RNA VIRUSESRNA VIRUSES

ALL SORTS OF STRATEGIES

RNA VirusesRNA 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 virusesPositive 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 VirusesNegative 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 dsRNA viruses - conservative replicationreplication

• 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 challengesTranscription 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?

TranslationTranslation challengeschallenges:: Recognition by ribosomes Recognition by ribosomes and competition from hostand competition from host

• Synthesize own cap (Reo in cytoplasm)

• Steal from host (Influenza in nucleus)

• Use host enzymes

• IRES

Transcription strategies: TogavirusesTranscription 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 Coronaviruses: frame-shifts and subgenomic RNAsRNAs

• 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 strandInfluenza virus - segmented negative strandantigenic drift (mutations) vs shift (reassortment)antigenic drift (mutations) vs shift (reassortment)

Influenza - negative strand virusInfluenza - 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 genomesAmbisense 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: Nonsegmented negative strand viruses: Mononegavirales (rhabdo, filo, paramyxo)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 Retroviruses: diploid ssRNA with repeats at endsrepeats 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 integrationTranscription occurs after integration

• Uses host RNA-P

• May require host factors to enhance (cell tropism)

• Polyprotein and splicing strategies

HIV is a more complex retrovirusHIV 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 REV binds to REV Response Element (RRE) in message(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

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