rna virus replication/ trnslation strategies sadia anjum virology lecture 5 1

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RNA VIRUS REPLICATION/ TRNSLATION STRATEGIES

Sadia Anjum

Virology lecture 5

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RNA VIRUS STRATEGIES

RNA -> RNARNA-dependent RNA polymerase

RNA -> DNARNA-dependent DNA polymerase- reverse transcriptase

Host cell DNA -> RNADNA-dependent RNA polymerase

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All animal RNA viruses code for a polymerase

Need to make mRNAPLUS (POSITIVE) SENSE RNA GENOMES

AAA

(+ve) sense mRNA

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NEED TO MAKE MRNA

MINUS (NEGATIVE) SENSE RNA GENOMES

AAA(+ve) sense mRNA

(-ve) sense genomic RNA

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NEED TO MAKE MRNAMINUS (NEGATIVE) SENSE RNA GENOMES

RNA polymerase must be packaged in virion.

AAA(+ve) sense mRNA

(-ve) sense genomic RNAIf used, RNA modifying enzymes arepackaged in virion.

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DOUBLE-STRANDED RNA GENOMES

AAA(+ve) sense mRNA

Double-stranded genomic RNAIf used, RNA modifying enzymes arepackaged in virion.

RNA polymerase must be packaged in virion.

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RETROVIRUSES

DSDNA

+VE RNA

DSDNA

Reverse transcriptase must be packaged in virion.

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RNA VIRUSES THAT DO NOT HAVE A DNA PHASE

TranscriptionNon-infectiousYesDouble-stranded RNA

TranscriptionNon-infectiousYesNegative-stranded RNA

TranslationInfectiousNoPlus-stranded RNA

Initial event in cell

Infectivity of RNA

RNA-dependent RNA polymerase

(=transcriptase) IN VIRION

Genome

TranscriptionNon-infectiousYesDouble-stranded RNA

TranscriptionNon-infectiousYesNegative-stranded RNA

TranslationInfectiousNoPlus-stranded RNA

Initial event in cell

Infectivity of RNA

RNA-dependent RNA polymerase

(=transcriptase) IN VIRION

Genome

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RETROVIRUSESGenome RNA-

dependent DNA polymerase (=reverse transcriptase) in virion

Infectivity of RNA

Initial event in cell

Plus-stranded RNA

Yes Non-infectious Reverse transcription

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THE MONOCISTRONIC MRNA PROBLEM

Make one monocistronic mRNA per protein Make a primary transcript and use alternative splicing Make a large protein and then cut it into smaller

proteins Include special features in the mRNA which enable

ribosomes to bind internally

AAAAAAAA

RIBOSOMES

mRNA

PROTEIN

AAAAAAAA

Eukaryotes

Prokaryotes/viruses

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POLYCISTRONIC VS MONOCISTRONIC

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RNA GENOME SIZE

TAUTOMERIZATION PROOF READING GENOME SIZE

RNA VIRUSES HAVE SMALL GENOMES~10,000 NUCLEOTIDES(herpesvirus DNA ~200,000)

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GENOME SIZE POLYMERASE ATTACHMENT PROTEIN CAPSID PROTEIN RELATIVELY FEW OTHER PROTEINS VIRION SIZE MAY BE QUITE LARGE BUT

LIMITED NUMBER OF PROTEINS

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POSITIVE-STRANDED RNA VIRUSES

EXAMPLES

PICORNAVIRUSES

TOGAVIRUSES

FLAVIVIRUSES

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PICORNAVIRUS FAMILY (PICORNAVIRIDAE)

SMALL ICOSAHEDRAL POSITIVE SENSE RNA NON-ENVELOPED

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INTERNAL RIBOSOME ENTRY SITE (IRES)

GENOMIC (+ SENSE) RNAAAAAAVPg

IRES

start codonfor translation

stop codonfor translation

POLYPROTEIN

.

