Amino Acid Residues in the N-Terminal Region of the PA Subunit of Influenza A Virus RNA Polymerase Play a Critical Role in Protein Stability, Endonuclease Activity, Cap Binding, and Virion RNA Promoter Binding

Abstract

The RNA-dependent RNA polymerase of influenza virus is a heterotrimer formed by the PB1, PB2, and PA subunits. Although PA is known to be required for polymerase activity, its precise role is still unclear. Here, we investigated the function of the N-terminal region of PA. Protease digestion of purified recombinant influenza virus A/PR/8/34 PA initially suggested that its N-terminal region is folded into a 25-kDa domain. We then systematically introduced point mutations into evolutionarily conserved amino acids in the N-terminal region of influenza virus A/WSN/33. Most alanine-scanning mutations between residues L109 and F117 caused PA degradation, mediated by a proteasome-ubiquitin pathway, and as a consequence interfered with polymerase activity. Three further PA mutations, K102A, D108A, and K134A, were investigated in detail. Mutation K102A caused a general decrease both in transcription and replication in vivo, whereas mutations D108A and K134A selectively inhibited transcription. Both the D108A and K134A mutations completely inhibited endonuclease activity in vitro, explaining their selective defect in transcription. K102A, on the other hand, resulted in a significant decrease in both cap binding and viral RNA promoter-binding activity and consequently inhibited both transcription and replication. These results suggest that the N-terminal region of PA is involved in multiple functions of the polymerase, including protein stability, endonuclease activity, cap binding, and promoter binding.

Oligomerization of the influenza virus polymerase complex in vivo

“…PB2 is responsible for cap recognition during transcription initiation and PA is a phosphoprotein with protease activity involved in RNA replication (reviewed by Elton et al, 2005;Palese & Shaw, 2006). The virus polymerase complex is a very compact structure (Area et al, 2004;Torreira et al, 2007) and mutations in the various subunits may alter either transcription or replication (Fodor et al, 2002;Gastaminza et al, 2003;Hara et al, 2006). The first step in virus gene expression is the primary transcription of virion RNPs.…”

Nouvelle stratégie pour cibler la réplication du virus de la grippe

“…Toutefois, la distance séparant les deux résidus acides (acide aspartique, D) du motif PDLYDYK de PA-Nter est inhabituellement courte. De plus, la lysine « catalytique » (Lys-134) [7] a migré à une autre position dans la séquence primaire de la protéine. Enfin, la structure tridimensionnelle souligne la présence, au sein du domaine, de deux atomes de manganèse coordonnés de manière similaire à ceux pré-sents dans la structure de EcoRV [6].…”

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.…”

“…The polymerase of the influenza virus consists of three different subunits: PA, PB1, and PB2. The PA subunit can be cleaved by limited tryptic digestion into two functional domains, which are in the N-terminal domain and C-terminal domain, respectively (Guu et al, 2008;Hara et al, 2006). The N-terminal domain is responsible for the catalysis of the endonuclease activity (Dias et al, 2009;Yuan et al, 2009), while the C-terminal domain is responsible for the binding to the PB1 subunit (He et al, 2008;Obayashi et al, 2008)].…”