover for

48 hours (16). To create cells containing the vector control (MRC5-vector), MRC5 cells were transduced

with pYD-C755-derived vector lentivirus. Transduced cells were then selected with 1 µg/mL puromycin,

and then maintained with 0.5 µg/mL puromycin.

To reconstitute recombinant MCMV or HCMV viruses, confluent MEF10.1 or MRC5 cells were

electroporated with corresponding MCMV or HCMV BAC DNA (see below), respectively. Recombinant

MCMV SMgfp and SMflag92 were reconstituted in MEF10.1 cells. SMin92 was reconstituted in 10.1-

flagM92 cells. To reconstitute recombinant HCMV, BAC-HCMV DNA, pp71-expression plasmid, and

G403-expression plasmid were co-transfected into MRC5-UL92flag cells by electroporation (62). Cells

were plated on a 10 cm plate, medium was changed 24 hours post transfection, and virus was harvested

by collecting cell-free culture medium after the entire monolayer of cells was lysed. Alternatively, virus

stocks were produced by collecting cell-free supernatant from infected culture at a multiplicity of infection

(MOI) of 0.01-0.001. Virus titers were determined in duplicate by a tissue culture infectious dose 50

(TCID50) assay in the appropriate cell type. In experiments where comparative analysis was performed

between SMin92 or ADinUL92 with other recombinant viruses, titers of all viruses were determined in

10.1-flagM92 or MRC5-UL92flag cells, respectively.

BAC recombineering. Recombinant BAC clones in this study were created using the linear

recombination-based BAC recombineering protocol that we have previously established (40).

Recombination was carried out in E. coli strain SW105 that harbored either the MCMV or HCMV BAC

116

clone, and expressed an arabinose inducible Flippase gene for transient expression of Flp recombinase

(57).

Recombinant MCMV BAC clones were derived from the parental clone pSM3fr that carried a full

length genome of the MCMV Smith strain (56). pSMgfp, used as the wild-type clone in this study,

contained the green fluorescent protein (GFP) expression cassette at the C-terminus of the IE2 loci,

which has been shown to be dispensable for MCMV infection in vivo and in vitro (8, 9, 36). The clone

pSMin92 carried a frame-shift mutation in the MCMV gene M92 (Fig. 1A). To construct pSMin92, the

FRT-bracketed GalK/kanamycin cassette was PCR amplified from pYD-C630 and recombined into

pSMgfp at nucleotide (nt) 358 of the M92 coding sequence. The selection cassette was then removed by

arabinose induction of Flp recombinase and subsequent Flp-FRT recombination (40), leaving an 88-nt

insert within M92 to create a frame-shift mutation. The clone pSMflag92 contained an N-terminally

3×FLAG-tagged M92 coding sequence (Fig. 1A). To construct pSMflag92, a fragment containing the

FRT-bracketed GalK/kanamycin selection cassette preceded by a 3×FLAG sequence was PCR amplified

from pYD-C746 and recombined into the N-terminus of the M92 coding sequence of pSMgfp. The

selection cassette was subsequently removed by Flp-FRT recombination, resulting in the 3×FLAG fused

in frame with the M92 coding sequence. Recombinant HCMV BAC clones were derived from the parental

clone pAD/Cre that carried the full length genome of HCMV strain AD169 (62). pADgfp, used as the wild-

type clone in this study, had a GFP gene in place of the viral US4-US6 region (54, 62). The clone

pADinUL92 was created in a similar manner to that of pSMin92, except that the insertion replaced nt 125-

406 of the UL92 coding sequence carried in pADgfp. All of the BAC clones were validated by restriction

digestion, PCR analysis, and direct sequencing as previously described (60).

Viral growth analysis. MEF10.1 or MRC5 cells were seeded in 12-well plates overnight to produce a

confluent monolayer. Cells were inoculated with recombinant viruses for 1 hour at an MOI of 2 for single

step or 0.01 for multistep growth analysis. The inoculum was removed, infected monolayer was rinsed

with phosphate-buffered saline (PBS), and fresh medium was replenished. At various times post infection,

cell-free virus was collected in duplicate by harvesting medium from infected cultures. Cell-associated

virus was collected by rinsing infected cells once with PBS and scraping cells into fresh medium. Cells

117

were lysed by one freeze-thaw cycle followed by sonication. Lysates were cleared of cell debris by low

speed centrifugation and supernatants were saved as cell-associated virus. Virus titers were determined

by TCID50 assay.

DNA and RNA analysis. Intracellular DNA was measured by quantitative PCR (qPCR) as previously

described (10). Briefly, cells were collected in a lysis buffer (200 mM NaCl, 20 mM Tris [pH 8.0], 20 mM

EDTA, 0.2 mg/ml proteinase K, 0.4% sodium dodecyl sulfate [SDS]), and lysed by incubation at 55°C

overnight. DNA was extracted with phenol-chloroform and treated with RNase A (100 µg/ml) at 37oC for 1

hour. Samples were extracted again with phenol-chloroform, precipitated with ethanol, and resuspended

in nuclease-free water (Ambion). Viral DNA was quantified by qPCR using SYBR Advantage qPCR

Premix (Clonetech) and a primer pair specific for the MCMV IE1 gene (10). Cellular DNA was quantified

using a primer pair specific for the mouse actin gene (10). A standard curve was generated using serially

diluted pSMgfp BAC DNA or cellular DNA, and was used to calculate relative amounts of viral or cellular

DNA in a sample. The amount of viral DNA was normalized by dividing IE1 equivalents over actin gene

equivalents. The normalized amount of viral DNA in SMgfp infected cells at 10 hours post infection (hpi)

was set at 1.

Intracellular RNA was determined by reverse transcription-coupled qPCR (RT-qPCR) as






Advantage qPCR Premix (Clonetech) and primer pairs specific for viral genes or cellular β-actin (Table 1).

A standard curve was generated for each gene using serially diluted cDNA from infected cells, and was

used to calculate the relative amount of a transcript in each sample. The amounts of viral transcript were

normalized by dividing viral transcript equivalents over actin equivalents. The normalized amount of


118

Protein analysis. Protein accumulation was analyzed by immunoblotting. Cells were washed and lysates






Intracellular localization of proteins of interest was analyzed by immunofluorescence assay. Cells

were seeded onto cover-slips 24 hours prior to infection. At 24 hours post infection, cells were washed

with PBS, fixed and permeabilized with methanol (-20oC) for 10 minutes, and blocked with 5% FBS in

PBS at room temperature for 1 hour. Cells were incubated with primary antibodies for 30 minutes at room

temperature and subsequently labeled with secondary antibodies coupled to Alexa Fluor 488 or Alexa

Fluor 594 (Invitrogen-Molecular Probes). Cells were counterstained with TO-PRO3 and mounted on

slides with Prolong Gold antifade reagent (Invitrogen-Molecular Probes). Confocal microscopic images

were captured by a Zeiss LSM510 Meta confocal laser scanning microscope.

Protein interactions were analyzed by co-immunoprecipitation. For MCMV, MEF 10.1 cells

transiently expressing C-terminally HA tagged M38 or M79 were infected with SMflag92 at an MOI of 2.

Cells were collected at 48hpi and lysed by incubation with extraction buffer (50 mM Tris pH 8.0, 300 mM

NaCl, 0.5% NP-40) for 15 minutes. In the meantime, 1 µg FLAG-antibody was conjugated to 25µL

Dynabeads (Novex, Life technologies) by incubation in conjugation buffer (0.02% Tween 20 in PBS, pH

7.2) for 20 minutes at room temperature. Lysates were cleared by centrifugation, pellet of cellular debris

was saved, and supernatant was incubated with FLAG antibody-conjugated Dynabeads in the presence

of endonuclease Benzonase (800 U/mL) (that digested DNA and prevented DNA-mediated, nonspecific

interactions among DNA-binding proteins). After an overnight incubation, beads were washed four times

with extraction buffer, and supernatant was saved as flow-through. Washed beads were mixed with

NuPAGE LDS sample buffer (Invitrogen) and boiled to elute FLAG-associated proteins. Pellets, flow-

through, and eluted samples were analyzed by a SDS-PAGE gel followed by immunoblotting. For HCMV,

HFF cells were infected with ADgfp or ADflagUL79 (41) at an MOI of 3. Infected cells were collected and

lysed at 72 hpi, and immunoprecipitation was performed as described above. After elution, the samples

119

were resolved on a SDS-PAGE gel, and protein bands were visualized by silverstain using the

ProteoSilver Plus Silver Stain Kit (Sigma).

120

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TABLE 1. Primers used in PCR analysis Primer Name Primer Sequence

MCMV IE1 forward 5'-CAGGGTGGATCATGAAGCCT-3'

MCMV IE1 reverse 5'-AGCGCATCGAAAGACAACG-3'

MCMV M34 forward 5’-TACTCTGATCGCGAACAGCA-3’

MCMV M34 reverse 5’-GCGTTGGTGGTTCGTGTCTG-3’

MCMV M37 forward 5’-ATGACGGCGGTCCTCTCCAT-3’

MCMV M37 reverse 5’-ACGTGTACGTCTTCCGTGCC-3’

MCMV M45 forward 5’-GGAACTCCTTGGTCATGCGA-3’

MCMV M45 reverse 5’-TTCCCCAAGTTCCCTAAGAG-3’

MCMV M46 forward 5’-CTGAACCTATAGCGCAGGAC-3’

MCMV M46 reverse 5’-ACTATAATAGCGATCGGCAG-3’

MCMV M55 (gB)forward 5'-GCGATGTCCGAGTGTGTCAAG-3'

MCMV M55 (gB) reverse 5'-CGACCAGCGGTCTCGAATAAC-3'

MCMV M74 forward 5’-AGGAGGCTGTGACTTTGAAA-3’

MCMV M74 reverse 5’-CTCATCAGCCGTTACTCGAG-3’

MCMV M85 forward 5’-TTTCATGAGGAGCATGTTGC-3’

MCMV M85 reverse 5’-CTGCATCTCGCTCTCCATGA-3’

MCMV M92 forward 5'-AAACCCACGGAGAATGCGAT-3'

MCMV M92 reverse 5'-ACGAACAGCTGACCTATGACGC-3'

MCMV M96 forward 5’-TCGAGGCGTTCGGTCCTGAT-3’

MCMV M96 reverse 5’-CGCATTCTCGGATACTCGCT-3’

MCMV M102 forward 5’-AGACCAGTACGGCGATCCAG-3’

MCMV M102 reverse 5’-AGCTTCCTGTAGGCGTGCGT-3’

MCMV M112/113 (E1) forward 5'-GAATCCGAGGAGGAAGACGAT-3'

MCMV M112/113 (E1) reverse 5'-GGTGAACGTTTGCTCGATCTC-3'

MCMV M116 forward 5’-TCCTTGGTGGTGATGGCGGT-3’

MCMV M116 reverse 5’-GCATCCCGTACCTGACCACA-3’

Mouse actin forward 5'-GCTGTATTCCCCTCCATCGTG-3'

Mouse actin reverse 5'-CACGGTTGGCCTTAGGGTTCA-3'

M92 forward a 5’-ATGTTCTCACACGGCCGAGA-3’

M92 reverse a 5’-CTAGCGGCCTGTTCGAAACG-3’

UL92 forward a 5-ATGTGCGACGCCTCGGGCGCCTG-3’

UL92 reverse a 5’-AACGCCAGATCCGAATACAGGTG-3’ a primers used in PCR analysis of cellular DNA from pM92- or pUL92-expressing MRC5 cells.

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Figure 1. pM92 is essential for MCMV replication in fibroblasts. (A) Diagram depicting the

construction of MCMV BACs used in this study by BAC recombineering. To create pSMin92, an 88-nt

insert was introduced (indicated by the black bar) at nt 358 of the M92 coding sequence, resulting in a

frame-shift mutation. pSMflag92 was created by fusing a 3×FLAG tag in frame at the N-terminus of the

M92 coding sequence (indicated by the shaded bar). (B) Growth of SMin92 virus in MEF 10.1 cells

expressing FLAG-tagged pM92 (10.1-flagM92). Left panels are fluorescent images of virus-driven GFP

expression in MEF 10.1 cells or 10.1-flagM92 cells, both of which were transfected with pSMin92. Images

were taken at 7 days post transfection. Right panels are titers of cell free virus at 72 hours post infection

(hpi) from 10.1-flagM92 cells infected with either SMgfp or SMin92 at a multiplicity of infection (MOI) of 2.

