Virology 5.3, 2015 RNA Virus Gene Expression and Replication Issues, Problems, Strategies for + RNA Viruses Continued

Some Numbers for Poliovirus RNA Genomic RNA is 7433 nucleotides w/o poly A tail 5’UTR is 741 nucleotides (long) 5’ region contains 8 AUG codons The 8 th one is followed by a long ORF Poly C tract 3’ UTR is 71 (short) Poly A tail is 62 nucleotides (variable) Secondary structures in UTRs

Protein Synthesis Is Bimodal in Infected Cells Short 35 S 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 Overcome Two Big Issues and Problems: Translation initiation on an uncapped RNA (preferential) See Ch. 11 pp Expression of downstream cistrons in a polycistronic 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 Eukaryotes Cap-Dependent Initiation of Protein Synthesis in Eukaryotes

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 IRES Textbook pp 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 2A Modification of initiation factor favors viral translation

Translation produces a single giant protein-the polyprotein (Textbook pp ) The polyprotein is 246 kDa in polio infected cells It is cut into smaller pieces as it is being synthesized Proteolytic cleavage-the peptide backbone is cut Proteolytic Processing of the Poliovirus Polyprotein

Proteolytic cleavages are autocatalytic Two viral enzymes make the cuts: 2A pro and 3C pro Also L pro in some picornaviruses Cleavage sites are TYR-GLY and GLN-GLY in correct context Cuts are either cis or trans P1-P2, P2-3 cleavages are primary (2A pro and 3C pro ); others are secondary (3C pro ) Proteolytic activity not dependent on size 3CDpro may be active as proteinase Final cleavages may be maturation cleavages

Viral Proteinases Resemble Cellular Enzymes Similar secondary structures Similar catalytic mechanisms

Protein-priming of minus strand  Membrane environment  Host “factors”  Circular template  Proteolysis  3B = VPg  3AB -> 3A + VPg  VPg converted to VPg- pUUU  Primer for negative strand synthesis

Poliovirus’ Strategies and Solutions to Overcome Two Major Issues or Problems It ensures that its own RNA is translated in a host cell by……….. It ensures the expression of downstream cistrons by…….. THE SOLUTIONS HAVE CONSEQUENCES

Polyprotein Processing in Picornaviruses No real regulation of gene expression All genes are always 100% on or 100% off No timing of expression What does the stoichiometry tell us?

Sites  cornaviridae (general) cornaviridae cornaviridae  33.html (brief) 33.html 33.html

Other Viruses Use Polyproteins Animal Viruses: Flaviviridae Caliciviridae (also sg mRNAs) Plant Viruses Comoviridae Potyviridae (VPg may aid translation) Zucchini Yellow Mosaic Virus-infected fruit