ALL SORTS OF STRATEGIES
RI replication intermediate • RNA dependent RNA polymerase of viral origin but may need host factors • Termini contain recognition signals for replicase
• All synthesize through a double stranded intermediate .
.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” .
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
• Uncoating activates enzymes that produce mRNA • + RNA also gets packaged • Then complementary RNA is produced • No dsRNA free in cell • Protects against IF induction
.dsRNA viruses .
• 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?
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
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
.Transcription strategies: Togaviruses
• NS at 5’ end .
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 .Coronaviruses: frame-shifts and subgenomic RNAs
• Genome translated into replicase • Antigenome produced • Subgenomic mRNAs represent a nested set of RNAs .7 base sequence in various parts of genome – Get recombinant viruses with mixed infections – DI particles are common
segmented negative strand antigenic drift (mutations) vs shift (reassortment)
.Influenza virus .
adds poly A tail
.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.
• One gene per segment except for two segments producing spliced mRNAs in two different reading frames yielding two proteins
• 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
• Hypothesis: Start-stop • Template 3’ end start point for virion L (RNA_P) and goes to termination signal and mRNA release . filo.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
.Nonsegmented negative strand viruses: Mononegavirales (rhabdo.
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
pol.REV binds to REV Response Element (RRE) in message
• Early messages are highly spliced and produce mainly TAT. env
. there is less splicing • Leads to synthesis of gag. REV and Nef • When REV increases and binds to message.