Mutational analysis of the influenza virus A Victoria/3/75 PA protein: Studies of interaction with PB1 protein and identification of a dominant negative mutant

The RNA polymerase activity and PB1 binding of influenza virus PA mutants were studied using an in vivo-reconstituted polymerase assay and a two hybrid system, Deletions covering the whole PA protein abolished polymerase activity, but the deletion of the 154 N-terminal amino acids allowed PB1 binding, indicating that the PA protein N terminus is not absolutely required for this interaction, Further internal or C-terminal deletions abolished PB1 interaction, suggesting that most of the protein is involved in this association, As a novel finding we showed that a single amino acid insertion mutant, PAI672, was responsible for a temperature-sensitive phenotype, Mutant PAS509, which had a serine insertion at position 509, bound to PB1 like wild-type PA but did not show any polymerase activity, Over-expression of PAS509 interfered with the polymerase activity of wild-type PA, identifying PAS509 as a dominant negative mutant.