title: Influenza virus polymerase basic protein 1 interacts with influenza virus polymerase basic protein 2 at multiple sites.
abstract: Three polymerase proteins of influenza type A virus interact with each other to form the active polymerase complex. Polymerase basic protein 1 (PB1) can interact with PB2 in the presence or absence of polymerase acidic protein. In this study, we investigated the domains of PB1 involved in complex formation with PB2 in vivo, using coexpression and coimmunoprecipitation of the PB1-PB2 complex with monospecific antibodies. Results show that PB1 possesses at least two regions which can interact independently and form stable complexes with PB2. Both of these regions are located at the NH2 terminus of PB1; the COOH-terminal half of PB1 is not involved in interacting with PB2. Deletion analysis further demonstrated that the interacting regions of PB1 encompass amino acids (aa) 48 to 145 and aa 251 to 321. Linker insertions throughout the PB1 sequences did not affect complex formation with PB2. Deletion and linker-insertion mutants of PB1 were tested for polymerase activity in vivo. For this analysis, we developed a simplified assay for viral polymerase activity that uses a reporter chloramphenicol acetyltransferase gene containing the 5' and 3' ends of influenza viral promoter and nontranslating regions (minus sense) of the NS gene joined to a hepatitis delta virus ribozyme at its 3' end. This assay demonstrated that all deletion mutants of PB1 exhibited either background or greatly reduced polymerase activity irrespective of the ability to interact with PB2 and that all linker-insertion mutants except one at the extreme COOH end (L-746) of PB1 were also negative for viral polymerase activity. These results show that compared with complex formation of PB1 with PB2, the polymerase activity of PB1 was extremely sensitive to structural perturbation.
authors.affiliation: Department of Microbiology and Immunology and Jonsson Comprehensive Cancer Center, UCLA School of Medicine, Los Angeles, California 90024-1747, USA.
authors.forename: S K
authors.initials: SK
authors.lastname: Biswas
authors.forename: D P
authors.initials: DP
authors.lastname: Nayak
mesh_terms: D001665:Binding Sites; D017353:Gene Deletion; D007118:Immunoassay; D009980:Influenza A virus / Q000201:enzymology*; D011485:Protein Binding; D012324:RNA-Dependent RNA Polymerase; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: C057027:PB2 protein, Influenzavirus A; D014764:Viral Proteins; C077346:influenza virus polymerase basic protein 1; D012324:RNA-Dependent RNA Polymerase