RNA VIRUS REPLICATION/ TRNSLATION STRATEGIES Sadia Anjum Virology lecture 5 1

RNA VIRUS STRATEGIES 2 RNA -> RNA RNA-dependent RNA polymerase RNA -> DNA RNA-dependent DNA polymerase - reverse transcriptase Host cell DNA -> RNA DNA-dependent RNA polymerase

3 All animal RNA viruses code for a polymerase Need to make mRNA PLUS (POSITIVE) SENSE RNA GENOMES AAA (+ve) sense mRNA

N EED TO MAKE M RNA 4 MINUS (NEGATIVE) SENSE RNA GENOMES AAA (+ve) sense mRNA (-ve) sense genomic RNA

N EED TO MAKE M RNA 5 MINUS (NEGATIVE) SENSE RNA GENOMES RNA polymerase must be packaged in virion. AAA (+ve) sense mRNA (-ve) sense genomic RNA If used, RNA modifying enzymes are packaged in virion.

6 DOUBLE-STRANDED RNA GENOMES AAA (+ve) sense mRNA Double-stranded genomic RNA If used, RNA modifying enzymes are packaged in virion. RNA polymerase must be packaged in virion.

7 RETROVIRUSES DS DNA +VE RNA DS DNA Reverse transcriptase must be packaged in virion.

RNA VIRUSES THAT DO NOT HAVE A DNA PHASE 8

RETROVIRUSES 9 GenomeRNA- dependent DNA polymerase (=reverse transcriptase) in virion Infectivity of RNA Initial event in cell Plus-stranded RNA YesNon-infectious Reverse transcription

T HE MONOCISTRONIC M RNA 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 10 AAAAAAAA RIBOSOMES mRNA PROTEIN AAAAAAAA Eukaryotes Prokaryotes/viruses

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P OLYCISTRONIC VS M ONOCISTRONIC 12

RNA GENOME SIZE TAUTOMERIZATION PROOF READING GENOME SIZE RNA VIRUSES HAVE SMALL GENOMES ~10,000 NUCLEOTIDES (herpesvirus DNA ~200,000) 13

GENOME SIZE POLYMERASE ATTACHMENT PROTEIN CAPSID PROTEIN RELATIVELY FEW OTHER PROTEINS VIRION SIZE MAY BE QUITE LARGE BUT LIMITED NUMBER OF PROTEINS 14

POSITIVE-STRANDED RNA VIRUSES EXAMPLES PICORNAVIRUSES TOGAVIRUSES FLAVIVIRUSES 15

PICORNAVIRUS FAMILY (PICORNAVIRIDAE ) 16 SMALL ICOSAHEDRAL POSITIVE SENSE RNA NON-ENVELOPED

INTERNAL RIBOSOME ENTRY SITE (IRES) 17 GENOMIC (+ SENSE) RNA AAAAAVPg IRES start codon for translation stop codon for translation POLYPROTEIN.

V PG 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 18

I NHIBITION OF HOST CELL TRANSLATION 19 HOST mRNA AAAAACAP Uninfected cell Factors+40S ribosomal subunit HOST mRNA AAAAACAP Altered factors+40S ribosomal subunit Infected cell

V IRAL TRANSLATION 20 GENOMIC (+ SENSE) RNA AAAAA IRES VPg Factors+40S ribosomal subunit Altered factors+40S ribosomal subunit GENOMIC (+ SENSE) RNA AAAAA IRES VPg Uninfected cell Infected cell

21 GENOMIC (+ SENSE) RNA AAAAA translation

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RNA REPLICATION 23 GENOMIC (+ SENSE) RNA 3’VPg (- SENSE) RNA 3’ VPg GENOMIC (+ SENSE) RNA 3’VPg

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 24

ASSEMBLY 25 VP0, VP1, VP3 VP2, VP4, VP1, VP3 VPg VPg-RNA enters, VP0 is cleaved

NON-SEGMENTED NEGATIVE STRAND VIRUSES 26 Examples: Rhabdovirus family (Rhabdoviridae) Paramyxovirus family (Paramyxoviridae) Filovirus family (Filoviridae)

RHABDOVIRUSES 27 helical nucleocapsid (RNA plus N protein) G glycoprotein SPIKES lipid bilayer membrane polymerase complex M protein

28 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.