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VPG VPg (viral protein genome-linked) is a protein that is

covalently attached to the 5′ end of positive strand viral RNA and acts as a primer during RNA synthesis in a variety of virus families including Picornaviridae and Caliciviridae.

The primer activity of VPg occurs when the protein becomes uridylylated, providing a free hydroxyl that can be extended by the virally encoded RNA-dependent RNA polymerase.

Aside from functioning as a primer, VPg also has a role in translation initiation by acting like a 5' mRNA cap

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INHIBITION OF HOST CELL TRANSLATION

HOST mRNAAAAAACAP

Uninfected cell

Factors+40S ribosomal subunit

HOST mRNAAAAAACAP

Altered factors+40S ribosomal subunit

Infected cell

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VIRAL TRANSLATION

GENOMIC (+ SENSE) RNAAAAAA

IRES

VPg

Factors+40S ribosomal subunit

Altered factors+40S ribosomal subunit

GENOMIC (+ SENSE) RNAAAAAA

IRES

VPg

Uninfected cell

Infected cell

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GENOMIC (+ SENSE) RNAAAAAA

translation

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RNA REPLICATION

GENOMIC (+ SENSE) RNA 3’VPg

(- SENSE) RNA 3’ VPg

GENOMIC (+ SENSE) RNA 3’VPg

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RNA REPLICATION viral RNA polymerase (replicase) host factors also involved as accessory

proteins new plus strands

packaged templates for more replication templates for more translation

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ASSEMBLY

VP0, VP1, VP3

VP2, VP4, VP1, VP3VPg

VPg-RNA enters, VP0 is cleaved

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NON-SEGMENTED NEGATIVE STRAND VIRUSES

Examples:

Rhabdovirus family (Rhabdoviridae)

Paramyxovirus family (Paramyxoviridae)

Filovirus family (Filoviridae)

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RHABDOVIRUSES

helical nucleocapsid (RNA plus N protein)

G glycoprotein SPIKES

lipid bilayer membrane

polymerase complex

M protein

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MINUS (NEGATIVE) SENSE RNA GENOME

RNA polymerase must be packaged in virion.

AAA(+ve) sense mRNA

(-ve) sense genomic RNA

RNA modifying enzymes are packaged in virion.

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RHABDOVIRUSES

EXAMPLESvesicular stomatitis

virus (VSV)

rabies virus

Dr FS Murphyhttp://www.vetnet.ucdavis.edu/fam_graphics/download.html

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ADSORPTION AND PENETRATION

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CYTOPLASMIC REPLICATION

GENOMIC RNA REMAINS IN NUCLEOCAPSID FORM

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RNA SYNTHESIS

3’ 5’genome (-ve)

3’ 5’ (-ve)

full-length copy

5’ 3’ (+ve)

replication (protein synthesis is a pre-requisite)

transcription

mRNAs (+ve sense)

AAA

AAA

AAA

AAA

AAA

= cap

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NEW MINUS STRAND

PACKAGED TEMPLATE FOR

MORE REPLICATION TEMPLATE FOR

MORE TRANSCRIPTION

3’ 5’genome (-ve)

3’ 5’ (-ve)

5’ 3’ (+ve)

AA

AA

AA

AA

AA

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SOME POINTS TO NOTE ABOUT RHABDOVIRUSES

ENTIRE CYCLE OCCURS IN CYTOPLASM

RNA POLYMERASE AND RNA MODIFICATION

ENZYMES ARE:

VIRALLY CODED

PRESENT IN THE VIRION

THERE IS NO EARLY/LATE DIVISION

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MEMBERS INCLUDE

PARAINFLUENZA VIRUS

MUMPS VIRUS

MEASLES VIRUS

RESPIRATORY SYNCYTIAL VIRUS

PARAMYXOVIRUSES

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PARAMYXOVIRUSES

M protein

helical nucleocapsid (RNA plusNP protein)