(C-D) Growth kinetic analysis of MCMV recombinant viruses used in this study. MEF 10.1 cells were

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infected with indicated viruses at an MOI of 2 (for single-step growth analysis) or 0.001 (for multi-step

growth analysis). Cell free and cell associated viruses were collected at indicated times and titers were

determined by tissue culture infectious dose 50 (TCID50) assay in 10.1-flagM92 cells. The detection limit

of the TCID50 is indicated with a dashed line.

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Figure 2. pM92 accumulates abundantly at late times of infection and localizes to viral nuclear

replication compartments. (A) Accumulation of the M92 protein product during MCMV infection. MEF

10.1 cells were infected with SMflag92 virus at an MOI of 2. Total cell lysates were harvested at indicated

times post infection and analyzed by immunoblotting. The M92 protein was detected with the mouse anti-

FLAG antibody, and actin was included as a loading control. (B) Accumulation of the M92 transcript

during MCMV infection. MEF 10.1 cells were infected with SMgfp at an MOI of 2 in the presence or

absence of viral DNA synthesis inhibitor phosphonoacetic acid (PAA) (200 µg/ml). Total RNA was

isolated at indicated times post infection and the M92 transcript was measured by reverse transcription-

coupled quantitative PCR (RT-qPCR) analysis with the primers listed in Table 1. The values were

normalized to β-actin, and the normalized amount of M92 transcript at 10 hpi in the absence of PAA was

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set to the value of 1. (C-D) The M92 protein localizes to nuclear replication compartments during

infection. MEF 10.1 cells were mock infected or infected with SMgfp or SMflag92 at an MOI of 2. At 24

hpi, cells were permeablized and fixed with methanol , and stained with either rabbit polyclonal (C) or

mouse monoclonal (D) anti-FLAG antibody to detect the FLAG-tagged M92 protein (green). In (C), cells

were also stained with mouse antibody to pM44, which served as a marker for replication compartments.

Cells were counterstained with TO-PRO3 to visualize the nuclei. Scale bars are equivalent to 20 µm.

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Figure 3. pM92 is dispensable for viral DNA synthesis but is required for efficiently accumulation

of M55 late gene products during infection. (A) Accumulation of representative viral proteins during

pM92-defcient virus infection. MEF 10.1 cells were infected with SMgfp or SMin92 at an MOI of 2. Cell

lysates were harvested at indicated times and analyzed by immunoblotting. (B) Accumulation of viral

transcripts during pM92-deficient virus infection. MEF 10.1 cells were infected with SMgfp in the presence

or absence of viral DNA synthesis inhibitor PAA (200 µg/mL), or with SMin92, at an MOI of 2. At 10 and

20 hpi, total RNA was harvested and specific transcripts were quantified by RT-qPCR analysis using the

primers listed in Table 1. The values were normalized to β-actin, and the normalized amount of transcript

during SMgfp infection at 10 hpi in the absence of PAA was set to the value of 1. (C) Accumulation of viral

DNA during pM92-deficient virus infection. MEF 10.1 cells were infected as described in (A), total DNA

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was isolated from infected cells at the indicated times, and viral DNA synthesis was analyzed by

quantitative PCR (qPCR). The values were normalized to β-actin and the quantity of DNA during SMgfp

infection at 10 hpi was set to the value of 1.

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Figure 4. pM92 is required for efficient accumulation of a panel of late transcripts. MEF 10.1 cells

were infected with SMgfp in the presence or absence of viral DNA synthesis inhibitor PAA, or with

SMin92, at an MOI of 2. At indicated times, total RNA was harvested and then representative early

transcripts (A) and late transcripts (B) were quantified by RT-qPCR analysis using the primers listed in

Table 1. The values were normalized to β-actin, and the normalized amount of transcript during SMgfp

infection at 10 hpi in the absence of PAA was set to the value of 1.

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Figure 5. CMV UL92/M92 proteins interact with UL79/M79 proteins during infection. MEF10.1 cells

expressing either HA-tagged M79 (pM79ha) or HA-tagged M38 (pM38ha) were infected with SMflag92 at

an MOI of 2 and cell lysates were collected at 48 hpi. Cell lysates were separated into insoluble cell

debris (pellet, “P’) and supernatant by centrifugation. Supernatant was then immunoprecipitated with the

mouse anti-FLAG antibody, washed, and eluted. Cell debris (“P”), flow-through fraction collected by wash

(“FT”), and eluted fraction (“E”) were analyzed by immunoblotting using anti-FLAG and anti-HA antibody.

Molecular weight markers (in kDa) are also shown.

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Figure 6. pM92 trans complements the growth of pUL92-deficient HCMV virus. (A) Coding sequence

alignment of MCMV M92 with its homologues HCMV UL92 and MHV-68 ORF31. (B) Diagram depicting

the construction of pUL92-deficient recombinant HCMV BAC, pADinUL92, by BAC recombineering.

pADinUL92 carried an 88-nt insertion (indicated by black bar) at nt 124 of the UL92 ORF to replace a

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282-nt segment of the coding sequence. (C-D) Growth of ADinUL92 virus in MRC5 cells expressing C-

terminally 3×FLAG-tagged pUL92 or pM92. MRC5 cells expressing tagged pUL92 (MRC5-UL92flag),

tagged pM92 (MRC5-M92flag), or the empty vector (MRC5-vector) were infected with ADinUL92 at an

MOI of 0.01. At 14 dpi, cells were examined under a fluorescent microscope for virus-driven GFP

expression (C), and titers of cell free virus were determined by TCID50 assay in MRC5-UL92flag cells (D).

The detection limit of the TCID50 is indicated with a dashed line. (E) Cellular DNA of MRC5-UL92flag and

MRC5-M92flag cells was isolated, and the presence of UL92 and M92 DNA in these cells was

determined by PCR analysis using the primers listed in Table 1.

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Chapter IV

MCMV Late Transcription Regulator pM92 Interacts with pM79 and RNAP II During Infection

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Preface

TJC designed and performed the majority of experiments, analyzed the data, and generated the figures

presented in this chapter. ARF provided reagents, advice, and support for completion of the study, DY

supervised the studies and greatly contributed to the design and analysis of the experiments. The final

draft of this chapter was written and edited by TJC.

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Introduction

Human cytomegalovirus (HCMV), the prototypical member of the betaherpesvirus subfamily, is a

ubiquitous, species specific pathogen that infects 90% of the general population (40). Due to its broad cell

tropism, virus can be found in most organs and cell types after infection. In immuno-competent hosts, the

infection is typically asymptomatic. However, in immuno-compromised hosts, HCMV infection causes

significant morbidity and mortality (40, 56). HCMV is the leading viral cause of birth defects in perinatally

infected infants, and is the major cause of retinitis and blindness in AIDS patients (1, 21, 56). It is also a

prominent cause of disease in patients under immuno-suppressants, such as bone marrow and solid-

organ transplant recipients, or cancer patients receiving chemotherapy (58). HCMV infection has also

been associated with cardiovascular disease and proliferative diseases such as cancer (54). After the

resolution of acute infection, HCMV establishes persistent life-long infections characterized by alternate

stages of virus productivity and latency (40). Lytic infection, both during primary infection and after

reactivation from latency, can cause severe disease. There is no vaccine to HCMV, and antiviral

therapies have poor toxicity scores, low availability, and are quickly becoming outdated by resistant

viruses (6). For these reasons, it is important to understand the role of viral genes involved in lytic

infection, as they may yield new targets for anti-viral therapies.

Due to species restriction and a slow replication cycle, studies with HCMV can be difficult. Murine

CMV (MCMV) is a model to study HCMV biology due to its conservation with regard to virion structure,

genome organization, gene expression, and tissue tropism. MCMV infection recapitulates many clinical

and pathohistological features of HCMV infection (18, 49, 50, 53). In addition MCMV contains many

functionally conserved genes and provides a tractable small-animal model for experimental MCMV

infection in vivo. The study of conserved lytic genes in MCMV will significantly contribute to the

understanding of their roles in viral replication and in the pathogenesis of HCMV.

Viral gene expression during lytic infection is temporally regulated and sequentially divided into

immediate early, early, and late phases (40). The Immediate early (IE) genes are expressed first requiring

only the incoming virion associated proteins and/or cellular transcription factors. The IE genes

transactivate the early genes (E) and modulate the cellular microenvironment to permit viral genome

replication. These proteins localize to a subnuclear domain, termed the viral replication compartment,

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where viral DNA synthesis, late gene transcription, and viral genome encapsidation take place (40).

Following viral DNA replication, late genes (L) coding for structural proteins are expressed. On a finer

scale, an intermediate category of L genes is defined and classified as early-late (also called delayed-

early or leaky-late), depending on their requirement for viral DNA replication. Early-late genes are

expressed preceding viral DNA replication, and their transcription rate is enhanced considerably during

DNA synthesis. True-late genes absolutely require that viral DNA replication occurs before expression

(40).

Though the regulation of IE and E gene expression has been studied extensively, very little is

known regarding the regulation of late gene expression during MCMV infection. Previous studies have

shown that the activation of both MCMV and HCMV late gene promoters is dependent on the origin of

viral DNA synthesis (ori-lyt) in cis (2, 14, 42). This further supports the notion that late gene transcription

is tightly coupled to viral DNA synthesis. However, additional levels of regulation for viral late gene

expression remain poorly defined.

During cytomegalovirus infection, host cell RNA polymerase II (RNAP II) and associated basal

transcription machinery direct the transcription of CMV genes. RNAP II is a 12-subunit DNA-dependent

RNA polymerase that is responsible for transcribing nuclear genes encoding messenger RNAs and

several small nuclear RNAs (45). RNAP II and its general initiation factors assemble on promoter DNA,

creating a large multi-protein DNA complex that supports accurate transcription initiation. Transcriptional

activators and coactivators, regulate the rate of RNA synthesis from each gene in response to various

developmental and environmental signals. Host cell transcription factors are influenced by virus-encoded

transactivators that modulate expression of both viral and host genes during infection (51, 59, 68). It has

been shown in HSV-1 that ICP4 can recruit RNAP II via accessory proteins to viral promoters and aid in

the process of transcription (7, 31, 55, 61). It has also been shown that proteins ICP22 and UL13 are

required for changing the phosphorylation status of RNAP II, which presumably has an impact on the

transcription of viral genes (19, 36). ICP22 and ICP4 also play roles in regulating the transcription

accessory proteins during transcription, as ICP4 and ICP22 are involved in the recruitment of TAF and

acetylated histones to viral promoters (24, 31, 52, 55). Likewise, it has been shown that ORF30 and

ORF34 are involved in recruiting RNAP II to the promoter of gammaherpesviruses genes during infection

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(66). This result indicates a critical role of ORF30 and ORF34 in stimulating the assembly of the

transcription complex on the late gene promoters. However, the role of RNAP II in gammaherpesviruses

transcription has not been further explored, and ORF30 and ORF34 do not have homologues in HSV-1.

Analysis of the RNAP II complex during CMV infection has not been done. Thus, the mechanism by

which RNAP II is recruited to viral promoters during CMV infection is still unknown.

Previously, it has been demonstrated that HCMV encodes five essential proteins, UL79, UL87,

UL91, UL92, and UL95, which were shown to be key regulators of late gene expression during infection

(28, 47, 48). In the absence of these genes, transcription of known late genes was abrogated during

HCMV infection; however, DNA replication was unaffected (48). The homologues of UL79 and UL92 in

MCMV, M79 and M92 respectively, have also been shown to be required for late gene expression (9, 11).

It was shown that M92 can complement HCMV deficient in UL92, implying that the mechanism of function

is similar between the human and mouse virus. Homologs of UL79, UL87, UL91, UL92, and UL95 are

conserved in beta and gamma herpesviruses, including HHV-6, EBV, KSHV, and MHV68. In MHV68

these homologues, ORF18, ORF24, ORF30, ORF31, and ORF34, respectively, have been found to

regulate late gene transcript accumulation during infection (3, 30, 65, 66).These proteins do not have

homologues in HSV-1, and the mechanism by which these genes function has not been determined (41,

46). Understanding how these late transactivators function during MCMV infection will be important to

understand how late genes are regulated during HCMV infection and will give insight into how

gammaherpesviruses regulate late gene expression.

