Lecture 16: Processing of viral pre-mRNA

1. Lecture 16: Processing of viral pre-mRNA BSCI437Flint et al., Chapter 10

2. GENERAL OVERVIEW • Viral mRNAs are translated by cellular protein synthetic apparatus • They must conform to the requirements of host cell translation system • A series of covalent modifications allow this to occur: RNA processing • After processing, mRNAs are translated in the cytoplasm • For viral mRNAs produced in the nucleus, they must be exported to the cytoplasm • Once in the cytoplasm, gene expression is a balance between the translatability of an mRNA and its stability. • Viral mRNAs have evolved to be very stable.

3. GENERAL OVERVIEW (Fig. 10.1)

4. COVALENT MODIFICATIONS DURING VIRAL PRE-mRNA PROCESSING • Pre-mRNA modification of cellular mRNAs is performed in the nucleus • Addition of 5’ 7Methyl-Gppp caps • Addition of 3’ poly-adenylated tails • Splicing • RNA editing (in some cases. Not discussed in this lecture).

5. Capping of cellular pre-mRNA 5’ ends 5’ 7MeGppp caps discovered by Shatkin using Reovirus Cellular mRNAs capped cotranscriptionally in the nucleus by action of 5 enzymes Functions of the cap: • Protect 5’ end from exonucolytic attack • Interact with translation initiation apparatus • Mark mRNAs as “self”

6. Capping of viral pre-mRNA 5’ ends • Synthesized by host cell enzymes. e.g. Retroviruses, Adenoviruses • Synthesized by viral enzymes, e.g. Poxviruses, Reoviruses • Cap snatching: virus steal caps from host mRNAs. e.g. Influenza • Note: many RNA viruses have evolved around requirement for cap. • Proteins covalently bound to 5’ end substitute for caps. e.g. Picornaviruses • Translation initiated internally on mRNA at IRES elements. e.g. Hepatitis C virus.

7. Synthesis of 3’ polyA tails: cellular mRNAs. • All cellular mRNAs have non-templated polyA tails attached to their 3’ ends. • PolyA tails also first discovered using viral systems • PolyA tails are post-transcriptionally added to cellular mRNAs using a series of cis-acting sequences on the mRNA and trans-acting ribonucleoprotein factors • PolyA tails interact with PolyA-binding protein: important for translation. (Fig. 10.3)

8. Synthesis of 3’ polyA tails: viral mRNAs • Viral mRNAs can be polyadenylated by host or viral enzymes • By Host enzymes: • Occurs like host mRNAs. • Post-transcriptionally • Examples: retroviruses, herpesviruses, adenoviruses.

9. Synthesis of 3’ polyA tails: viral mRNAs • By Viral enzymes • Can occur co-transcriptionally: • Copying of a long polyU stretch in template RNA: picornaviruses, M virus of yeast • Reiteritive copying of short U stretches in template RNA: Ortho- and Paramyxoviruses • Can occur post-transcriptionally • Example: poxviruses • Note: many viruses have dispensed with polyA tails altogether. Rather, they trick polyA-binding protein to interact with complex 3’ mRNA structures.

10. Splicing of pre-mRNA Background • hnRNA: heterogeneous nuclear RNA • Larger than mRNA • Has same 5’ and 3’ UTRs as mRNA • Conclusion: both sides are preserved in mRNA but somehow information in-between is lost

11. Splicing of pre-mRNA Sharp and Roberts (1993 Nobel Prize). • The Adenovirus late major mRNA • Contains sequence derived from 4 different blocks of genomic sequence • Precursor late major RNA has the 4 sequence blocks + all sequence in between. • Conclusion: in between sequences are “spliced out” of the pre-mRNA to make the mature mRNA.

12. Discovery of splicing. R-looping: hybridize mRNA with DNA: note that DNA sequences looped out. Compare sequences of cDNA versus gDNA with in situ-hybridization/EM staining.

13. Splicing: evolutionary implications • Exons contain protein coding information • Shuffling of exons can be used to create new functional arrangements • Reflected in modular arrangement of many proteins. • Introns facilitate transfer of genetic information between cellular and viral genomes

14. Constitutive splicing: Every intron is spliced out; Every exon is spliced in Alternative splicing: All introns spliced out; Only selected exons spliced in Result: mRNAs having different coding information derived from a single gene Constitutive vs. Alternative splicing • (Fig. 10.8)

15. Alternative splicing and viruses • Allows expansion of the limited coding capacity of viral genomes • Can be employed to temporally regulate viral gene expression • Can control balance in the production of different regulatory units. • Can control balance in production between spliced and unspliced RNAs.

16. Spliced RNAs: mRNAs encoding 3’ information. E.g. splicing of retroviral mRNAs produces mRNAs endoding env gene Unspliced RNAs: Can encode 5’ genes, e.g. gag-pol of retroviruses Can be used as ‘genomes’ for packaging inside of nascent viral particles (e.g. retroviruses) Alternative splicing and viruses • (Fig. 10.11)

17. POSTTRANSCRIPTIONAL REGULATION BY VIRAL PROTEINS • In general: the presence of an mRNA is not equivalent to the presence of its encoded protein. • The extent of translation of an mRNA can be regulated post-transcriptionally through: • Regulation of initiation • Regulation of mRNA stability • Viral proteins can regulate translation of either • Viral mRNAs, or • Cellular mRNAs

18. Temporal control of gene expression Regulation of alternative polyadenylation • Polyadenylation is required to translate most mRNAs • e.g.: Bovine papillomavirus late mRNA • Always present • But, only polyadenylated (and therefore expressed) late in life cycle.

19. Temporal control of gene expression Regulation of splicing • Control of alternative splicing is a way to regulate gene expression • e.g. Influenza A M1 mRNA (Fig. 10.19) • Early: Splicosome recognizes M3 splice site…makes M3 mRNA • Late: Viral P proteins recruit cellular SR protein, directing splisosome to M3 splice site to make M2 mRNA.

20. Inhibition of cellular mRNA production by viral proteins General notion: • In the battle between viruses and host cell, viruses can gain an upper hand by shutting down cellular functions. • One approach is to inhibit production of translation competent cellular mRNAs

21. Inhibition of polyadenylation and splicing • Influenza NS1 protein inhibits both polyadenylation and splicing of cellular mRNAs resulting in preferential translation of viral mRNAs

22. Inhibition of polyadenylation and splicing • HSV ICP27 protein mislocalizes splicosome components, resulting in inhibition of host pre-mRNA splicing.

23. Regulation of mRNA stability by a viral protein • Protein expression = (rate of translation initiation) x (mRNA half-life) • The more stable an mRNA is, the more protein can be synthesized from it • Many viruses encode proteins that preferentially destabilize cellular mRNAs • e.g. virion host shutoff protein (Vhs) of Herpes simplex encodes an RNase H that degrades mRNAs