This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hara, K.
Right arrow Articles by Brownlee, G. G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hara, K.
Right arrow Articles by Brownlee, G. G.

 Previous Article  |  Next Article 

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,{dagger} Florian I. Schmidt,{ddagger} 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.

* Corresponding author. Mailing address: Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, United Kingdom. Phone: 44 (01865) 275559. Fax: 44 (01865) 275556. E-mail: george.brownlee{at}path.ox.ac.uk.

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

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

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

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.



This article has been cited by other articles:

  • Song, M.-S., Pascua, P. N. Q., Lee, J. H., Baek, Y. H., Lee, O.-J., Kim, C.-J., Kim, H., Webby, R. J., Webster, R. G., Choi, Y. K. (2009). The Polymerase Acidic Protein Gene of Influenza A Virus Contributes to Pathogenicity in a Mouse Model. J. Virol. 83: 12325-12335 [Abstract] [Full Text]  
  • Zhao, C., Lou, Z., Guo, Y., Ma, M., Chen, Y., Liang, S., Zhang, L., Chen, S., Li, X., Liu, Y., Bartlam, M., Rao, Z. (2009). Nucleoside Monophosphate Complex Structures of the Endonuclease Domain from the Influenza Virus Polymerase PA Subunit Reveal the Substrate Binding Site inside the Catalytic Center. J. Virol. 83: 9024-9030 [Abstract] [Full Text]  
  • Wise, H. M., Foeglein, A., Sun, J., Dalton, R. M., Patel, S., Howard, W., Anderson, E. C., Barclay, W. S., Digard, P. (2009). A Complicated Message: Identification of a Novel PB1-Related Protein Translated from Influenza A Virus Segment 2 mRNA. J. Virol. 83: 8021-8031 [Abstract] [Full Text]  
  • Hemerka, J. N., Wang, D., Weng, Y., Lu, W., Kaushik, R. S., Jin, J., Harmon, A. F., Li, F. (2009). Detection and Characterization of Influenza A Virus PA-PB2 Interaction through a Bimolecular Fluorescence Complementation Assay. J. Virol. 83: 3944-3955 [Abstract] [Full Text]  
  • Bradel-Tretheway, B. G., Kelley, Z., Chakraborty-Sett, S., Takimoto, T., Kim, B., Dewhurst, S. (2008). The human H5N1 influenza A virus polymerase complex is active in vitro over a broad range of temperatures, in contrast to the WSN complex, and this property can be attributed to the PB2 subunit. J. Gen. Virol. 89: 2923-2932 [Abstract] [Full Text]  
  • Li, C., Hatta, M., Watanabe, S., Neumann, G., Kawaoka, Y. (2008). Compatibility among Polymerase Subunit Proteins Is a Restricting Factor in Reassortment between Equine H7N7 and Human H3N2 Influenza Viruses. J. Virol. 82: 11880-11888 [Abstract] [Full Text]  
  • Vreede, F. T., Gifford, H., Brownlee, G. G. (2008). Role of Initiating Nucleoside Triphosphate Concentrations in the Regulation of Influenza Virus Replication and Transcription. J. Virol. 82: 6902-6910 [Abstract] [Full Text]  
  • Jorba, N., Area, E., Ortin, J. (2008). Oligomerization of the influenza virus polymerase complex in vivo. J. Gen. Virol. 89: 520-524 [Abstract] [Full Text]  
  • Finkelstein, D. B., Mukatira, S., Mehta, P. K., Obenauer, J. C., Su, X., Webster, R. G., Naeve, C. W. (2007). Persistent Host Markers in Pandemic and H5N1 Influenza Viruses. J. Virol. 81: 10292-10299 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»