HN/H/G glycoprotein SPIKES

polymerase complex

lipid bilayer membrane

F glycoprotein SPIKES

pleomorphic

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PARAMYXOVIRIDAE

PARAMYXOVIRUS SUBFAMILY

PARAMYXOVIRUS

RUBULAVIRUS

MORBILLIVIRUS

PNEUMOVIRUS SUBFAMILY

PNEUMOVIRUS GENUS

METAPNEUMOVIRUS GENUS

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GENUS GLYCOPROTEINS TYPICAL MEMBERS

Paramyxovirus HN, F HPIV1, HPIV3Rubulavirus HN, F HPIV2, HPIV4, mumps virusMorbillivirus H, F measles virus

Pneumovirus G, F respiratory syncytial virusMetapneumovirus G, F metapneumoviruses

PARAMYXOVIRUS FAMILY SURFACE GLYCOPROTEINS

PARAMYXOVIRUS SUBFAMILY

PNEUMOVIRUS SUBFAMILY

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TRANSCRIPTION, TRANSLATION, REPLICATION

3’ 5’genome (-ve)

full-length copy

5’ 3’ (+ve)

3’ 5’ (-ve)

replication (protein synthesis is a pre-requisite)

transcription

mRNAs (+ve sense)

AAA

AAA

AAA

AAA

AAA

= cap

AAA

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SOME DIFFERENCES BETWEEN RHABDOVIRUSES AND PARAMYXOVIRUSES

two:

one attachment

one fusion

one (has both attachment and fusion activities)

glycoproteins

neutral

physiological

acidicfusion pH

round

pleomorphic

bullet

bacilliform

shape

PARAMYXOVIRUSESRHABDOVIRUSES

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ORTHOMYXOVIRUSES

M1 protein

helical nucleocapsid (RNA and NP protein)

HA - hemagglutinin

polymerasecomplex

lipid bilayer membrane

NA - neuraminidase

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ADSORPTION AND PENETRATION

NUCLEUS

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TRANSCRIPTION (MRNA SYNTHESIS)

AAAA

viral endonuclease

A

host cell mRNA

influenza mRNA

=cap

influenza virus nucleocapsidAU

AU

viral polymerase

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RNA SYNTHESIS

Endonuclease is virally coded and packed in the

virion

RNA polymerase is virally coded and packed in

the virion

Poly(A) polymerase is virally coded and in the

virion

Why doesn’t the virus use host cell enzymes?

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RNA SPLICING

Several segments give rise to transcripts which can have one of two fates

AAAAAA

AAAAAA

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RNA REPLICATION nuclear

authentic copies

replicase and

transcriptase probably

same enzyme

coated with protein as

made

no clear early/late

3’ 5’genomic (-ve)

3’ 5’ genomic (-ve)

5’ 3’ (+ve)

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NUCLEUS

acid pH inside endosome

- HA conformational change, now active in membrane fusion

Infection of next cell

HA cleavage by host cell enzymes

Flu Virus

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PARAMYXOVIRIDAE ORTHOMYXOVIRIDAE

genome nonsegmented segmented

RNA synthesis cytoplasmic nuclear

need for RNA primer

no yes

hemagglutinin, neuraminidase

if both, part of same protein (HN)

Influenza A and B viruses have both, but on different proteins (HA and NA)

syncytial formation

yes (F functions at physiol. pH)

no (HA functions at acid pH)

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REOVIRUS FAMILYdouble stranded RNA genome

outer capsid

inner capsid

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DOUBLE-STRANDED RNA GENOMES

AAA(+ve) sense mRNA

Double-stranded genomic RNARNA modifying enzymes arepackaged in virion.

RNA polymerase must bepackaged in virion.

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REOVIRUS FAMILYdouble stranded RNA genome

outer capsid

inner capsid

RNA polymerase plus 5’ endmodification enzymes

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REOVIRUS FAMILY INCLUDES

REOVIRUSES ROTAVIRUSES

IMPORTANT HUMAN PATHOGENS ORBIVIRUSES

INCLUDE COLORADO TICK FEVER VIRUS

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Annual Review of Microbiology 51:227 RF Ramig