title: Association of the influenza virus RNA polymerase subunit PB2 with the host chaperonin CCT.
abstract: The RNA polymerase of influenza A virus is a host range determinant and virulence factor. In particular, the PB2 subunit of the RNA polymerase has been implicated as a crucial factor that affects cell tropism as well as virulence in animal models. These findings suggest that host factors associating with the PB2 protein may play an important role during viral replication. In order to identify host factors that associate with the PB2 protein, we purified recombinant PB2 from transiently transfected mammalian cells and identified copurifying host proteins by mass spectrometry. We found that the PB2 protein associates with the cytosolic chaperonin containing TCP-1 (CCT), stress-induced phosphoprotein 1 (STIP1), FK506 binding protein 5 (FKBP5), alpha- and beta-tubulin, Hsp60, and mitochondrial protein p32. Some of these binding partners associate with each other, suggesting that PB2 might interact with these proteins in multimeric complexes. More detailed analysis of the interaction of the PB2 protein with CCT revealed that PB2 associates with CCT as a monomer and that the CCT binding site is located in a central region of the PB2 protein. PB2 proteins from various influenza virus subtypes and origins can associate with CCT. Silencing of CCT resulted in reduced viral replication and reduced PB2 protein and viral RNA accumulation in a ribonucleoprotein reconstitution assay, suggesting an important function for CCT during the influenza virus life cycle. We propose that CCT might be acting as a chaperone for PB2 to aid its folding and possibly its incorporation into the trimeric RNA polymerase complex.
authors.affiliation: Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom.
authors.forename: Tatiana
authors.initials: T
authors.lastname: Fislová
authors.forename: Benjamin
authors.initials: B
authors.lastname: Thomas
authors.forename: Katy M
authors.initials: KM
authors.lastname: Graef
authors.forename: Ervin
authors.initials: E
authors.lastname: Fodor
mesh_terms: D001665:Binding Sites; D002460:Cell Line; D056404:Chaperonin Containing TCP-1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D009980:Influenza A virus / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D007251:Influenza, Human / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: C502893:PB2 protein, influenza virus; D014764:Viral Proteins; D056404:Chaperonin Containing TCP-1