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Journal of Virology, August 2006, p. 7789-7798, Vol. 80, No. 16
0022-538X/06/$08.00+0     doi:10.1128/JVI.00600-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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

Koyu Hara, Florian I. Schmidt, Mandy Crow, and George G. Brownlee^*

Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom

Received 24 March 2006/ Accepted 23 May 2006

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.

Present address: Department of Virology, Kurume University School of Medicine, Fukuoka, Japan.

Present address: Department of Chemistry, Technical University, Munich, Germany.

Present address: University of Manchester, Oxford Road, Manchester M13 9PL, United Kingdom.

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