ALL SORTS OF STRATEGIES RNA VIRUSES ALL SORTS OF STRATEGIES

RNA Viruses All synthesize through a double stranded intermediate - RI - replication intermediate RNA dependent RNA polymerase of viral origin but may need host factors Termini contain recognition signals for replicase

Positive strand viruses Begin with translation to produce replicase Makes more positive than negative strand Limiting factor or rapid packaging so can’t act as template Poliovirus uses VPg linked to nucleotides as “primer” - like Ad

Negative Strand Viruses Contain enzymes for transcription in virion Make mRNA prior to antigenome Message gets capped; genome does not Plus strand is template for minus strand genome Makes more minus than plus strand

dsRNA viruses - conservative replication Uncoating activates enzymes that produce mRNA + RNA also gets packaged Then complementary - RNA is produced No dsRNA free in cell Protects against IF induction

Transcription challenges Less temporal control than in DNA viruses Monogenic problem Segmented genomes usually have individual genes Polyprotein cleavage What would expect to see on gel in early stages of infection? As infection progresses? What if you performed a pulse-chase experiment?

Synthesize own cap (Reo in cytoplasm) Translation challenges: Recognition by ribosomes and competition from host Synthesize own cap (Reo in cytoplasm) Steal from host (Influenza in nucleus) Use host enzymes IRES

Transcription strategies: Togaviruses NS at 5’ end - S at 3’ In vitro only synthesize NS proteins; stop signal leads to polyprotein In vivo get shorter mRNA only after minus strand synthesis that codes for S polyprotein Internal transcription site on minus strand Minus is template for mRNA and for genome S message is more abundant than NS as genome gets packaged

Coronaviruses: frame-shifts and subgenomic RNAs Genome translated into replicase Antigenome produced Subgenomic mRNAs represent a nested set of RNAs - all share short 5’ sequence and a 7 base sequence but have unique AUG site and share 3’ end of genome May be produced by jumping polymerase - 7 base sequence in various parts of genome Get recombinant viruses with mixed infections DI particles are common

Influenza virus - segmented negative strand antigenic drift (mutations) vs shift (reassortment)

Influenza - negative strand virus Replication in nucleus using viral enzymes but need host RNA-P to function Virion enzyme cleaves cap from host mRNA and uses it to extend; adds poly A tail

One gene per segment except for two segments producing spliced mRNAs in two different reading frames yielding two proteins

Ambisense genomes Bunyaviridae such as Hantavirus Genome is used to make short positive mRNA Genome is replicated and antigenome (plus strand) is used to make second mRNA Antigenome does not act as message

Nonsegmented negative strand viruses: Mononegavirales (rhabdo, filo, paramyxo) Hypothesis: Start-stop Template 3’ end start point for virion L (RNA_P) and goes to termination signal and mRNA release - then cap and poly A added Some polymerase reinitiates at next initiation signal and goes to termination; process repeats Each subsequent RNA may be produced at a lower frequency (20-30% less) Replication requires N capsid and NS proteins to read through to complete copy

Retroviruses: diploid ssRNA with repeats at ends RT needs a primer - uses tRNA at primer binding site Synthesized to end and jumps to 3’ end of strand Uses PPT as template for second strand Makes another jump Results in dsDNA with Long Terminal Repeats Needed for integration Contains promotor and regulatory regions Poly A site

Transcription occurs after integration Uses host RNA-P May require host factors to enhance (cell tropism) Polyprotein and splicing strategies

HIV is a more complex retrovirus Transactivator protein (TAT) needed for high level of transcription TAT binds to TAR RNA and causes readthrough beyond 5’ region

REV binds to REV Response Element (RRE) in message Early messages are highly spliced and produce mainly TAT, REV and Nef When REV increases and binds to message, there is less splicing Leads to synthesis of gag, pol, env