Functional analysis of PA binding by influenza a virus PB1: Effects on polymerase activity and viral infectivity

Influenza A virus expresses three viral polymerase (P) subunits-PB1, PB2, a nd PA-all of which are essential for RNA and viral replication. The functio ns of P proteins in transcription and replication have been partially eluci dated, yet some of these functions seem to be dependent on the formation of a heterotrimer for optimal viral RNA transcription and replication. Althou gh it is conceivable that heterotrimer subunit interactions may allow a mor e efficient catalysis, direct evidence of their essentiality for viral repl ication is lacking. Biochemical studies addressing the molecular anatomy of the P complexes have revealed direct interactions between PBI and PB2 as w ell 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 f or binding PA. We report here the refined mapping of the amino acid sequenc es 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 s ite-directed mutagenesis to identify the critical amino acid residues of PB I for interaction with PA in vivo. The first 12 amino acids of PB1 were fou nd to constitute the core of the interaction interface, thus narrowing the previous boundaries of domain a. The role of the minimal PB1 domain alpha i n influenza virus gene expression and genome replication was subsequently a nalyzed by evaluating the activity of a set of PB1 mutants in a model repor ter minigenome system. A strong correlation was observed between a function al PA binding site on PB1 and P activity. Influenza viruses bearing mutant PB1 genes were recovered using a plasmid-based influenza virus reverse gene tics system. Interestingly, mutations that rendered PB1 unable to bind PA w ere either nonviable or severely growth impaired. These data are consistent with an essential role for the N terminus of PBI in binding PA, P activity , and virus growth.