Lecture 16: Processing of viral pre-mRNA BSCI437 Flint et al., Chapter 10

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.

GENERAL OVERVIEW (Fig. 10.1)

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).

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”

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.

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)

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.

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.

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

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.

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.

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

Constitutive vs. Alternative splicing 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 (Fig. 10.8)

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.

Alternative splicing and viruses 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) (Fig. 10.11)

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

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.

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.

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

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

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

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