Structural basis of an essential interaction between influenza polymerase and Pol II CTD

Abstract

The heterotrimeric influenza polymerase (FluPol), comprising subunits PA, PB1 and PB2, binds to the conserved 5' and 3' termini (the 'promoter') of each of the eight single-stranded viral RNA (vRNA) genome segments and performs both transcription and replication of vRNA in the infected cell nucleus. To transcribe viral mRNAs, FluPol associates with cellular RNA polymerase II (Pol II), which enables it to take 5'-capped primers from nascent Pol II transcripts. Here we present a co-crystal structure of bat influenza A polymerase bound to a Pol II C-terminal domain (CTD) peptide mimic, which shows two distinct phosphoserine-5 (SeP5)-binding sites in the polymerase PA subunit, accommodating four CTD heptad repeats overall. Mutagenesis of the SeP5-contacting basic residues (PA K289, R454, K635 and R638) weakens CTD repeat binding in vitro without affecting the intrinsic cap-primed (transcription) or unprimed (replication) RNA synthesis activity of recombinant polymerase, whereas in cell-based minigenome assays the same mutations substantially reduce overall polymerase activity. Only recombinant viruses with a single mutation in one of the SeP5-binding sites can be rescued, but these viruses are severely attenuated and genetically unstable. Several previously described mutants that modulate virulence can be rationalized by our results, including a second site mutation (PA(C453R)) that enables the highly attenuated mutant virus (PA(R638A)) to revert to near wild-type infectivity. We conclude that direct binding of FluPol to the SeP5 Pol II CTD is fine-tuned to allow efficient viral transcription and propose that the CTD-binding site on FluPol could be targeted for antiviral drug development.

The RNA Exosome Syncs IAV-RNAPII Transcription to Promote Viral Ribogenesis and Infectivity

“…Although one cannot formally exclude that nucleoplasmic cap-snatching could occur (perhaps facilitated by active inhibition of the CBC complex by viral proteins or cellular decapping enzymes), chromatin targeting of viral polymerase via ChIP and the loss of signal in our RAP experiments in uncrosslinked cells favors the fact that the majority of cap snatching occurs on chromatin. In agreement with our results, a recent analysis indicates that the interaction of viral polymerase and RNAPII’s CTD is increased by phosphorylation of the CTD at its serine 5 position - a chemical mark mainly present at transcriptionally engaged RNAPII at promoters (Lukarska, et al 2017). Moreover, if cap-snatching could occur in the nucleoplasm, it’s hard to imagine why less cap-snatching occurs when more ncRNA caps are available upon exosome deficiency.…”

Anin vitrofluorescence based study of initiation of RNA synthesis by influenza B polymerase

“…So how does the polymerase ‘switch’ from transcription (capped RNA primed) to replication (unprimed) at the same vRNA promoter? A plausible explanation is based on the fact that influenza polymerase is known to be associated with host Pol II CTD (50,51), which likely gives it access to nascent transcripts for pirating (52). It is thus possible that Pol II CTD sites become saturated as infection proceeds, due to both increasing numbers of polymerase complexes and decreasing amounts of Pol II resulting from degradation (53) and, in the absence of available primers, RNPs replicate de novo instead.…”

MultiBac: from protein complex structures to synthetic viral nanosystems

“…2). Noteworthy in this context are the highresolution structures of influenza polymerase-the longelusive complex that the influenza virus uses to replicate and transcribe its genome [32][33][34][35]. Influenza polymerase consists of three subunits, PA, PB1 and PB2, with many functions, including cap-snatching and endonuclease activities.…”

“…Flu polymerase structures. Crystal structures of MultiBac-produced polymerases of the three major influenza subtypes, fluA, fluB and fluC, have been determined [32][33][34][35]. For fluA and fluB, a polyprotein approach was used to balance stoichiometry.…”