PMID:16873236

Citation	Hara, K, Schmidt, FI, Crow, M and Brownlee, GG (2006) 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. J. Virol. 80:7789-98
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.
Links	PubMed PMC1563815 Online version:10.1128/JVI.00600-06
Keywords	Amino Acid Sequence; Amino Acid Substitution; Endonucleases/chemistry; Endonucleases/metabolism; Enzyme Stability; Gene Expression Regulation, Viral; Influenza A virus/enzymology; Influenza A virus/genetics; Molecular Sequence Data; Mutagenesis; Mutation; Promoter Regions, Genetic; Protein Folding; Protein Structure, Tertiary; Protein Subunits/chemistry; Protein Subunits/genetics; Protein Subunits/metabolism; RNA Caps/metabolism; RNA Replicase/chemistry; RNA Replicase/genetics; RNA Replicase/metabolism; RNA, Viral/metabolism; Transcription, Genetic; Trypsin/chemistry; Viral Proteins/chemistry; Viral Proteins/genetics; Viral Proteins/metabolism; Virion/genetics
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
I34A1:PA	Contributes to	GO:0004519: endonuclease activity	ECO:0000315:		F		The experiment focused on the precise role of PA subunit of RNA-dependent RNA Polymerase in transcription. Previous research has suggested that mutant H510A in PA subunit has impaired endonuclease activity and selective inhibited transcription. They used three mutant phenotypes of PA subunit: K102A, D108A, and K134A to determine the effect on polymerase activity. No significant endonuclease activity was determined for mutations which indicates that PA is involved in endonuclease activity (Fig. 5D and G).	complete CACAO 13286

References

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