In this study, we will further investigate the molecular bases for gene functions by proteomic

analysis to identify binding partners of pM79 and pM92. We use an unbiased approach for this objective

by utilizing the co-immunoprecipitation (co-IP)-coupled mass spectroscopy strategy. This analysis

revealed that both pM79 and pM92 interacted with a panel of viral and host proteins, including host

proteins involved in transcription and putative viral late transactivators. The mass spectroscopy analysis

was confirmed by directed co-IP in which pM79 and pM92 were tested for their interaction with RNAP II

during infection. It was found that RNAP II copurified with both pM79 and pM92. However, the interaction

between pM92 and RNAP II appeared to be at least partially facilitated by pM79, as pM79 was required

for this interaction. To get a better understanding of the mechanism by which pM92 functions we propose

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to try to identify the functional domains of pM92 by creating a series of pM92 mutants and screening them

for their ability to maintain interaction with binding partners RNAP II and pM79. Though these studies are

not complete, we can at least conclude that during late times of infection pM79 and pM92 form a complex

with RNAP II. Analyzing the interactome of these late gene transactivators and identifying the domains

necessary for late gene expression will provide important insights into how late gene regulation occurs

during betaherpesvirus infection.

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Results

Recover proteins bound to late transcription regulators by mass spectrometry analysis. In an initial

effort to identify proteins associated with pM79 and pM92 during MCMV infection, we first created a

recombinant MCMV in which the M79 or M92 coding sequence was tagged with the 3×FLAG sequence.

The M79 ORF was tagged on the C-terminus (SM79flag) and the M92 ORF was tagged on the N-

terminus (SMflag92) so that protein complexes containing either recombinant protein could be isolated by

co-immunoprecipitation (co-IP) with an anti-FLAG antibody. It has been shown previously that both

single-step and multi-step growth curve analyses in MEF 10.1 fibroblasts indicated that SM79flag and

SMflag92 grew similarly to SMgfp (wild-type), suggesting that the 3×FLAG tag did not interfere with the

function of the tagged gene or neighboring genes (9, 11). We then infected MEF 10.1 cells with SM79flag

or SMflag92 at an MOI 2 and collected lysates at 48hpi for co-IP with the anti-FLAG antibody. The lysates

were incubated with an extraction buffer designed to isolate whole cell proteins, the lysates were clarified

by centrifugation, and the supernatant was incubated with magnetic beads conjugated to anti-FLAG

antibody. The same procedure was performed for cells infected with SMgfp as a negative control. The

endonuclease Benzonase was also added to each sample in an effort to reduce non-specific binding of

proteins that are only indirectly associated with pM79 and pM92 via DNA interactions (57).

Immunoprecipitated proteins were resolved by SDS-PAGE and visualized by silver-staining (Fig. 1).

Silver-stain analysis revealed a number of protein bands that were unique to the pM79 and pM92

containing samples that could not be seen in the SMgfp control lane (Fig. 1). We extracted the protein

bands that were the most easily visualized, and determined their identities by mass spectrometry. As a

negative control, we also extracted gel bands from the SMgfp sample with migrating positions

corresponding to those of the SM79flag and SMflag92 specific protein bands. Table 1 and Table 2 lists

the full set of cellular proteins that were identified by this approach and unique to SM79flag and SMflag92

co-IP, respectively. Table 3 lists the full set of viral proteins that were identified by this approach and

unique to SM79flag and SMflag92 Co-IP samples.

It can be seen that pM79 and pM92 share many interaction partners during infection. This is not

surprising since viruses deficient for either of these proteins have similar phenotypes. The list of cellular

proteins can be categorized into several functional groups. The prominent group of cellular proteins that

143

copurify with the viral transactivators are proteins involved in transcription. That includes RNA helicases,

histones, and most notably, three out of the twelve RNA polymerase II (RNAP II) subunits were identified

(Table 1 and 2). The largest subunit, Rpb1, has been shown to be very important for regulation of viral

transcription in other herpesviruses, primarily through regulation of the C-terminal domain, which can be

phosphorylated to regulate transcription activity and interaction with various transcription accessory

proteins. In Table 3 it can be seen that several viral proteins that are conserved in beta and

gammaherpesviruses were found to complex with pM79 and pM92. In HCMV pUL87 and pUL95 together

with pUL79 or pUL92, all of which are homologous to the MCMV proteins of similar name, have been

shown to be essential for viral late gene expression (15, 28, 47, 67). The functions of pM87 and pM95 are

not known in MCMV, but it is likely that they interact with pM79 and pM92 to facilitate late gene

expression. Interestingly, proteins involved in viral DNA synthesis, including the pM112/113 and the viral

polymerase pM54, were found to interact with pM79 or pM92 (41). This suggests that these late gene

transactivators may partially act by regulating viral DNA replication. Finally, several cellular proteins

involved in post-transcription mRNA activities, including proteins involved in splicing and translation were

also copurified with pM79 and pM92. The list of proteins that copurified with pM79 and pM92 suggests

that these two viral proteins are directly involved in late gene transcription during infection.

pM79 and pM92 interact with RNAP II during virus infection. We next validated the mass-spec

analysis to confirm the interaction of pM79 and pM92 with RNAP II. MEF 10.1 cells were infected with

either SM79flag, SMflag92, or SMgfp (negative control), cell lysates were collected at 48hpi, and lysates

were subjected to co-IP using anti-FLAG antibody. The IP elutions were resolved by SDS-PAGE gel and

immunoblotted with both FLAG and RNAP II antibody to the Rpb1 subunit. The results indicate that

RNAP II copurifies with both pM79 and pM92 (Fig. 2 and data not shown). This interaction was specific

for the FLAG tagged protein as RNAP II did not co-immunoprecipitate in the SMgfp control sample. In

addition, the use of benzonase in this assay rules out the possibility that the observed interaction is a

byproduct of DNA/RNA binding by any of these proteins. Taken together, these results indicated that

pM79 and pM92 interact with RNAP II during infection, and that this association is not mediated by

nucleic acids.

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We then determined whether the viral proteins could interact with RNAP II independent of

additional viral factors. To achieve this, we transfected MEF10.1 cells with a plasmid expressing either HA

tagged pM79 (pM79ha) or FLAG tagged pM92 (pflagM92). Lysates were collected at 48hpi, and

subjected to co-IP using anti-HA or anti-FLAG antibody. The IP elutions were resolved by SDS-PAGE gel

and immunoblotted with both FLAG and RNAP II antibody to the Rpb1 subunit. Cells transfected with

unrelated viral protein M38 with an HA tag (pM38ha or M44 with a FLAG tag (pM44flag) were used as

negative controls. RNAP II copurified with pM79ha in the absence of viral factors, an interaction that was

specific due to a failure for RNAP II to copurify with pM38ha (data not shown). Interestingly, RNAP II did

not copurify with pflagM92. This implies that pM79 and pM92 are functioning in late gene regulation at

different capacities.

pM92 requires viral accessory proteins to facilitate interaction with pM79. Though many viral

proteins were common partners for pM92 and pM79, these late gene regulators were not identified as

interacting partners for each other. This could be due to an extraction issue (both proteins run near the

IgG band) or / and a sensitivity issue (proteins could be present in low abundance). Thus, an experiment

was set up in which a vector containing M79 C-terminally tagged with HA (pM79ha) was transfected into

MEF 10.1 cells. These cells were subsequently infected with SMflag92 at an MOI 2. Cell lysates were

collected at 48hpi and subjected to co-IP using an anti-FLAG antibody. The IP eluted proteins were

resolved by SDS-PAGE gel and immunoblotted with both anti-FLAG and anti-HA antibodies. Cells

transfected with unrelated viral protein pM38 with an HA tag (pM38ha) were used as negative controls.

The blot revealed that pM79ha copurified with SMflag92 (Fig. 3A). This interaction was specific due to a

failure of pM38ha to copurify with SMflag92. This data suggests that pM79 and pM92 are found in a

complex during virus infection.

Next we wanted to determine if pM92 and pM79 could interact independent of accessory viral

proteins. We transfected MEF 10.1 cells with a vector containing pM79ha and a vector containing

pflagM92. These cells were allowed to incubate for 36 hours before cell lysates were collected and

subjected to co-IP using either anti-HA or anti-FLAG antibody. The IP elutions were resolved by SDS-

PAGE gel and immunoblotted with both anti-FLAG and anti-HA. Cells transfected with pM79ha and

145

pM44flag or pflagM92 and pM38ha were used as negative controls. The blot revealed that pM79 and

pM92 were unable to complex in the absence of viral infection (Fig. 3B and data not shown). Thus, viral

accessory proteins are required for complex formation between pM92 and pM79.

Creation of M92 mutants. We propose to follow up the pM92 interaction studies by better characterizing

the interaction between pM92, pM79, and RNAP II. We intend to do this by creating a series of M92

mutant proteins. Mutagenesis will be done at highly conserved residues of the M92 ORF to determine

which regions are important for function, interaction with pM79, and interaction with RNAP II. This type of

analysis will help to map the functional domains of pM92 by potentially identifying regions that are

differentially important for binding pM79, and for maintaining its function as a transcriptional regulator.

With luck, we will be able to indentify mutants that maintain binding to pM79, but are unable to

complement SMin92 growth, giving us insight into the downstream functions of pM92 (ex. domains

involved in the recruitment of other proteins).

In an effort to minimize the mutagenesis efforts, we constructed two retro viral vectors; each

containing a truncated version of the M92 ORF. One vector contained 110aa of the N-terminal portion of

M92, and the second vector contained 130aa of the C-terminal portion of M92. Both fragments of M92

were tagged with the FLAG epitope for downstream immunoassays. Each vector was transfected

individually into fibroblasts, along with a vector containing HA tagged M79 (pM79ha). A vector containing

the full length M92 protein was also transfected into fibroblasts with the M79 containing vector as a

positive control. The cell lysates were then collected and subjected to co-IP using an anti-FLAG antibody.

We then performed a western blot on the collected samples, blotting with an antibody to the HA tag. The

blot revealed that neither pM92 fragment could pull-down pM79ha (data not shown). Western blot

analysis of the pM92 fragments revealed that the N-terminal fragment was not expressed in transfected

cells; likely due to rapid degradation. However, the C-terminal fragment was expressed at abundant

levels (data not shown). Thus, it appears that the C-terminal fragment is not sufficient for direct interaction

with pM79.

Since our initial analysis implies that the N- and C-terminal domains of pM92 are insufficient to

complement pM92 function, a more specific mutagenesis strategy will be required to parse out the

146

domains of pM92 required for its function. We analyze the M92 ORF and identified all of the residues that

are conserved between representative homologues. The alignment of the M92 homologues from

betaherpesviruses RhCMV, MCMV, HCMV, RCMV, HHV-6, and gammaherpesviruses MHV68, EBV, and

KSHV revealed that there are 15 residues that are identical between all homologues (Fig. 4).

QuickChange mutagenesis was designed to replace each of the first nine conserved residues with an

alanine. The primers designed for this purpose are listed in Table 4. There are five cysteine residues

among these mutants which are known to be important for protein structure; so it is very likely that we will

see a phenotype with one of these mutants that will inform us of the function of the N-terminus of pM92.

For the remaining six conserved residues, a single gene was synthesized that contained all six mutants.

The gene synthesis was done by the company Genewiz. This will tell us whether any of the conserved

residues are important in the C-terminus without having to design a QuickChange mutagenesis strategy

for each residue. It is possible that a single residue mutation will not disrupt the function of pM92 enough

to see a phenotype, which is why we choose to group the mutations in the C-terminus.

147

Future directions

At the time of writing this dissertation, the planned experiments for this study are not completed.

Though they will be completed at a later time, for the purposes of this dissertation, a description of the

planned experiments will have to suffice.

Impact of M92 mutants on protein-protein interactions. The mutants that were created for pM92 will

be tested for their ability to complement the growth of SMin92 mutant virus. The mutant M92 ORFs have

an N-terminal 3×FLAG tag in a retro-viral vector (pRetroflagM92). Retrovirus will be made for each of the

mutant containing vectors, and then 10.1 MEF cells will be transduced with one of these retroviruses. The

expressing cells will then be infected with SMin92, and tested for their ability to complement the mutant

virus growth. Any M92 mutant expressing cells that are able to complement the growth of SMin92 will be

deemed uninteresting for this study. Though those M92 mutant proteins may have other important

phenotypes under different conditions, we are only interested in M92 mutants that recapitulate the

essential phenotype of SMin92. Following the initial screen, we will test for the ability of the candidate

M92 mutant ORFs to produce protein. This is an important check because we are only interested in

mutant proteins that are stably expressed and folded during infection. Since we are mutating many

cystiene residues, it is likely that we will find some pM92 mutants that are misfolded and degraded. Such

proteins will not be informative about pM92 functional domains.

