Virology 5 4 2014
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Virology 5.4 2014. RNA Virus Gene Expression and Replication Negative Sense RNA Viruses Influenza Virus. Influenza A Genome. Ss – RNA Segmented genome Nuclear replication. Influenza A Replication Diagram. Synthesis of viral RNAs occur in the nucleus “Nuclear options” available

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Virology 5.4 2014

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Virology 5 4 2014

Virology 5.4 2014

RNA Virus Gene Expression and Replication

Negative Sense RNA Viruses

Influenza Virus

Influenza a genome

Influenza A Genome

Ss – RNA

Segmented genome

Nuclear replication

Influenza a replication diagram

Influenza A Replication Diagram

Synthesis of viral RNAs occur in the nucleus

“Nuclear options” available

Genome in RNP form

Virology 5 4 2014

Three types of RNA formed: mRNA(+), vRNA(-), cRNA(+)

Virology 5 4 2014

Types of Influenza RNA found in the nucleus

How does influenza a ensure its mrnas are expressed key points

How does Influenza A ensure its mRNAs are expressed?Key Points

Viral mRNAs “mimic” cellular mRNAs with a stolen cap


“Stuttering” produces poly A tail

Transcripts of RNA 7 & 8 exist in both spliced and unspliced forms: “alternate splicing”

Virology 5 4 2014

Cap snatching by influenza polymerase

Stuttering makes polyA tail

Replication vs expression

“Replication” vs. “Expression”

Switch from mRNA synthesis to + cRNA

Same enzyme responsible for both but

products very different

+ cRNA copied to form new vRNA segments

Does NP availability control the switch?

Negative strand rna viruses rhabdoviruses

Negative Strand RNA Viruses-Rhabdoviruses

What strategies do they use to deal with their issues and problems?

Virology 5 4 20141

Virology 5.4 2014

RNA Virus Gene Expression and Replication

Negative Sense RNA Viruses


Vsv vesicular stomatitis virus vesiculovirus genus of rhabdoviridae best studied rhabdovirus

VSVVesicular Stomatitis Virus: Vesiculovirus genus of Rhabdoviridae(best-studied rhabdovirus)

Rhabdoviridae are – ssRNA with a “bullet” shape


Wide host range among rhabdoviridae

Virology 5 4 2014

Infected Cow Showing Ruptured Vesicles on Tongue

Virology 5 4 2014

Electron Micrograph of Virus

Membrane & Nucleocapsid Core

Rhabdovirus Structure-Vesicular Stomatis Virus


Lipid membrane - derived from the host cell.

G Protein - Glycoprotein trimers protrude from the membrane as spikes.

M Protein - Matrix protein located at inner membrane surface. Binds C-terminus of the G protein & the RNP Core.

(~1200 G proteins & 1825 M proteins/Virion)


- Infectious & transcriptionally active.

- Synthesizes (+) sense mRNAs in vitro.

N Protein - Nucleocapsid protein (50 kD) that encapsidates the genomic & antigenomic RNA.

P Protein - Phosphoprotein (50 kD) is chaperoneand helps regulate transcription & replication.

L Protein - RdRp protein (175 kD) that

functions in mRNA transcription &

replication.(~1260 N, 470 P & 50 L


L is a.k.a. Transcriptase

Virology 5 4 2014

VSV Genome Organization and mRNA Transcription

G protein

M Protein

P Protein

N Protein

L Protein

  • Gene order is 3’ N, P, M, G, L, 5’.

  • Short leader (46 nt) and trailer (64) nt sequences flank coding regions.

  • Each gene is separated by a short gene junction (GJ) sequence.

4) RDRP starts transcription of the leader RNA at 3’ end of genomic RNA

in nucleoprotein form.

5) The leader RNA terminates at the GJ sequence. Two nucleotides are skipped & N mRNA is transcribed.

Diagram of Virus Structure

Virology 5 4 2014

Transcription of Rhabdovirus mRNAs is Polar

The RDRP transcribes the N gene to produce

N mRNA (+).

RdRp is responsible for capping each VSV


As the RDRP approaches the N:P gene-junction it slows down to copy a series of

U residues near the end of the gene.

About 25% of the time RdRp falls off the template after copying the U’s and about

75% of the time it releases the template but reinitiates and continues into

the next gene. (mechanism called transcriptional attenuation)

With this mechanism, decreasing amounts of capped & polyadenylated P, M, G &

L mRNAS are synthesized. Leader RNA is also made but not capped or


The mRNAs are naked and not associated with the N, P or L proteins because the

only nucleation site for the core proteins is at the 3’ end of the genomic

and antigenomic RNAs.

Synthesis of new viral proteins is not essential for mRNA transcription.

How does the rdrp switch from transcription to replication

How does the RdRp Switch From Transcription to Replication?

Synthesis of viral proteins N and P is required

N and P are translated from VSV mRNAs.

The proteins bind to (“encapsidate”) the leader sequence.

This change in template structure allows RdRp to ignore termination signals.

The enzyme becomes more “processive”. It is not clear whether there is a change in the structure of the RdRp.

Synthesizes full length + and – RNAs (asymmetric).

Replication is linked to expression through the synthesis of the N and P proteins.

Virology 5 4 2014

Uses of VSV Recombinant Derivatives

  • Can be used to study functions of proteins.

  • Useful for assessing pathogenicity determinants.

  • Rearranged genes have use as attenuated vaccines.

  • Foreign gene expression can protect against other viruses.

  • Medicinal proteins can modified for diagnostic and theraupic purposes.

Virology 5 4 2014

How do rhabdoviruses ensure that their mRNAs are expressed?

How do rhabdoviruses make sure that downstream genes are expressed?

How do rhabdoviruses fine tune gene expression?

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