1. Virology 5.4 2015 RNA Virus Gene Expression and Replication Negative Sense RNA Viruses Influenza Virus

2. Influenza A Genome Ss – RNA Segmented genome Nuclear replication

3. Influenza A Replication Diagram Synthesis of viral RNAs occur in the nucleus “Nuclear options” available Genome in RNP form

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

5. Types of Influenza RNA found in the nucleus

6. How does Influenza A ensure its mRNAs are expressed? Key Points Viral mRNAs “mimic” cellular mRNAs with a stolen cap “Cap-snatching” “Stuttering” produces poly A tail Transcripts of RNA 7 & 8 exist in both spliced and unspliced forms: “alternate splicing”

7. Cap snatching by influenza polymerase Stuttering makes polyA tail

8. “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?

9. Negative Strand RNA Viruses- Rhabdoviruses What strategies do they use to deal with their issues and problems?

10. VSV Vesicular Stomatitis Virus: Vesiculovirus genus of Rhabdoviridae (best-studied rhabdovirus) Rhabdoviridae are – ssRNA with a “bullet” shape enveloped enveloped Wide host range among rhabdoviridae

11. Infected Cow Showing Ruptured Vesicles on Tongue

12. Rhabdovirus Structure-Vesicular Stomatis Virus Electron Micrograph of Virus Membrane & Nucleocapsid Core VIRAL ENVELOPE 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) 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

13. 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 RNA in 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.

14. 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 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 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.

15. 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.

16. 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.

17. 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?