Virology 5.3, 2015Virology 5.3, 2015

RNA Virus Gene Expression and RNA Virus Gene Expression and ReplicationReplication

Issues, Problems, Strategies for + RNA Issues, Problems, Strategies for + RNA Viruses Continued Viruses Continued

Some Numbers for Poliovirus RNASome Numbers for Poliovirus RNA

Genomic RNA is 7433 nucleotides w/o poly A tailGenomic RNA is 7433 nucleotides w/o poly A tail5’UTR is 741 nucleotides (long)5’UTR is 741 nucleotides (long)5’ region contains 8 AUG codons5’ region contains 8 AUG codonsThe 8The 8thth one is followed by a long ORF one is followed by a long ORFPoly C tractPoly C tract3’ UTR is 71 (short)3’ UTR is 71 (short)Poly A tail is 62 nucleotides (variable)Poly A tail is 62 nucleotides (variable)Secondary structures in UTRsSecondary structures in UTRs

Protein Synthesis Is Bimodal in Infected Cells

Short Short 3535S Methionine PulseS Methionine Pulse

Protein synthesis shows rapid decline 30 min postinfection.After two hours synthesis is lowest but increases rapidly.By four hours most synthesis is poliovirus protein.Synthesis again declines rapidly near end of replication.

Polio and Other Picornaviruses Polio and Other Picornaviruses Overcome Two Big Issues and Overcome Two Big Issues and

Problems:Problems:

Translation initiation on an uncapped RNATranslation initiation on an uncapped RNA(preferential) See Ch. 11 pp 379-389.(preferential) See Ch. 11 pp 379-389.

Expression of downstream cistrons in a Expression of downstream cistrons in a polycistronic mRNApolycistronic mRNA

The 40S complex then scans down the 5’ untranslated region to the first AUG codon.A GTP hydrolysis step by eIF5 triggers GDP binding of eIF2 and released of eIF factors.The 60S subunit joins to form the 80S ribosome translation of the ORF is initiated. HOST CELL mRNAs ARE CAPPED BUT PICORNA RNA IS NOT!!!!!

An initiation complex forms at the cap with the 40S ribosomal subunit and elongation initiation factors (eIF) are added. These include a polyA binding protein that interacts with the 3’ poly(A) of eukaryotic mRNAs.

Cap-Dependent Initiation of Protein Synthesis in Cap-Dependent Initiation of Protein Synthesis in EukaryotesEukaryotes

Internal Ribosome Entry Site (IRES) for Cap-Independent Initiation

Left: Poliovirus with Type I IRES, Right: Type II IRES of EMC virus;Common motifs = GNRA sequence, Y-rich region

Picornavirus mRNAs have no cap (pUp) at their 5’ terminus and no VPg.Ribosomal binding occurs at an Internal Ribosomal Entry Site (IRES). The IRES contains a high level of secondary structure in the UTR that mediates ribosome 40 S subunit binding

and initiation of translation.eIF-3, eIF-4G and eIF-4a promote IRES assembly. A host factor X is also required (poly C binding protein PCB or pyrimidine tract binding protein PTB?).•Eighth AUG used

Translation Initiation on an IRES

Truncated Version of eIF4G Directs Initiation at IRESTextbook pp. 402-404

Very first events of infection are unclear. How is the FIRST round of translation initiated.Later in infection the eIF4G is cleaved by viral protease 2AModification of initiation factor favors viral translation

Translation produces a single giant protein-the Translation produces a single giant protein-the polyprotein (Textbook pp. 392-394)polyprotein (Textbook pp. 392-394)

The polyprotein is 246 kDa in polio infected cellsThe polyprotein is 246 kDa in polio infected cellsIt is cut into smaller pieces as it is being synthesizedIt is cut into smaller pieces as it is being synthesized

Proteolytic cleavage-the peptide backbone is cutProteolytic cleavage-the peptide backbone is cutProteolytic Processing of the Poliovirus PolyproteinProteolytic Processing of the Poliovirus Polyprotein

Proteolytic cleavages are autocatalyticProteolytic cleavages are autocatalytic

Two viral enzymes make the cuts: 2ATwo viral enzymes make the cuts: 2Apropro and 3C and 3Cpropro

Also LAlso Lpropro in some picornaviruses in some picornavirusesCleavage sites are TYR-GLY and GLN-GLY in correct contextCleavage sites are TYR-GLY and GLN-GLY in correct context

Cuts are either cis or transCuts are either cis or transP1-P2, P2-3 cleavages are primary (2AP1-P2, P2-3 cleavages are primary (2Apropro and 3C and 3Cpropro); others are secondary (3C); others are secondary (3Cpropro))

Proteolytic activity not dependent on sizeProteolytic activity not dependent on size3CDpro may be active as proteinase 3CDpro may be active as proteinase

Final cleavages may be maturation cleavagesFinal cleavages may be maturation cleavages

Viral Proteinases Resemble Cellular EnzymesViral Proteinases Resemble Cellular Enzymes

Similar secondary structuresSimilar secondary structuresSimilar catalytic mechanismsSimilar catalytic mechanisms

Protein-priming of minus strandProtein-priming of minus strand Membrane environmentMembrane environment Host “factors”Host “factors” Circular templateCircular template ProteolysisProteolysis 3B = VPg3B = VPg 3AB -> 3A + VPg3AB -> 3A + VPg

VPg converted to VPg-pUUUVPg converted to VPg-pUUU Primer for negative strand Primer for negative strand

synthesissynthesis

Poliovirus’ Strategies and Solutions Poliovirus’ Strategies and Solutions to Overcome Two Major Issues or to Overcome Two Major Issues or

ProblemsProblems

It ensures that its own RNA is translated in a It ensures that its own RNA is translated in a host cell by………..host cell by………..

It ensures the expression of downstream It ensures the expression of downstream cistrons by……..cistrons by……..

THE SOLUTIONS HAVE CONSEQUENCESTHE SOLUTIONS HAVE CONSEQUENCES

Polyprotein Processing in Polyprotein Processing in PicornavirusesPicornaviruses

No real regulation of gene expressionNo real regulation of gene expression

All genes are always 100% on or 100% offAll genes are always 100% on or 100% off

No timing of expressionNo timing of expression

What does the stoichiometry tell us?What does the stoichiometry tell us?

SitesSites

http://microbewiki.kenyon.edu/index.php/Picornaviridae (general) (general)

http://viralzone.expasy.org/all_by_species/33.html (brief) (brief)

Other Viruses Use PolyproteinsOther Viruses Use Polyproteins

Animal Viruses: Animal Viruses: FlaviviridaeFlaviviridaeCaliciviridae (also sg Caliciviridae (also sg mRNAs)mRNAs)

Plant VirusesPlant VirusesComoviridaeComoviridaePotyviridae (VPg may Potyviridae (VPg may aid translation)aid translation)

Zucchini Yellow Mosaic Virus-infected fruit