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Journal of Virology, September 2001, p. 8127-8136, Vol. 75, No. 17
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.17.8127-8136.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Functional Analysis of PA Binding by Influenza A Virus PB1: Effects on Polymerase Activity and Viral Infectivity†

Daniel R. Perez1,2 and Ruben O. Donis1,*

Department of Veterinary and Biomedical Sciences, University of Nebraska---Lincoln, Lincoln, Nebraska 68583-0905,1 and Department of Virology and Molecular Biology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105-27942

Received 27 February 2001/Accepted 25 April 2001

Influenza A virus expresses three viral polymerase (P) subunits---PB1, PB2, and PA---all of which are essential for RNA and viral replication. The functions of P proteins in transcription and replication have been partially elucidated, yet some of these functions seem to be dependent on the formation of a heterotrimer for optimal viral RNA transcription and replication. Although it is conceivable that heterotrimer subunit interactions may allow a more efficient catalysis, direct evidence of their essentiality for viral replication is lacking. Biochemical studies addressing the molecular anatomy of the P complexes have revealed direct interactions between PB1 and PB2 as well as between PB1 and PA. Previous studies have shown that the N-terminal 48 amino acids of PB1, termed domain alpha , contain the residues required for binding PA. We report here the refined mapping of the amino acid sequences within this small region of PB1 that are indispensable for binding PA by deletion mutagenesis of PB1 in a two-hybrid assay. Subsequently, we used site-directed mutagenesis to identify the critical amino acid residues of PB1 for interaction with PA in vivo. The first 12 amino acids of PB1 were found to constitute the core of the interaction interface, thus narrowing the previous boundaries of domain alpha . The role of the minimal PB1 domain alpha  in influenza virus gene expression and genome replication was subsequently analyzed by evaluating the activity of a set of PB1 mutants in a model reporter minigenome system. A strong correlation was observed between a functional PA binding site on PB1 and P activity. Influenza viruses bearing mutant PB1 genes were recovered using a plasmid-based influenza virus reverse genetics system. Interestingly, mutations that rendered PB1 unable to bind PA were either nonviable or severely growth impaired. These data are consistent with an essential role for the N terminus of PB1 in binding PA, P activity, and virus growth.

* Corresponding author. Mailing address: Department of Veterinary and Biomedical Sciences, 202 VBS, University of Nebraska---Lincoln, Fair St. and East Campus Loop, Lincoln, NE 68583-0905. Phone: (402) 472-6063. Fax: (402) 472-9690. E-mail: rdonis{at}unlnotes.unl.edu.

dagger This is publication no. 12905 of the Agricultural Research Division, IANR, University of Nebraska---Lincoln.

Journal of Virology, September 2001, p. 8127-8136, Vol. 75, No. 17
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.17.8127-8136.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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