Once we have a candidate set of pM92 mutants that fail to complement SMin92 but are stably

expressed, then we are ready to test the mutant proteins for their ability to facilitate late gene

transcription. This function will be tested by infecting mutant expressing cells with SMin92, isolating RNA

by Trizol extraction, and performing RT-qPCR on representative transcripts from each kinetic class of

expression. We expect that many of the M92 mutant proteins will fail to compensate the late transcription

defect seen in SMin92. If it is observed that transcript accumulation is unaffected with some of these

pM92 mutants, then we’ll know that pM92 has functions beyond transcription which are important for the

lytic replication of MCMV. However, following up on such mutant proteins is outside of the scope of the

proposed work.

148

We will focus on those pM92 mutants that fail to complement the transcription defect of SMin92,

and analyze them for their functional interactions. We know that pM92 can interact with RNAP II during

infection (Fig 2). We will test whether the selected pM92 mutants can still interact with RNAP II. Mutants

that are able to maintain the interaction with RNAPII, but unable to restore late gene transcript

accumulation, will inform us that the interaction with RNAP II is not sufficient for the function that pM92

plays in late gene transcription. This may imply that pM92 recruits additional factors to the transcription

complex, or that pM92 has an enzymatic activity that is required for transcription initiation but dispensable

for protein binding.

Mutants that fail to bind RNAP II will be tested for their ability to bind to pM79. We saw that pM79

facilitates the interaction between pM92 and RNAP II (data not shown). Thus, it would be interesting to

determine if there are pM92 mutants that are able to bind pM79 despite a failure to associate with RNAP

II. This would suggest that the pM92-pM79 complex actually has additional functions outside of RNAP II

activity. Since we know that pM79 can directly interact with RNAP II, the ability to maintain association

with pM92 in the absence of RNAP II implies that the two proteins form multiple complexes together

which are likely involved in multiple processes important for lytic replication. This is supported by the fact

that both pM79 and pM92 have multiple overlapping binding partners not known to be involved in

transcription (Table 1 and 2).

In this proposed study, we will elucidate the many functional domains of the pM92 protein. All of

this information will no doubt increase the resolution of our understanding about late gene expression in

CMV, and about how these late transactivators function during infection.

149

Discussion

The expression of late genes is a critical step in the lytic infection of cytomegalovirus. Key

proteins involved in this process could be attractive targets for specific antiviral strategies. In this study,

we discovered a novel regulatory mechanism of viral transcription mediated by MCMV protein pM79 and

pM92. We identified cellular RNA polymerase II (RNAP II) as a key factor that interacted with these two

viral late transactivators. Co-immunoprecipitation (co-IP)-coupled mass spectroscopy studies

demonstrated that pM92 and pM79 interact with a wide array of cellular proteins involved in transcription.

Of these, RNAP II was confirmed by subsequent co-IP analysis. The interaction between pM79 and

RNAP II is a direct interaction, but pM92 requires viral accessory proteins to mediate complex formation

with RNAP II. We also showed that pM92 and pM79 interact during viral infection, but not when

expressed together in trans without viral infection. This suggests that pM92, pM79, and RNAP II form a

viral transcription complex that requires additional viral proteins to facilitate the interaction. The fact that

neither a M92 deficient virus nor a M79 deficient virus completes the viral replication cycle implies that

both members of the complex are required to facilitate transcription. This suggests that even though

pM79 directly binds to RNAP II, this interaction is not sufficient to facilitate transcription. How this complex

functions and what is the protein composition of the complex remain interesting questions.

We also set out to identify the pM92 residues required for its function as a viral transcription factor.

Though these studies were not completed at the time of this dissertation, the experiments have been

planned and will be completed in the near future.

How is late gene transcription regulated? It is known that herpes virus genomes associate with

histones during infection, and require epigenetic regulation for gene expression (8, 12, 22, 31, 37, 43, 44).

Thus, it’s possible that pM92 and pM79 activate late gene transcription by remodeling the chromatin

structure of the viral genome. In this way, it’s easy to imagine that pM92 and pM79 could play a role in

recruiting chromatin-remodeling complexes to the late gene promoters making them more accessible for

transcription. Such activity has already been observed for HSV-1 late gene trans factors (13, 26).

Another distinct possibility is that pM92 and pM79 are involved directly in transcription. Herpesvirus

transcription is driven by RNAP II, and RNAP II requires a host of accessory scaffold proteins and

enzymes for its function (5, 35, 60, 62). It’s easy to imagine that pM92 and pM79 could be involved in the

150

recruitment or assembly of these accessory components on late gene promoters. This behavior has been

observed in HSV-1, and has been suggested as a mechanism for gammaherpesvirus late gene regulation

(31, 66). It has been shown that late promoters of beta and gammaherpesviruses contain a non-canonical

TATA box sequence (23, 27, 64). In HCMV, several characterized viral late promoters contain the TATT

non-canonical TATA sequence, which is presumably recognized by the UL87 TATA box binding protein

(20, 29, 32, 34, 39, 63). Studies of the UL87 homologue in EBV, ORF24, suggest that this family of

proteins preferentially bind to non-canonical TATA sequences. Therefore, pM79 and pM92 may function

to guide transcription machinery to assemble around the UL87 binding site.

There is clearly much more to learn about pM92 and its role in late gene regulation. A few areas

of future research can be suggested. First, following up on some of the other cellular and viral factors that

interact with pM92 could provide significant insights into the protein’s function. In HSV-1 it has been

shown that Dead-box helicases play an important role in late gene regulation during infection (38).

Exactly what role dead-box helicases play during herpesvirus infection is still under investigation. Second,

further analysis of the pM92 mutant proteins will be important to identify the comprehensive list of pM92

functions during infection. We only identified the domains important for binding to RNAP II and pM79, it is

likely that other domains of pM92 have additional functions. Lastly, it would be interesting to see how

pM79 and pM92 participate in viral DNA synthesis. As both transactivators interact with viral DNA

synthesis proteins by mass-spec analysis (Table 3), it is possible that pM92 and pM79 regulate gene

expression by modulating viral DNA replication.

In summary, we have used a systematic proteomic approach to identify RNAP II as a binding

partner for both pM79 and pM92, and are on our way to determining what residues of pM92 are important

for both of these interactions. Further study of pM92 and its relationship to pM79 and RNAP II is required

to elucidate the molecular mechanism governing late gene regulation, potentially presenting new targets

for therapeutic intervention in CMV related disease. More importantly, this protein will serve as a gateway

into studying a viral process that is little defined and critical to the viral life cycle of both beta and

gammaherpesviruses.

151


Plasmids, antibodies, and chemicals. pYD-C433, pYD-C569, pYD-C245, and pYD-C618 were

retroviral vectors derived from pRetro-EBNA (33). pYD-C433 and pYD-C569 contained the C-terminally

HA tagged M38 and M79 coding sequences, respectively. pYD-C245 expressed the red fluorescent

protein (DsRed) (4) from an internal ribosome entry site (IRES). pYD-C618 was derived from pYD-C245,

and carried the N-terminally 1×FLAG tagged M92 coding sequence that was expressed together with

DsRed as a bicistronic transcript. pYD-C755 (gift from Roger Everett, University of Glasgow Center for

Viral Research), pYD-C678, and pYD-C780 were pLKO.1-based lentiviral expression vectors that carried

a puromycin resistance marker (16, 17). pYD-C780 was derived from pYD-C755, and carries the C-

terminally 3×FLAG tagged M92 coding sequence.

The primary antibodies included: anti-FLAG polyclonal rabbit antibody (F7425) and monoclonal

mouse antibody (F1804) (Sigma); rat anti-HA (11867423001, Roche); and rabbit anti-RNAP II (sc-899X,

Santa Cruz). The secondary antibodies used for immunoblotting were horseradish peroxidase (HRP)-

conjugated goat anti-mouse IgG, goat anti-rabbit IgG, and goat anti-rat IgG (Jackson Laboratory).


Aldrich); Dyanbeads (Novex, Life technologies); Benzonase (Novagen, Fisher Scientific).

Cells and viruses. Mouse embryonic fibroblast 10.1 cells (MEF10.1) (25) were propagated in Dulbecco

modified Eagle medium supplemented with 10% fetal bovine serum, nonessential amino acids, 1 mM

sodium pyruvate and 100 U/mL penicillin-streptomycin. Cells were maintained at 37oC and 5% CO2 in a

humidified atmosphere. To create cell lines expressing N-terminally 3×FLAG tagged pM92, MEF10.1 cells

were transduced three times with pYD-C618-derived retrovirus and allowed to recover for 48 hours.

Transduced cells were then selected with 1 µg/mL puromycin, and then maintained with 0.5 µg/mL

puromycin. Expressing cells were tested for their ability to produce virus upon transfection with the mutant

MCMV SMin92 (see below).

Virus stocks were produced by collecting cell-free supernatant from infected culture at a

multiplicity of infection (MOI) of 0.01-0.001. Virus titers were determined in duplicate by a tissue culture

152

infectious dose 50 (TCID50) assay in the appropriate cell type. MCMV SMgfp and SMflag92 were

reconstituted in MEF10.1 cells. SMin92 was reconstituted in 10.1-flagM92 cells.

Viral growth analysis. MEF10.1 cells expressing various vectors containing M92 mutant ORFs were

seeded in 12-well plates overnight to produce a confluent monolayer. Cells were inoculated with

recombinant viruses for 1 hour at an MOI of 2. The inoculum was removed, infected monolayer was

rinsed with phosphate-buffered saline (PBS), and fresh medium was replenished. At various times post

infection, cell-free virus was collected in duplicate by harvesting medium from infected cultures. Cell-

associated virus was collected by rinsing infected cells once with PBS and scraping cells into fresh

medium. Cells were lysed by one freeze-thaw cycle followed by sonication. Lysates were cleared of cell

debris by low speed centrifugation and supernatants were saved as cell-associated virus. Virus titers

were determined by TCID50 assay.

RNA analysis. Intracellular RNA was determined by reverse transcription-coupled qPCR (RT-qPCR) as






Advantage qPCR Premix (Clonetech) and primer pairs specific for viral genes or cellular β-actin (Table 1).

A standard curve was generated for each gene using serially diluted cDNA from infected cells, and was

used to calculate the relative amount of a transcript in each sample. The amounts of viral transcript were

normalized by dividing viral transcript equivalents over actin equivalents. The normalized amount of


Protein analysis. Protein accumulation was analyzed by immunoblotting. Cells were washed and lysates



153




Protein interactions were analyzed by co-immunoprecipitation. For MCMV, MEF 10.1 cells

transiently expressing C-terminally HA tagged M38 or M79 were infected with SMflag92 at an MOI of 2.

Cells were collected at 48hpi and lysed by incubation with extraction buffer (50 mM Tris pH 8.0, 300 mM

NaCl, 0.5% NP-40) for 15 minutes. In the meantime, 1 µg FLAG-antibody was conjugated to 25µL

Dynabeads (Novex, Life technologies) by incubation in conjugation buffer (0.02% Tween 20 in PBS, pH

7.2) for 20 minutes at room temperature. Lysates were cleared by centrifugation, pellet of cellular debris

was saved, and supernatant was incubated with FLAG antibody-conjugated Dynabeads in the presence

of endonuclease Benzonase (800 U/mL) (that digested DNA and prevented DNA-mediated, nonspecific

interactions among DNA-binding proteins). After an overnight incubation, beads were washed four times

with extraction buffer, and supernatant was saved as flow-through. Washed beads were mixed with

NuPAGE LDS sample buffer (Invitrogen) and boiled to elute FLAG-associated proteins. Pellets, flow-

through, and eluted samples were analyzed by a SDS-PAGE gel followed by immunoblotting.

For silver stain analysis, after the elution, the samples were resolved on a SDS-PAGE gel, and

protein bands were visualized by silverstain using the ProteoSilver Plus Silver Stain Kit (Sigma).

