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

• Published in: Journal of general virology 1996-08, Vol.77 (8), p.1745-1749
• Main Authors: Zurcher, Thomas, de la Luna, Susana, Sanz-Ezquerro, Juan J, Nieto, Amelia, Ortin, Juan
• Format: Article
• Language: English
• Subjects: Animals; Binding Sites; Cell Line, Transformed; Cercopithecus aethiops; DNA-Directed RNA Polymerases - genetics; DNA-Directed RNA Polymerases - metabolism; Genes, Dominant; Humans; influenza A virus; Influenza A virus - enzymology; Mutation; Sequence Deletion; Viral Proteins - metabolism
• ISSN: 0022-1317; 1465-2099
• DOI: 10.1099/0022-1317-77-8-1745

Centro Nacional de Biotecnologia (CSIC), Universidad Autonoma, Cantoblanco, 28049 Madrid, Spain 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. Present address: National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK Received 6 February 1996; accepted 23 April 1996.