title: Phosphorylation of the influenza A virus protein PB1-F2 by PKC is crucial for apoptosis promoting functions in monocytes.
abstract: The 11(th) influenza A virus (IAV) protein PB1-F2 is encoded by an alternative reading frame of the PB1 polymerase gene and found in the nucleus, cytosol and at the mitochondria of infected cells, the latter is consistent with experimental evidence for its pro-apoptotic function. Here, the function of PB1-F2 as a phosphoprotein was characterized. PB1-F2 derived from isolate IAV(PR8) and synthetic fragments thereof were phosphorylated in vitro by purified protein kinase C (PKC) and cellular extract. Constitutively active PKCalpha interacts with PB1-F2 in yeast two-hybrid assays. (32)P radiolabelling of transfected 293T cells revealed that phosphorylation of PB1-F2 is sensitive to inhibitors of PKC and could be increased by the PKC activator PMA. ESI-MS analysis and cellular expression of PB1-F2 mutants identified the positions Ser-35 as the major and the Thr-27 as an alternative PKC phosphorylation site. Infection of MDCK cells with recombinant IAV(PR8) lacking these PKC sites abrogated phosphorylation of PB1-F2 in vivo. Furthermore, infection of primary human monocytes with mutant viruses lacking these PB1-F2 phosphorylation sites resulted in impaired caspase 3 activation and reduced progeny virus titres, indicating that the integrity of the identified phosphorylation sites is crucial for a cell-specific function of PB1-F2 during virus replication.
authors.affiliation: Institute of Clinical and Molecular Virology, University of Erlangen-Nuremberg, Germany.
authors.forename: David
authors.initials: D
authors.lastname: Mitzner
authors.forename: Sabine Eva
authors.initials: SE
authors.lastname: Dudek
authors.forename: Nicole
authors.initials: N
authors.lastname: Studtrucker
authors.forename: Darisuren
authors.initials: D
authors.lastname: Anhlan
authors.forename: Igor
authors.initials: I
authors.lastname: Mazur
authors.forename: Josef
authors.initials: J
authors.lastname: Wissing
authors.forename: Lothar
authors.initials: L
authors.lastname: Jänsch
authors.forename: Ludmilla
authors.initials: L
authors.lastname: Wixler
authors.forename: Karsten
authors.initials: K
authors.lastname: Bruns
authors.forename: Alok
authors.initials: A
authors.lastname: Sharma
authors.forename: Victor
authors.initials: V
authors.lastname: Wray
authors.forename: Peter
authors.initials: P
authors.lastname: Henklein
authors.forename: Stephan
authors.initials: S
authors.lastname: Ludwig
authors.forename: Ulrich
authors.initials: U
authors.lastname: Schubert
mesh_terms: D017209:Apoptosis; D002460:Cell Line; D006801:Humans; D009980:Influenza A virus / Q000472:pathogenicity*; D009000:Monocytes / Q000276:immunology*; D010766:Phosphorylation; D025941:Protein Interaction Mapping; D011493:Protein Kinase C / Q000378:metabolism*; D012441:Saccharomyces cerevisiae; D020798:Two-Hybrid System Techniques; D014764:Viral Proteins / Q000378:metabolism*
chemical_list: C442816:PB1-F2 protein, Influenza A virus; D014764:Viral Proteins; D011493:Protein Kinase C