154

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Table 1. Mass Spectrometry Identities for SM79flag Immunoprecipitation Score Expectation Protein Name Function MW %Coverage

1829 0 DNA-directed RNA polymerase II subunit RPB1

Subunit of RNA polII 217039 32.3

1522 0 DNA-directed RNA polymerase II subunit RPB2


374 1.90E-33 DNA-directed RNA polymerase II subunit RPB3


248 7.90E-21 Isoform 2 of Heterogeneous nuclear ribonucleoprotein M

splicing 73692 13.6

165 1.70E-12 Histone H1.2 Histone 21254 19.3

145 1.50E-10 60S ribosomal protein L4 ribosomal protein 47124 15.8

140 5.60E-10 Uncharacterized protein Other 30030 23.3

120 4.60E-08 ATP-dependent RNA helicase DDX3X RNA helicase 73056 8.2


103 0.0000027 A-kinase anchor protein 8 RNA helicase 76246 3.6

72 0.0034 Histone H1.5 Histone 22562 14.3

66 0.012 60S ribosomal protein L8 ribosomal protein 28007 10.5

65 0.017 Protein 2210010C04Rik Other 26405 8.1

64 0.021 ATPase family AAA domain-containing protein 1

Other 40718 4.4

59 0.066 ATP-dependent RNA helicase A RNA helicase 149596 2.9

41 4 Cardiotrophin-like cytokine factor 1 Other 25245 3.6

38 8.8 Isoform 2 of Serine/arginine-rich splicing factor 7

splicing 27361 16.8

37 9.5 Isoform 2 of Heterogeneous nuclear ribonucleoprotein F

splicing 43658 6.8

34 21 Glyceraldehyde-3-phosphate dehydrogenase

Transcription? 36519 20.1

33 27 Protein Gm8225 ribosomal protein 31486 7.9

32 31 Neurofilament medium polypeptide Other 95859 0.8

31 38 Nocturnin Other 48270 1.9

30 53 Dual specificity protein phosphatase 10 Other 52498 2.1

30 55 Isoform 2 of Protein prune homolog 2 Other 37105 5.2

27 110 Isoform 2 of Coiled-coil domain-containing protein 132

Other 107106 3.6

26 130 cAMP-specific 3',5'-cyclic phosphodiesterase 4D (Fragment)

Other 85498 5.8

160

Table 2. Mass Spectrometry Identities for SMflag92 Immunoprecipitation Score Expectation Protein Name Function MW %Coverage

2109 0 DNA-directed RNA polymerase II subunit RPB1 Subunit of RNA polII 217039 35.9

1382 0 DNA-directed RNA polymerase II subunit RPB2 Subunit of RNA polII 133825 37.1

625 1.50E-58 Heat shock cognate 71 kDa protein splicing 70827 32.8

260 5.50E-22 DNA-directed RNA polymerase II subunit RPB3 Subunit of RNA polII 31424 36.4


175 1.70E-13 Voltage-dependent anion-selective channel protein 2 (Fragment)

Mitochondria 30427 16.6

166 1.30E-12 Histone H1.3 Histone 22086 17.6

118 7.50E-08 Uncharacterized protein Other 30030 18.4

88 0.000073 Voltage-dependent anion-selective channel protein 3

Mitochondria 30832 8.1

85 0.00016 ADP/ATP translocase 2 Mitochondria 32910 19.8

81 0.0004 Heterogeneous nuclear ribonucleoprotein M ribosomal protein 72022 8.5

78 0.00077 DEAD (Asp-Glu-Ala-Asp) box polypeptide 5 RNA helicase 69223 5.7

74 0.0021 Histone H1.5 Histone 22562 8.5


66 0.014 Glyceraldehyde-3-phosphate dehydrogenase Transcription? 36519 4.4

66 0.011 Isoform Mt-VDAC1 of Voltage-dependent anion-selective channel protein 1

Mitochondria 30737 11

65 0.015 Myeloid leukemia factor 2 Other 28037 11.3

61 0.036 ATPase family AAA domain-containing protein 1 Other 40718 4.4


53 0.26 Isoform 2 of UAP56-interacting factor RNA export 32271 4.6

51 0.41 Putative ATP-dependent RNA helicase Pl10 RNA helicase 73095 3.5

48 0.84 A-kinase anchor protein 8 RNA helicase 76246 8.4

48 0.84 Prohibitin-2 Transcripton/ Mitochondria?

33276 7.4

47 1.1 Protein 2210010C04Rik Other 26405 8.1

46 1.4 Mitochondrial carrier homolog 2 Mitochondria 32323 4.8

43 2.3 Uncharacterized protein Other 29152 12.1

33 28 Isoform 2 of RNA-binding protein 39 splicing 58647 13.7

28 73 Trifunctional enzyme subunit alpha, mitochondrial Mitochondria 82617 2.8

27 98 Isoform 2 of Serine/arginine-rich splicing factor 7 splicing 27361 7.1

25 160 60S ribosomal protein L7 (Fragment) ribosomal protein 32507 17.6

22 280 Myosin light chain 3 Other 22407 9.3

15 1600 Isoform 1 of Paired amphipathic helix protein Sin3b transcription 109325 3

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Table 3. Mass Spectrometry Identities for Viral Proteins Score Expectation Protein Name Function MW %Coverage

SM79flag

3112 0 M87 Potential Transcription 102346 49.9


1008 2.70E-99 M79 Transcription 29354 66.3

539 2.00E-52 m139 unknown 71713 19.3

439 2.40E-42 M112andM113 Transcription/ DNA replication 65988 15.7

340 5.20E-29 IE3 viral transactivator 68061 23.9

156 4.90E-14 M69 RNA export 92977 9.3

114 7.10E-10 M25 Unknown 103152 7.4

86 4.10E-07 M86 Major capsid protein 151329 5.9

76 0.0000039 m138 Unknown 63024 3.2

67 0.000033 M31 Unknown 56622 6.2

56 0.00045 M50 Unknown 34691 8.9

29 0.21 M49 Unknown 60761 2.8

23 0.95 M54 DNA polymerase 123759 1.1

21 1.3 m25.2 Unknown 35360 3.7

SMflag92


687 3.40E-67 M112andM113 alternative splice site

Transcription/ DNA replication 34568 53.9

417 3.70E-40 M112andM113 Transcription/ DNA replication 65988 21.1

274 6.50E-26 m139 Unknown 71713 13.7

209 2.10E-19 M87 Potential Transcription 102346 11.9

184 7.80E-17 M92 Viral transactivator 25430 23

138 2.60E-12 M49 Transcription 60761 4.9

106 4.80E-09 M25 Unknown 103152 8.3

72 0.000011 M31 Unknown 56622 3.5

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TABLE 4. Primers used in QuickChange Mutagenesis

Primer Name Primer Sequence

AA 58 Cysteine forward 5'-GGAATATGTACGTGGCCGTGCGCTGTCACCG-3'

AA 58 Cysteine reverse 5'-CGGTGACAGCGCACGGCCACGTACATATTCC-3'

AA 61 Cysteine forward 5'-CGTGTGCGTGCGCGCTCACCGCACTCACCTCTGCG-3'

AA 61 Cysteine reverse 5'-CGCAGAGGTGAGTGCGGTGAGCGCGCACGCACACG-3'

AA 65 Histidine forward 5'-GCTGTCACCGCACTGCCCTCTGCGATCTACG-3'

AA 65 Histidine reverse 5'-CGTAGATCGCAGAGGGCAGTGCGGTGACAGC-3'

AA 67 Cysteine forward 5'-GCTGTCACCGCACTCACCTCGCCGATCTACGGCACG-3'

AA 67 Cysteine reverse 5'-CGTGCCGTAGATCGGCGAGGTGAGTGCGGTGACAGC-3'

AA 73 Cysteine forward 5'-CGATCTACGGCACGACGCTGTGGTGGTCCACACG-3'

AA 73 Cysteine reverse 5'-CGTGTGGACCACCACAGCGTCGTGCCGTAGATCG-3'

AA 84 Cysteine forward 5'-CCACACGCAGGACGGCTCCGTCGCCATCAAGACGGGCCTCACC-3'

AA 84 Cysteine reverse 5'-GGTGAGGCCCGTCTTGATGGCGACGGAGCCGTCCTGCGTGTGG-3'

AA 87 / 88 Threonine / Glycine forward 5'-GCTCCGTCTGCATCAAGGCGGCCCTCACCTACGGCTCC-3'

AA 87 / 88 Threonine / Glycine reverse 5'-GGAGCCGTAGGTGAGGGCCGCCTTGATGCAGACGGAGC-3’

AA 160 Phenylalanine forward 5'-GGAGAATGCGATCTATTTTACGGCCAACCGCGTCTTCAAGCAGC-3'

AA 160 Phenylalanine reverse 5'-GCTGCTTGAAGACGCGGTTGGCCGTAAAATAGATCGCATTCTCC-3'

Red text indicates Alanine residues designed to replace the original sequence

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Figure 1. Proteomic analysis to identify binding partners of pM79 and pM92. Identification of pM79

and pM92 interacting partners. MEF 10.1 cells were infected with SMwt, SM79flag, or SMflag92 at MOI 2,

collected at 48 hpi, and were immunoprecipitated in the presence of benzonase with FLAG antibody.

Eluted proteins were run on an SDS-containing polyacrylamide gel and silver stained. The bands

indicated with an arrow were extracted for mass spectrometry. Potential alignment of proteins identified

by mass spec with candidate bands. The silver stained gel has been labeled with potential alignment of

proteins identified from the mass-spec results.

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Figure 2. Immunoprecipitation analysis to confirm RNAP II interactions. (A) FLAG-tagged pM79

interacts with RNAP II during MCMV infection. MEF 10.1 cells were infected with SMwt or SM79flag at

MOI 2, collected at 48 hpi, and lysates were subjected to co-immunoprecipitation with FLAG mouse

monoclonal antibody. Cell pellets, lysates, and eluted proteins were analyzed by immunoblotting with

indicated antibodies. Blots were cropped to save space but were from the same lane and exposed film.

(B) FLAG-tagged pM92 interacts with HA-tagged pM79 in MCMV infection. MEF 10.1 cells were

transfected with a vector expressing HA-tagged pM79. 24 hours after transfection, cells were infected

with SMflag92 virus at MOI 2. Lysates were collected at 48hpi and subjected to co-immunnoprecipitaiton

with FLAG antibody. Cell pellets (P), lysates (FT), and eluted proteins (E) were analyzed by

immunoblotting with indicated antibodies.

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Figure 3. pM92 requires viral accessory proteins to facilitate interaction with pM79. (A) pM92

interacts with pM79 during infection. MEF10.1 cells expressing either HA-tagged M79 (pM79ha) or HA-

tagged M38 (pM38ha) were infected with SMflag92 at an MOI of 2 and cell lysates were collected at 48

hpi and subjected to co-immunoprecipitation with anti-FLAG antibody. Cell pellets, lysates, and eluted

proteins were analyzed by immunoblotting with anti-FLAG and anti-HA antibody. Molecular weight

markers (in kDa) are also shown. (B) pM92 does not interact with pM79 independent of viral infection.

Lysates from MEF 10.1 cells expressing HA-tagged M79 and FLAG-tagged M92 were collected and

analyzed as in (A).

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Figure 4. Amino acid alignment of the UL92 protein family. Amino acid alignment of UL92 proteins

from mouse, rat, human, and rhesus, CMVs, with HHV6, MHV68, KSHV, and EBV. Amino acids

highlighted in red are conserved across all species.

