Influenza A Virus Polymerase: Structural Insights into Replication and Host Adaptation Mechanisms

Abstract

The heterotrimeric RNA-dependent RNA polymerase of influenza viruses catalyzes RNA replication and transcription activities in infected cell nuclei. The nucleotide polymerization activity is common to both replication and transcription processes, with an additional cap-snatching function being employed during transcription to steal short 5-capped RNA primers from host mRNAs. Cap-binding, endonuclease, and polymerase activities have long been studied biochemically, but structural studies on the polymerase and its subunits have been hindered by difficulties in producing sufficient quantities of material. Recently, because of heightened effort and advances in expression and crystallization technologies, a series of high resolution structures of individual domains have been determined. These shed light on intrinsic activities of the polymerase, including cap snatching, subunit association, and nucleocytoplasmic transport, and open up the possibility of structureguided development of new polymerase inhibitors. Furthermore, the activity of influenza polymerase is highly host-and cell type-specific, being dependent on the identity of a few key amino acid positions in the different subunits, especially in the C-terminal region of PB2. New structures demonstrate the surface exposure of these residues, consistent with ideas that they might modulate interactions with host-specific factors that enhance or restrict activity. Recent proteomic and genome-wide interactome and RNA interference screens have suggested the identities of some of these potential regulators of polymerase function.

Cap-snatching mechanism in yeast L-A double-stranded RNA virus

“…This allowed us to specifically label the 5′ end of the viral transcript (acceptor) and examine its fate during cap transfer. In the absence of CTP, 7 GpppG, part of transcripts (ca. 20%) was converted to a BAP-resistant 17-mer (Fig.…”

“…Here we report a unique cap-snatching mechanism by which the yeast double-stranded RNA totivirus L-A furnishes its transcript with a cap structure derived from mRNA. Unlike influenza virus, L-A transfers only m 7 Gp from the cap donor to the 5′ end of the viral transcript, thus preserving the 5′ α-and β-phosphates of the transcript in the triphosphate linkage of the final product. This in vitro capping reaction requires His154 of the coat protein Gag, a residue essential for decapping of host mRNA and known to form m 7 Gp-His adduct.…”

“…Unlike influenza virus, L-A transfers only m 7 Gp from the cap donor to the 5′ end of the viral transcript, thus preserving the 5′ α-and β-phosphates of the transcript in the triphosphate linkage of the final product. This in vitro capping reaction requires His154 of the coat protein Gag, a residue essential for decapping of host mRNA and known to form m 7 Gp-His adduct. Furthermore, the synthesis of capped viral transcripts in vivo and their expression were greatly compromised by the Arg154 mutation, indicating the involvement of Gag in the cap-snatching reaction.…”

Multiple amino acid mutations in viral RNA polymerase may synergistically enhance the transmissibility and/or virulence of the 2009 pandemic influenza (H1N1) virus

“…The V14I mutation is located on the α-helix in the N-terminal domain of PA (Fig. 2a), which cleaves 10-15 nucleotides (nt) downstream of the 5΄-terminal 7-methyl- guanosine cap of the pre-mRNA (Boivin et al, 2010;Fodor et al, 2002;Hara et al, 2006). Since this α-helix is in the proximity of the active site of the N-terminal domain of PA, it may play a role in enhancing the stability of the active site by altering the steric hindrance.…”

Early host response and immune signaling to 2009 pandemic influenza A (H1N1) viruses in primary cell culture models.

“…More recent studies have shown a differential dependence on a specific importin-alpha proteins, with avian IAVs dependent on importin-alpha3 and mammalian IAVs dependent on importin-alpha7 [142]. Regardless, once in the nucleus, mRNA and complementary viral RNA (cRNA) are transcribed from the vRNA template Viral transcription of the viral mRNAs is mediated by the heterotrimeric RdRp (PB1, PB2 and PA), NP and host-derived components (described below) [143,144]. First the RdRp complex recognizes and binds to newly transcribed host cellular mRNAs containing a 5' cap in the nucleus [145,146].…”