RHABDOVIRUSES EXAMPLES vesicular stomatitis virus (VSV) rabies virus 29 Dr FS Murphy

ADSORPTION AND PENETRATION 30

CYTOPLASMIC REPLICATION GENOMIC RNA REMAINS IN NUCLEOCAPSID FORM 31

RNA SYNTHESIS 32 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 = cap

NEW MINUS STRAND PACKAGED TEMPLATE FOR MORE REPLICATION TEMPLATE FOR MORE TRANSCRIPTION 33 3’5’ genome (-ve) 3’ 5’ (-ve) 5’3’ (+ve) AA

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 34

MEMBERS INCLUDE PARAINFLUENZA VIRUS MUMPS VIRUS MEASLES VIRUS RESPIRATORY SYNCYTIAL VIRUS 35 PARAMYXOVIRUSES

36 M protein helical nucleocapsid (RNA plus NP protein) HN/H/G glycoprotein SPIKES polymerase complex lipid bilayer membrane F glycoprotein SPIKES pleomorphic

PARAMYXOVIRIDAE PARAMYXOVIRUS SUBFAMILY PARAMYXOVIRUS RUBULAVIRUS MORBILLIVIRUS PNEUMOVIRUS SUBFAMILY PNEUMOVIRUS GENUS METAPNEUMOVIRUS GENUS 37

38 GENUSGLYCOPROTEINSTYPICAL MEMBERS ParamyxovirusHN, FHPIV1, HPIV3 RubulavirusHN, FHPIV2, HPIV4, mumps virus MorbillivirusH, Fmeasles virus PneumovirusG, Frespiratory syncytial virus MetapneumovirusG, Fmetapneumoviruses PARAMYXOVIRUS FAMILY SURFACE GLYCOPROTEINS PARAMYXOVIRUS SUBFAMILY PNEUMOVIRUS SUBFAMILY

TRANSCRIPTION, TRANSLATION, REPLICATION 39 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 = cap AAA

S OME DIFFERENCES BETWEEN RHABDOVIRUSES AND PARAMYXOVIRUSES 40 two: one attachment one fusion one (has both attachment and fusion activities) glycoproteins neutral physiological acidicfusion pH round pleomorphic bullet bacilliform shape PARAMYXOVIRUSESRHABDOVIRUSES

ORTHOMYXOVIRUSES 41 M1 protein helical nucleocapsid (RNA and NP protein) HA - hemagglutinin polymerase complex lipid bilayer membrane NA - neuraminidase

ADSORPTION AND PENETRATION 42 NUCLEUS

TRANSCRIPTION ( M RNA SYNTHESIS ) 43 AAA A viral endonuclease A host cell mRNA influenza mRNA =cap influenza virus nucleocapsid A U A U viral polymerase

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? 44

RNA SPLICING 45 Several segments give rise to transcripts which can have one of two fates AAAAAA

RNA REPLICATION nuclear authentic copies replicase and transcriptase probably same enzyme coated with protein as made no clear early/late 46 3’5’ genomic (-ve) 3’5’ genomic (-ve) 5’3’ (+ve)

47 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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REOVIRUS FAMILY 49 double stranded RNA genome outer capsid inner capsid

50 DOUBLE-STRANDED RNA GENOMES AAA (+ve) sense mRNA Double-stranded genomic RNA RNA modifying enzymes are packaged in virion. RNA polymerase must be packaged in virion.

REOVIRUS FAMILY 51 double stranded RNA genome outer capsid inner capsid RNA polymerase plus 5’ end modification enzymes

REOVIRUS FAMILY INCLUDES REOVIRUSES ROTAVIRUSES IMPORTANT HUMAN PATHOGENS ORBIVIRUSES INCLUDE COLORADO TICK FEVER VIRUS 52

53 Annual Review of Microbiology 51:227 RF Ramig