M92 -MFSHGRDPRTGKPISAAG-------ARAHARGGGSGKCDGGGGCDMRNLCNPLTQELNLRNMYVCVRCHRTHLCDLRHDCVVVHTQDGSVCIKTGLT 90 PR92 MMFAHGRDPRTGKPLSAPGGAGGGGSALAHARGG-VGGCDDSGGCDMRNLCNPLTQELNLRNMYACVRCHRTHLCDLRHDCVVVHTQDGSVCAKTGLT 97 UL92 -------------------------------------MCDASGACDMRHVQNAFTEEIQLHSLYACTRCFRTHLCDLGSGCALVSTLEGSVCVKTGLV 61 Rh92 -MHAVMNHHPTGTGPPRHHRMHKKSKAAAAAAAGALDMCDVSGTCDMRHVQNVFTEEIQLHSLYVCTRCFRTHLCDLGSGCALVSTLEGSVCVKTGLV 97 HHV6 MELPRKYDRVTGRILTHKN----------------NQMCT-TECSQMYNLHNPITFELGLGNVFVCMRCLTVHHCDMQTDCTIVNTHEGYVCAKTGLF 81 ORF31 ---------------------------------MYKRCKRGQDKCMMRPLSNPLTEAYNVQNLYQCLNCDDYHLCNGSLNCDPLVDREGIVCTVTGKY 65 KSHV ----------------------------------------------MKSVASPLCQFHGVFCLYQCRQCLAYHVCDGGAECVLLHTPESVICELTGNC 52 EBV -MSDQGRLSLP-------------------RGEGGTDEPNPRHLCSYSKLEFHLPLPESMASVFACWGCGEYHVCDGSSECTLIETHEGVVCALTGND 78 : : : :: * * * *: * : :. :* ** M92 YGSVFPG-------GCVSALEPVTEPHVDEINVVGVIMSYVYTYLTRNADHYADVIGSVIEGG-WFNKPTENAIYFTFNRVFKQH-NALQKVPISVIG 179 PR92 YSSVFPG-------GRGDVLEPVSEPHVDEINMVGVVMSYVYTYLTRNAEHYADVIQSVIDDG-WFNKSTEDAIYFTFDRVFRHP-NSLQKIPLSVVG 186 UL92 YEALYPV-------ARSHLLEPIEEAALDDVNIISAVLSGVYSYLMTHAGRYADVIQEVVERD-RLKKQVEDSIYFTFNKVFRSM-HNVNRISVPVIS 150 Rh92 YEALYPV-------AREHLLEPIEETSLDDVNVIGAVLAEVYRYLMSHAARYADVIQEVVERD-RLKKQVEENIYFTFNKVFRSM-QNVNRISVPVIS 186 HHV6 YSGWMPA-------YADCFLEPICEPNIETVNVVVVLLSYVYSFLMENKERYAAIIDSIIKDG-KFIKNVEDAVFYTFNAVFTN--STFNKIPLTTIS 169 ORF31 IQCNVRESP---MCG-AACLDVDIHPE-DNTDCILRALFGDIVDIINKLDDIAEIKDKILAGD-SLNEHIKKKIECTFPMCANII-NCNSQG-YSILC 155 KSHV MLGNIQEGQ---FLGPVPYRTLDNQVDRDAYHGMLACLKRDIVRYLQTWPDTTVIVQEIALGD-GVTDTISAIIDETFGECLPVL-GEAQGG-YALVC 144 EBV MGPHFQPALRPWTEIRQDTQDQRDKWEPEQVQGLVQTVVNHLYHYFLNENVISGVSEALFDQDGALRPHIPALVSFVFPCCLMLFRGASSEKVVDVVL 176 . : . : : : : : . . : .* . : M92 QLFVQLVIGVHARVTKYDSTVIKVSRRKREDGLLKRMRFEYGNAPSFRTGR- 230 PR92 QLFVQLVIGVHARVTKYDATVIKVSRRKREDGLLKRMRFEYGNAPAFRAGRR 238 UL92 QLFIQLIIGIYSKQTKYDACVIKVSRKKREDALLKQMRSEYGNAPVFGSGV- 201 Rh92 QLFTQLIIGIYSKQTKYDSCVIKVSRKKREDALLKQMRSEYGNAPVFGLGV- 237 HHV6 RLFVQLIIGGHAKGTIYDSNVIRVSRRKREDSLLKKMRLEYGNALIL----- 216 ORF31 SMFVHIIMSIYANKTIYGHMLFKCTRNKKYDTIAKKIREKWMYLE------- 200 KSHV SMYLHVIVSIYSTKTVYNSMLFKCTKNKKYDCIAKRVRTKWMRMLSTKDT-- 194 EBV SLYIHVIISIYSQKTVYGALLFKSTRNKRYDAVAKRMRELWMSTLTTKC--- 225 :: ::::. :: * *. ::: ::.*: * : *::* :

167

Chapter V

Conclusions and Future Directions

The final draft of this chapter was written and edited by TJC.

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Conclusions

Functional profiling. Despite the ready availability of the MCMV Smith strain as an infectious BAC clone

and the wide use of MCMV as an animal model of CMV infection, the 78 open reading frames that are

conserved between HCMV and MCMV have never undergone comparative functional analysis. After

excluding the genes that are well studied in MCMV, or genes that have well studied homologues, we

were left with 28 genes of interest to target. Mutagenesis of the BAC-MCMV was achieved by PCR-based

linear recombination followed by FRT/FLP recombination. Twenty-two of the twenty-eight target genes

have been successfully mutated and confirmed via PCR, restriction digest, and sequencing. The 22 target

genes that have been successfully mutated have been reconstituted in fibroblasts and screened for a

phenotype via multi-step growth curve analysis. The screen identified seven genes that were either

essential for viral replication or augmenting for viral growth. Identification of essential and augmenting

genes that are conserved across cytomegaloviruses will help herpes virologists focus their effort on

proteins that may be involved in important conserved viral processes.

Late gene transactivators. MCMV is the commonly used model virus for HCMV, so revealing HCMV

genes that are functionally conserved in MCMV will allow the use of the robust mouse genetic system to

elucidate their role and test novel antivirals targeting these gene products. We have found that the

products of MCMV genes M79 and M92 regulate viral Late gene expression. In particular, we showed

that the encoded proteins, pM79 and pM92 respectively, were critical for MCMV to promote accumulation

of a set of Late gene transcripts (10, 13). Moreover, MCMV DNA was not only synthesized efficiently in

the absence of these Late gene transactivators, but in the case of pM79 deficient virus Pulse Field Gel

Electrophoresis analysis showed that the overall structure of replicating viral DNA was unimpaired and

that the development of viral replication compartments during mutant virus infection was indistinguishable

from those during wild-type virus infection. This body of evidence further excludes the involvement of

pM92 and pM79 in viral DNA synthesis or other events preceding viral transcription at late times of

infection. This phenotype has also been observed for the HCMV homologues UL79 and UL92 (66). We

also determined that pM79 accumulates with Early-Late kinetics in nuclear viral replication compartments

during infection, which suggests that it is expressed slightly earlier than the other lytic genes. It is likely

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that this Early-Late expression is required to initiate Late gene transcription. How pM79 expression is

regulated remains a mystery. pM92 is expressed at late times of infection, and is likely regulated by pM79

as other Late genes are during infection. Furthermore, we demonstrated that viral protein pUL92, the

HCMV homologue of pM92, was also essential for virus infection. Another group further showed that

pUL92 was important for viral transcript accumulation at late times of infection, a phenotype that mimics

what was seen for pM92 deficient MCMV (65). Importantly, pM92 could trans-complement the growth of

pUL92 deficient HCMV recombinant virus. These experiments do not specify whether the compensation

occurs at the transcriptional or translational level during HCMV infection, and further work is required to

define the exact mechanism at play. Regardless, this work suggests a conserved function for pM92

homologues among betaherpesviruses.

Our work, together with the reported role of its homologues, including pUL79 and pUL92 in

HCMV and ORF18 and ORF31 in MHV68 (3, 41, 43, 65, 66), suggests a common mechanism governing

Late transcription among beta and gammaherpesviruses. How viral Late transcription is regulated

remains largely unknown and viral/host factors involved are poorly defined. These late transcription

transactivator proteins represent an invaluable tool to gain insight into this key viral process.

Viral DNA synthesis. Our study also provides additional evidence that viral DNA synthesis is necessary

but not sufficient to drive viral Late gene expression during herpesvirus infection. Inhibition of the viral

polymerase by PAA abolishes the accumulation of Late gene transcripts (33, 34, 39, 79). This

dependence on viral DNA synthesis has been linked to the origin of lytic replication (ori-lyt), as the ori-lyt

sequence is required in cis for proper expression of Late transcripts in many herpes viruses (2, 14, 19,

45, 82). However, our previous data and the data presented here demonstrate that viral gene expression

at late times of infection depends not only on viral DNA replication in cis, but also on viral factors such as

pM92 and pM79 in trans (10, 11, 13). In the absence of these viral factors, DNA synthesis kinetics were

indistinguishable from wild-type virus despite a defect in Late gene transcript accumulation. Therefore,

pM79 and pM92 do not function as viral DNA synthesis proteins; rather they specifically act on gene

expression at late times of infection.

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Several theories have been proposed to explain why Late genes are dependent on DNA

replication. It has been shown in alpha and gammaherpesviruses that Late gene promoters can be

unusual in structure, sometimes not even containing a canonical TATA element (26, 40, 86, 89). These

unusual structures make it hard for the cellular transcription complex to assemble, since the viral

promoter region lacks some of the recognition sequences for transcription accessory proteins. Thus, a

popular hypothesis is that the transcription complex is recruited by the viral DNA replication complex to

Late gene promoters, bypassing the requirement for conventional activators. Such a mechanism would

be very effective to link viral DNA replication to the activation of Late gene transcription. This activity has

been seen in HSV-1, where ICP8, a component of the viral DNA replication complex, interacts with the

RNAP II complex to assist in recruitment to the replicating viral DNA, thereby stimulating Late gene

expression (15, 64, 91). This mechanism could be conserved in beta herpesviruses, especially since we

were able to copurify DNA replication components with the Late gene transactivators, pM79 and pM92.

Protein interactions. In this study, we demonstrated that pM92 interacted with pM79 during MCMV

infection; likewise pUL92 could interact with pUL79 during HCMV infection (13). This suggests that during

cytomegalovirus infection, a complex containing conserved virus-encoded factors forms to promote Late

gene expression. How this complex functions and what additional protein components are in this complex

remain important questions. Cytomegalovirus genes are transcribed by the cellular RNA polymerase II

(RNAP II), a 12-subunit multi-protein enzyme that requires a host of accessory scaffold and regulatory

proteins for its activity (4, 52, 80, 85). Our study also identified RNAP II as an interaction partner for both

pM92 and pM79 during infection. This implies that pM92 and pM79 play a direct role in transcription. The

pM92/pM79 complex could play an essential role in the recruitment or assembly of cellular transcription

components on viral Late gene promoters. Such a mechanism has been suggested for regulation of Late

gene expression in both alpha and gammaherpesviruses (47, 88). However in what capacity the

interaction of pM79 and pM92 with RNAP II aids in transcription is unknown. Further studies will need to

be done to determine how these proteins modulate transcription. One possibility is that pM79 and/or

pM92 modulate phosphorylation of the RNAP II C-terminal domain (CTD), which impacts initiation,

elongation, and termination of transcription. Secondly, pM79 and/or pM92 could recruit RNAP II to the

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promoters of Late genes, potentially by recruiting TAF or TFIID accessory factors. Third, pM79 and pM92

could modulate the architecture of the promoter by regulating methylation or acetylation of DNA or

histones in the area. Confirmation of any of these strategies will greatly advance our understanding of

herpesvirus Late gene transcription and regulation.

Latency. Finally, it is tempting to speculate that Late gene expression regulators such as pM92 and

pM79 could play a role in the establishment of latency. As both proteins are essential for the lytic viral life

cycle, regulation of their activity and/or expression may be a deciding factor for viral latency and

reactivation.

Implications. The work described here has many implications for herpesviruses, virology research, and

medicine. With the dearth of drugs available to treat HCMV, identifying new drug targets is important.

Despite being discovered 40 years ago, many of the fundamental pathways of the HCMV life cycle are

undefined. Here we identify major players in one of the most important steps for lytic replication in CMV.

The development of small molecule inhibitors of pM92, pM79, or their homologues may result in drugs

that effectively stop CMV at the lytic stages of infection, and can be combined with the acyclovir cocktail

to prevent the evolution of escape viruses. It is also important to note that this research effectively adds

an additional level of regulation to Late gene expression that succeeds viral DNA replication. We now

know that DNA synthesis is required but not sufficient for Late gene expression in cytomegalovirus

replication. Though the pathway has not been mapped out completely, this thesis comprises substantial

work toward elucidating the details of this process. Understanding how DNA replication and transcription

are related will give us a better understanding of how these crucial lytic steps can be inhibited during

infection. Understanding how the virus hijacks host machinery is also of great interest to the field of

virology. Though decent progress has been made for identifying important protein interactions for

alphaherpesviruses, little to no progress has been made for beta or gammaherpesviruses, both of which

are responsible for severe disease.

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Late gene transcription in other herpesviruses. Though we have identified a few important elements

for regulating Late gene transcription during cytomegalovirus infection, there are still many elements of

Late gene expression that need to be defined. In the studies moving forward, it will be important to

consider what is known about Late gene regulation in other herpesviruses. In this way, future experiments

can be directed at testing for strategies that are likely to be conserved in this family of viruses.

HSV-1 is the most extensively studied herpesvirus, and as a result there is much known about

the regulation of Late gene transcription. It is known that ICP4, ICP22, ICP27, ICP8, and ICP0 are

important for Late gene expression. ICP4 is the major activator of Early and Late genes and interacts with

the viral transcription complex (9, 59). ICP4 has a DNA binding domain that interacts with Early and Late

promoters. However, these specific sites aren’t required for activation of transcription (18, 21). The

sequence that is required for Late promoter activity is the INR which is downstream of the TATA box (27,

29, 30, 37). In fact, the INR can initiate transcription in the absence of a TATA box (51, 73). The INR

helps ICP4 stabilize RNAP II accessory protein, TFIID complex, which contains TBP (the TATA-binding

protein), that is required for transcription. Specifically, ICP4 interacts with the TAF250 subunit of the TFIID

complex (9). Interestingly, it was shown that RNAP II accessory protein TFIIA is required to stabilize TBP

on Early promoters, but was not required for Late promoters. At Late times of infection TFIIA is down

regulated, which may help control the switch between Early gene transcription and Late gene

transcription.

