Virology 5.4 2015Virology 5.4 2015

RNA Virus Gene Expression and RNA Virus Gene Expression and ReplicationReplication

Negative Sense RNA VirusesNegative Sense RNA Viruses

Influenza VirusInfluenza Virus

Influenza A GenomeInfluenza A Genome

Ss – RNASegmented genomeNuclear replication

Influenza A Replication DiagramInfluenza A Replication Diagram

Synthesis of viral RNAs occur in the nucleus“Nuclear options” availableGenome in RNP form

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

Types of Influenza RNA found in the nucleus

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

Key PointsKey Points

Viral mRNAs “mimic” cellular mRNAs with a stolen Viral mRNAs “mimic” cellular mRNAs with a stolen capcap

““Cap-snatching” Cap-snatching”

““Stuttering” produces poly A tailStuttering” produces poly A tail

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

Cap snatching by influenza polymerase

Stuttering makes polyA tail

““Replication” vs. “Expression”Replication” vs. “Expression”

Switch from mRNA synthesis to + cRNASwitch from mRNA synthesis to + cRNA

Same enzyme responsible for both butSame enzyme responsible for both but

products very differentproducts very different

+ cRNA copied to form new vRNA + cRNA copied to form new vRNA segmentssegments

Does NP availability control the switch?Does NP availability control the switch?

Negative Strand RNA Viruses-Negative Strand RNA Viruses-Rhabdoviruses Rhabdoviruses

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

VSVVSVVesicular Stomatitis Vesicular Stomatitis

Virus: Virus: Vesiculovirus genus of Vesiculovirus genus of

RhabdoviridaeRhabdoviridae

(best-studied rhabdovirus)(best-studied rhabdovirus)

Rhabdoviridae are – ssRNA with a Rhabdoviridae are – ssRNA with a “bullet” shape“bullet” shape

envelopedenveloped

Wide host range among rhabdoviridaeWide host range among rhabdoviridae

Infected Cow Showing Ruptured Vesicles on Tongue

Rhabdovirus Structure-Vesicular Stomatis Virus

Electron Micrograph of Virus

Membrane & Nucleocapsid Core

VIRAL ENVELOPELipid 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)

RIBONUCLEOPROTEIN CORE (RNP)- 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 chaperone and helps regulate transcription & replication.

L Protein - RdRp protein (175 kD) that

functions in mRNA transcription & replication. (~1260 N, 470 P &

50 L proteins/Virion) L is a.k.a. Transcriptase

VSV Genome Organization and mRNA Transcription

N Protein

P Protein

M Protein

L Protein

G protein

Diagram of Virus Structure

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

genomic RNAin nucleoprotein form.

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

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

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

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

Transcription of Rhabdovirus mRNAs is Polar

The RDRP transcribes the N gene to produce N mRNA (+).

RdRp is responsible for capping each VSV mRNA.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 about75% of the time it releases the template but reinitiates and continues intothe 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

tailed.

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 How does the RdRp Switch From Transcription to Replication?to Replication?

Synthesis of viral proteins N and P is requiredSynthesis 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.

Uses of VSV Recombinant Derivatives

1) Can be used to study functions of proteins.2) Useful for assessing pathogenicity

determinants.3) Rearranged genes have use as attenuated

vaccines.4) Foreign gene expression can protect against

other viruses.5) Medicinal proteins can modified for diagnostic

and theraupic purposes.

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?