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.
Zürcher
T
T
Centro Nacional de Biotecnologia (CSIC), Universidad Autonoma, Cantoblanco, Madrid, Spain.
de la Luna
S
S
Sanz-Ezquerro
J J
JJ
Nieto
A
A
Ortín
J
J
D000818:Animals; D001665:Binding Sites; D002461:Cell Line, Transformed; D002522:Chlorocebus aethiops; D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000378:metabolism; D005799:Genes, Dominant; D006801:Humans; D009980:Influenza A virus / Q000201:enzymology*; D009154:Mutation; D017384:Sequence Deletion; D014764:Viral Proteins / Q000378:metabolism*
D014764:Viral Proteins; C077346:influenza virus polymerase basic protein 1; D012321:DNA-Directed RNA Polymerases