ICP4 localizes to the nucleus in viral replication compartments where it can be found in a viral

transcription complex, which includes RNAP II and ICP22 (16, 17, 53). ICP22 is involved in HSV-1 Late

gene transcription. ICP22 binds to pTEFb and causes alterations to RNAP II phosphorylation, which

presumably regulates viral transcription (24, 28). It is unclear how ICP22 activity is achieved, but it may

have something to do with the binding partner pTEFb, which has the ability to phosphorylate RNAP II to

modulate transcription. ICP22 also interacts with circadian histone acetyltransferase CLOCK, which has

been shown to be important for viral gene expression (22, 32, 46, 47, 50). CLOCK interacts with ICP0,

which has been shown to displace HDACs from the REST/CoREST complex to enable Early and Late

gene transcription.

Unfortunately, the regulatory proteins in HSV-1 do not have homologues in the beta or

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gammaherpesviruses. So though the information from HSV-1 might shed light on some potential themes

to test, it is more likely that gammaherpesviruses will give us better insight into what HCMV might be

doing. As mentioned earlier, the homologues ORF18, ORF24, ORF30, ORF31, and ORF34 have a

similar phenotype as the HCMV homologues M79, M87, M91, M92, and M95 respectively (3, 41, 43, 65,

66, 87, 88). In fact, ORF30 and ORF34 have been implicated in recruiting RNAP II to the promoters of

MHV68 Late genes. Further studies will be needed to determine exactly how these proteins regulate

RNAP II, and what the impact is on late gene transcription.

Understanding how the expression of the late gene regulators themselves are regulated will also

be important to understanding how late gene transcription is regulated. In MHV68, some information has

been discovered about the regulation of ORF18 during infection. All gammaherpesviruses encode a gene

called replication and transcription activator (RTA). RTA controls the switch from latency to lytic infection.

It has been shown that RTA can bind directly to responsive elements (RREs) or can act in conjunction

with other binding partners (5, 6, 20, 74-76). It was found that ORF18 has a RRE element in its promoter,

which allows it to be activated by RTA (36). Since the ORF18 promoter is in the ori-lyt of MHV68, it’s

possible that DNA replication frees up the RRE so that RTA can bind and transactivate ORF18

expression, which starts the Late gene cascade. At this point a homologue to RTA does not exist in

betaherpesviruses, but it is possible that one of the late gene transactivators plays a similar role.

pM79/pUL79 is a good candidate for this role, as it is expressed at Early-Late times of infection.

In addition to the Late gene transactivators, MHV68 also regulates its Late gene transcription by

mRNA degradation, which is mediated by SOX endonuclease (1). SOX was shown to degrade viral

mRNA from all kinetic classes of expression, but a mutant in SOX had its primary defect due to missing

components of the tegument. The altered tegument composition resulted in the inability of progeny virus

to undergo lytic infection. Instead, all progeny virus defaulted to latent infection. Since tegumentation is a

process that occurs late in infection, it is assumed that degradation of Late gene transcripts is important

to maintain the appropriate balance of lytic gene expression during virus infection. The exact mechanism

in effect here has yet to be determined.

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Future directions

The work that has been done in this thesis has only touched the surface of understanding Late

gene expression and the regulation of CMV transcription. There are many more studies to be done, and

many directions with which to start.

Analyze various levels of Late gene expression. Our oligonucleotide tiled array analysis identified a

set of annotated genes whose transcription is substantially reduced at late stages of virus infection when

pM79 is abrogated. Comparative analysis of viral transcriptomes among MCMV infections with PAA,

without PAA, or in the absence of pM79 leads to two interesting observations. First, while viral DNA

synthesis is required for transcription from genomic regions containing many previously reported Late

genes, it also facilitates the continued transcription from genomic regions containing several previously

reported Early genes at Late times of infection. Second, viral transcripts that are dependent on viral DNA

synthesis also have a dependency on pM79 for their accumulation (10). There seems to be a hierarchy in

dependence, such that without pM79, some transcripts are markedly reduced, whereas others are

reduced to a much less extent. Thus, pM79 is a key viral regulator of Late gene transcription in MCMV

infection, but it is not the only regulator. In future studies, similar transcriptome analysis should be applied

to other viral genes known or predicted to be regulators of Late gene expression. Examples are HCMV

UL79 and MHV-68 ORF18, as well as MCMV M87 and M95 (homologues of HCMV UL87 and UL95) (3,

62, 65, 66). Such analysis will reveal whether these viral regulators control the expression of an

overlapping or distinct set of viral Late genes.

I think RNAseq analysis will yield a more high-throughput view of the cytomegalovirus

transcriptome. Comparing the abundance of transcripts between pM79 or pM92 deficient virus and wild-

type virus will let us know all of the genes affected by these Late gene transactivators. RNAseq is more

beneficial than an oligonucleotide array because the method does not rely on annotated open reading

frames for analysis. This is especially important in light of the recent discovery that HCMV produces over

700 transcripts during infection, most of which are not annotated (78). Thus, this experiment has the

potential to identify novel transcripts that are involved in MCMV lytic replication. It is important to know

exactly what genes are affected by pM79 and pM92 expression, and by what magnitude these genes are

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regulated. Such findings may reveal additional levels of lytic gene regulation, and may lead us to clues of

additional viral regulators involved in both Late gene transcription and viral DNA synthesis regulation.

Functional domain study of pM79 to define functionally important protein regions. How does pM79

regulate expression of Late gene transcripts? A Late gene transactivator could be involved in epigenetic

regulation of replicating viral DNA. For instance, viral DNA-associated histones are modified when

herpesviruses replicate their genomes, and this may render Late gene promoters accessible for

transcription (54, 55, 72). However, the pM79 coding sequence does not resemble those of histone

modifying enzymes, such as histone acetyltransferase or histone deacetylase (data not shown). If pM79

had a role in epigenetic regulation of gene expression, it would likely act indirectly, for instance, by

recruiting modification enzymes to histones associated with Late gene promoters. Late gene regulators

may also act as transcription factors. However, pM79 does not contain any identifiable putative DNA

binding domains (data not shown). Therefore, in this capacity pM79 would have to act as a modulator of

cellular and viral transcriptional regulators or RNAP II to facilitate viral Late gene transcription. To this

end, we have shown that pM79 can interact with RNAP II during infection (Chapter IV). Furthermore, our

research group has unpublished data that the HCMV homologue, pUL79, interacts with RNAP II during

infection. During HCMV infection, UL79 can modulate the phosporylation state of RNAP II to control

transcript elongation (Perng et al, unpublished). Determining what domains of pM79 are important for its

function during infection will lead us to answer some of the posed questions above, and further

understand the mechanism of its activity.

A mutagenesis strategy will be designed that targets the highly conserved residues of pM79 (Fig.

1). Much like the Quickchange mutagenesis of pM92 done in Chapter IV, conserved residues in pM79 will

be converted to alanine. These pM79 mutants will be tested for their ability to complement the growth of

SMin79 mutant virus. Any mutants that are able to complement the growth of SMin79 will be deemed

uninteresting. Though those mutant may have other important phenotypes under different conditions, we

are only interested in mutants that recapitulate the essential phenotype of a M79 deficient virus. pM79

mutants will also be tested for their ability to facilitate Late gene transcription. This function will be tested

by performing RT-qPCR on transcripts representative of genes from each kinetic class. pM79 mutants

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that fail to complement the transcription defect of SMin79 will be analyzed for functional interactions. We

would do co-IP-coupled mass spectrometry analysis to determine the difference in interaction partners

between all selected pM79 mutants and wild-type pM79. The results of this experiment would identify

cellular and viral factors that interact with pM79 in a domain specific way. This method could provide

significant insights into the function of pM79. Any and all results will no doubt increase the resolution of

our understanding about late gene expression in CMV, and about how pM79 functions during infection.

Continue the functional domain study for pM92 to define protein regions important for function.

Though some functions of pM92 have been elucidated, it is likely that this protein is multifunctional. It

would be interesting to do a different screen with the pM92 mutants to determine if the domains that were

dispensable for RNAP II and pM79 interaction are important for another interaction or process.

Furthermore, it is important to consider putting the pM92 mutants that had no phenotype during the in

vitro screen into mice. Mouse experiments may reveal additional functions of this Late gene transactivator

that are not observed in vitro.

Determine promoter sequence elements for Late genes that are important for expression. We know

that pM79 and pM92 interact with RNAP II, and we know that RNAP II is recruited to Late gene promoters

at late times of infection. However, why Late gene promoters are not engaged until late times of infection

by the transcription machinery is a mystery. Initiation of transcription requires the assembly of TFIIA, B, D,

E, F, H and RNAP II onto the core promoter elements (61). TFIID consists of the cellular TATA-binding

protein (TBP) and TBP-associated factors (TAFs). TAFs interact with initiator elements (INRs) to aid

transcription (48, 49). TFIID is stabilized by TFIIA and TFIIB. It is known that kinetic classes are regulated

in part by the promoter architecture (84, 90). In HSV-1 there is a decrease in promoter complexity from IE

to Early to Late genes (83, 85). IE gene promoters have viral and cellular cis acting sequences in the

promoter; Early gene promoters only have cellular elements (sp1 and CTF); Late gene promoters lack

any influential upstream cis-acting elements (23, 30, 35, 38, 44). For True Late gene promoters,

activators have not been described, but the main elements are the sequences downstream from the

TATA box that are important for Late gene regulation (27, 29, 30, 37, 38, 77). It has actually been shown

177

in HSV-1 that both Early and Late gene promoters are engaged by transcription factors at early times of

infection, yet they still have different kinetics for expression. Two theories are that there is relocalization of

key proteins to the viral replication compartments at late times of infection, or changes in the DNA

damage response at late times of infection could stimulate Late gene promoter transcription (16, 42, 56).

Similar themes have been seen in the gammaherpesviruses. Studies in EBV and MHV68 have

shown that unlike viral Early gene promoters, the promoters of Late genes map to the regions that lack

regulatory sequences upstream of the TATA box (23, 30, 35, 38, 44). However, the role of the TATA box,

and the sequence that defines the functional TATA box in gammaherpesviruses Late genes has only

recently been uncovered. It has been shown in EBV that the region comprising the Late gene promoter

includes a short core sequence required for expression that includes an unconventional TATT sequence,

rather than a canonical TATA box (2, 71). In KSHV, a region of 12 bp that also contained a TATT

sequence was found to be sufficient to support activation of a Late gene promoters (81).

The most striking characteristic of this atypical TATT sequence is a T at the fourth position, which

is only found in 10% of TATA box containing promoters of eukaryotic genes (8). The functional

significance of this fourth T has been examined for other herpesvirus Late gene promoters by creating a

T-to-A point mutation. It was shown that such mutation in the two Late gene promoters of EBV result in a

loss of promoter function (2, 71). However, these results are not universal for Late gene promoters,

because a study has been done to show that mutagenesis of the ORF52 Late promoter indicates that

both TATATA and TATTA/TA support the Late gene promoter activity, but TATAAA does not (86). This

implicates that nucleotides neighboring the TATA/TATT element are also important for Late gene

regulation.

Minimal analysis has been done to analyze the TATA elements of Late genes in the

cytomegalovirus genome. The UL44 Late gene promoter of HCMV has a noncanonical TATT sequence,

and a T-to-A point mutation greatly reduces the level of the UL44 Late gene transcript (40). Attempts

have also been made to map the promoter regions of Late genes in both HCMV and MCMV using

bioinformatic programs. It was observed that the sequences of TATTA/TA and TATA/TTA can been found

in the promoter regions of many Late genes, such as UL14, UL18, UL31, UL32, and UL48 of HCMV and

M25, M31, M34, M69, M97, M100, and M102 of MCMV (86). This analysis supports the theory that the

178

canonical TATA box is not likely to mediate transcription of herpesvirus Late gene promoters. The inability

of a canonical TATA box consensus sequence to function as a core element for the Late gene promoter

is an important piece of information in understanding the regulation of Late gene expression.

However, formal analysis of Late gene promoters is scarce in betaherpesvirus literature. Even

taking the above information into account, many questions remain. Is the required core sequence of CMV

similar to the 12bp KSHV core sequence (81)? How does such a short core sequence govern the

expression of Late genes during virus infection? How does the TATT sequence affect recruitment of

transcription initiation factors? To address these questions, it will be important to analyze the role of

individual nucleotides of Late gene promoters in controlling Late gene expression. To do this we would

utilize a luciferase reporter vector containing the CMV ori-lyt, which is essential for the activation of Late

promoters (63). We would decipher the cis elements required for activation of Late gene transcription by

putting Late gene promoter regions upstream of the luciferase in the reporter vector. Mutagenesis of this

promoter region would give insight into what sequences are important and sufficient for Late gene

expression and what effect the mutations have on transcription factor binding.

A study was done in MHV68 that analyzed the effects of promoter engagement by ORF24, the

homologue of UL87 in HCMV. ORF24 and UL87 are thought to be virally encoded TATA binding proteins

(62, 86, 89). The study showed that the binding of ORF24 to Late gene promoters was consistently

reduced upon the mutation of the TATT sequence. However, the mutation still allowed at least some

transcription from the Late gene promoter, which suggests that additional regulatory factors play a role in

Late gene expression. Thus, future studies in HCMV will likely reveal the importance of protein binding to

Late gene promoter sequences, and define how the actual sequence of the Late promoter regulates its

kinetic class.

Confirm the other interaction partners important for pM79/pM92 function. Though RNAP II is critical

for viral gene transcription, there are many other binding partners that copurified with pM79 and pM92

during infection. One such protein is a dead-box helicase. Though the function of this family of helicases

is not completely defined in virus infection, a recent study in HSV-1 showed that a dead-box helicase

could bind to actively replicating viral DNA and RNAP II during infection (60). The dead-box helicase and

179

its interaction with DNA and RNAP II were required for efficient expression of both Early and Late viral

genes. Thus, it would be interesting to see if dead-box helicases have a similar role in CMV infection and,

if so, to define what exactly that role is.

Furthermore, it would be interesting to define the role of the other mystery proteins that copurified

with pM79 or pM92. The identification of any of these proteins as having a role in Late gene transcription

will be informative for the field.

Determine the impact of mutations in pM79 and pM92 on the viral replication cycle in mice. MCMV

infection in mice is an important model for preclinical evaluation of antiviral compounds, most of which

have been directed at highly conserved viral DNA replication proteins. The Late transactivator proteins

present an attractive, alternative target for therapeutic intervention in CMV disease. The functional

conservation between pM79/pM92 and pUL79/pUL92 justifies MCMV as a credible model to test the

effect of targeted antivirals in vivo. However, none of the Late gene transactivator mutants have been

tested for their growth in mice. In HSV-1 a study was done where the ICP22 mutant was put into mice.

Interestingly, the mutant virus was able to grow to high titers in the mouse model. This was not surprising

as the ICP22 mutant has an essential phenotype that is cell type specific (reviewed in (70). Since most of

the studies for beta and gammaherpesvirus transactivators have been done in vitro and in the same cell

lines, it will be important to test what impact pM79 and pM92 mutants have on in vivo infection.

If these genes are essential for in vivo infection, then it is very possible that drugs could be

designed to target the transactivators during infection. It is also possible that transactivator mutants could

serve as vaccine candidates. However, this would depend on just how many lytic genes are down

regulated in the absence of a single Late gene transactivator. The immune response is primarily designed

to recognize lytic antigens; so a mutant virus that produces no Late genes would be a poor vaccine

candidate.

Determine what role pM79 may have in regulating latency. Finally, it is tempting to speculate that Late

gene expression regulators such as pM79 could play a role in the establishment of latency. As both

proteins are essential for the lytic viral life cycle, regulation of their activity and/or expression may be a

180

deciding factor for viral latency and reactivation. This concept is supported by the fact that RTA seems to

regulate the expression of ORF18 in MHV68 virus infection (36). Since RTA is the lytic switch for

gammaherpesviruses, it is possible that a similar mechanism is found in betaherpesviruses.

No molecular definition of CMV latency exists, but latency has been defined operationally as the

inability to detect infectious virus despite the presence of viral DNA. Both HCMV and MCMV infect a

variety of cell types in their respective host, but latency seems to be confined to cells of the myeloid

lineage (7, 25, 31, 67, 68). Similar to HCMV, chromatinization and recruitment of cellular repressors to the

viral DNA and to the major immediate early (MIE) gene locus are critically involved in the in

vivo establishment of MCMV latency (57, 58). The latent viral genome is associated with repressive

chromatin in immature myeloid cells, whereas virus reactivation is accompanied by chromatin remodeling

and initiation of transcription at the MIE locus during cell-differentiation (69). The IE genes regulated by

the MIE promoter (MIEP) act as essential transactivators of Early and Late genes (62). Therefore, MIEP

transcriptional activity is generally considered an important checkpoint in CMV latency and reactivation.

To test the involvement of pM79 in the reactivation of HCMV from latency, we would analyze the

chromatinization of the M79 promoter region, and assess how the state of chromatin repression is altered

after the induction of reactivation. This analysis would be followed by monitoring the cells for expression

of pM79. Since DNA replication is required for late gene expression during lytic infection, it would be

interesting to see if the requirement is altered during reactivation from latency. The fact that MCMV BAC

DNA can be reconstituted in the absence of tegument proteins makes it unclear what the requirement for

a Late gene transactivator would be during reactivation. However, it has not been determined exactly

what role pM79 may have in viral DNA synthesis, especially in light of the interaction data in Chapter IV.

181

Concluding Remarks

We have identified pM79 and pM92 as novel Late gene regulators in MCMV lytic infection, shown

their interaction with not only each other but with RNAP II, and demonstrated their conserved functions

with the HCMV homologs pUL79 and pUL92. We have published two papers that detail our research of

M79 and M92 (12, 13). pM79 and pM92 represent potential new targets for therapeutic intervention in

CMV disease, and a gateway into studying a largely uncharted viral process that is critical to the viral life

cycle. Our current efforts aim at a better mechanistic understanding of the role of pM92 in Late gene

regulation. With the mass spectrometry results (Chapter IV), we have identified cellular and viral factors

that interact with pM92. Though pM79 and RNAP II are proven partners of pM92, it is almost certain that

all additional partners will provide important insight into the function of pM92. Furthermore, genetic and

protein analysis to identify functional domains and structural elements of pM92 will be invaluable to

understand the mechanistic basis for its activity and to determine additional functions that pM92 may

have. We plan to publish an additional paper to document the mass spectrometry interaction data for

pM79 and pM92, and also to outline the functional domains of pM92. The work of this thesis has greatly

contributed to understanding a very important process in betaherpesvirus biology, and has given insights

that will help our understanding of novel host gene functions, and how nuclear replicating viruses regulate

transcription of their genome.

182

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Figure 1. Amino acid alignment of the UL79 protein family. Amino acid alignment of UL79 proteins

from mouse, rat, human, and rhesus, CMVs, with HHV6, MHV68, KSHV, and EBV. Amino acids

highlighted in red are conserved across all species.

M79 ---------------MVKTIRVGRFLHLSDD-NH-LILHITTKLLSGQPLSSMRLEELKIIRLACLLTLGRGIELLILRETVANNGVSDNTILNRKISPEFWRKMYE 90 PR79 MAQQEQRQRQRQRQRSADSARVGRFLQLRDD-NH-LILHITGKLLSGQPLSSMRLEELKIIRLACLLTLGRGIELLLLRETLANAGVSDNTILNRKVSPAFWLKVYD 105 UL79 ------MMARDEENPAVPRVRTGKFS-FTCA-NH-LILQISEKMSRGQPLSSLRLEELKIVRLICVLLFHRGLETLLLRETMNNLGVSDHAVLSRKTPQPYWPHLYR 98 Rh79 ------MSSPTDEA----RVRTGKFS-FTCA-NH-LILQISEKMSRGQPLNTLRLEELKILRLICVLLFNRGLETLLLRETTNNLGVSDNAVLSRKTPTSYWPHLYQ 94 HHV6 ------------------MTQIGQYIKLNDA-VPNLILHITTKLLRNENLTSFKQEELLLIQHVCTSMLSHGIKILLLRESLYNSGIGDIVILNRKISNNYWFRLFS 88 ORF18 -------MSTYPPTYIGASAMLGRYVIVGKP-MSPGLQALLSKILFKDKLNTLSSTELRLLHAILCHLYNFTTNCYLFKYALYNTGTYDNGVLGRKVPIQFWTLLYE 99 KSHV --------------------MLGKYVCETEP-LSPGLRRLMWRFLQNKNLNTFHAQELRFIHLVLCKMYNFGLNVYLLREATANAGTYDEVVLGRKVPAEVWKLVYD 86 EBV -------------------MLMGLGVRGDDARMTPGIHGLLLKMLKNWPLCALREDELRFLHLSLTKLLTLSVNFYLWREAVINTGNRTNRVLARKVPDEYWYLLYR 88 * : : :: * :: ** ::: : : : : * * :* ** . * :: M79 AMRAH-VPTETLH-RAFSERSAAALSVEMT--GSRACRALVSHLIRTE-TGLALSLPDELLSDGNIFFSLGTVYGHRLFRLLRFFNRHWGKEAHEPAIRTICQKVWF 192 PR79 ALRER-TPRRALA-GVFSERSAAALSAQLH--RSGAARALVLRLLRDE-TGLAIRPPRELTEDGNVLFSLGTVYGHRLFRLLRFFNAHWGREEFEPAVRVVCQKIWF 207 UL79 ELRQA-FPGLDFEAAVFDETRAARLSQRLC--HPRLSGGLLTRFVQRH-TGLPVVFPEDLARNGNILFSLGTLYGHRLFRLAAFFTRHWGAEAYEPLIRIICQKMWY 201 Rh79 ELRRT-FPQFDFH-VIFHEEQAAQLSQKLS--QPQLSTALLSRFVQRH-TGLLISFPEDLARNGNLLFSLGTLYGHRLFRLAAFFTRHWGTATYEPLIRIICQKMWY 196 HHV6 ILKQH-SDAELLR-HMFNESHSAYISKKLH--YSGNVSHIINFLFMDE-FGLSLKIPEEIICEGNIVFSVGAIYNHRLLKICRFFNRFWGDQEREPAVRLICKHLWF 190 ORF18 VCLESGLTTKDLT----DETVAASLWYRLNTADDGTLCGFCEKIFKKLGLTHHVALDLVNLRDGNLLFNLGSTIPCRLLMCLLFCLKNWGAQGLEPWVRHYVGKFFL 202 KSHV GLEEMGVSSEMLL----CEAYRDSLWMHLN-DKVGLLRGLANYLFHRLGVTHDVRIAPENLVDGNFLFNLGSVLPCRLLLAAGYCLAFWGSDEHERWVRFFAQKLFI 188 EBV ALARVGFPAEALR----PGNRSRSLCLFLH-DRPDVTGAVCACVWAQLGLRRAPDLSQVTLADGNLLFNLGSVLPNRLVVGVLYCLVHWGADEHETRVRARLRPLFV 190 : : : . . :**..*.:*: **. : ** * :* .: M79 FYLIAWKKLTVSPEAFSVQRSDHELGIFSFLIQDYLTFTGTLRRSTP-------------------------PMDKKEEGVIADLLSGALE---------------- 258 PR79 FYLIAWKKLSVSAEAFRVQRSEHQLGVFAFLIQDYLTFTGTLRRRPP-------------------------PLAAENERAIAGLLRGAIQD--------------- 274 UL79 FYLIGTGKMRITPDAFEIQRSRHETGIFTFIMEDYRTFAGTLSRHPHRPHPQQQQHHHPGPPHPPLSHPASSCLSPEAVLAARALHMPTLANDV------------- 295 Rh79 FYLISTGKMTISSEAFEIQRSRHEMGIFTFIIEDYKAFAGTLSLTPR------------------------MTLNPEAVASAKALHTPTACGDM------------- 266 HHV6 AYLIMFGKFEISTLAYNQQRAEHKAGLFSFLQNDFKVFCG-MSENPQ-------------------------LLDSSAIFDLTGISAEDLFSYE------------- 258 ORF18 LYLILAGYLLPRKECVEFAATQSYAGLMETICADILTFQGSQALSLP-------------------------PDNYLQELDQLFTFNNSFISYTTATVTGSAPNTQK 284 KSHV CYLIVSGRLMPQRSLLVWASETGYPGPVEAVCRDIRSMYGIRTYAVSG----------------------YLPAPSEAQLAYLGAFNNNAV---------------- 257 EBV AFLCLAGYLLLDR--AILSDAHDYEGLWHAVALSMAAWHGLTPLPETR----------------------DEKEAKPPCDDFIYLFANDPLQCEELAQLGATGEAR- 272 :* : * : . *

189

murine cytomegalovirus encodes proteins that regulate

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