title: Newly identified phosphorylation site in the vesicular stomatitis virus P protein is required for viral RNA synthesis.
abstract: The vesicular stomatitis virus (VSV) RNA-dependent RNA polymerase consists of two viral proteins; the large (L) protein is the main catalytic subunit, and the phosphoprotein (P) is an essential cofactor for polymerase function. The P protein interacts with the L protein and the N-RNA template, thus connecting the polymerase to the template. P protein also binds to free N protein to maintain it in a soluble, encapsidation-competent form. Previously, five sites of phosphorylation were identified on the P protein and these sites were reported to be differentially important for mRNA synthesis or genomic replication. The previous studies were carried out by biochemical analysis of portions of the authentic viral P protein or by analysis of bacterium-expressed, exogenously phosphorylated P protein by mutagenesis. However, there has been no systematic biochemical search for phosphorylation sites on authentic, virus-expressed P protein. In this study, we analyzed the P protein isolated from VSV-infected cells for sites of phosphorylation by mass spectrometry. We report the identification of Tyr14 as a previously unidentified phosphorylation site of VSV P and show that it is essential for viral transcription and replication. However, our mass spectral analysis failed to observe the phosphorylation of previously reported C-terminal residues Ser226 and Ser227 and mutagenic analyses did not demonstrate a role for these sites in RNA synthesis.
authors.affiliation: Department of Pathology, University of Virginia, Charlottesville, Virginia, USA.
authors.forename: Arindam
authors.initials: A
authors.lastname: Mondal
authors.forename: Ken G
authors.initials: KG
authors.lastname: Victor
authors.forename: R S
authors.initials: RS
authors.lastname: Pudupakam
authors.forename: Charles E
authors.initials: CE
authors.lastname: Lyons
authors.forename: Gail W
authors.initials: GW
authors.lastname: Wertz
mesh_terms: D020816:Amino Acid Motifs; D006801:Humans; D013058:Mass Spectrometry; D010750:Phosphoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D012367:RNA, Viral / Q000096:biosynthesis* / Q000235:genetics; D012694:Serine / Q000235:genetics / Q000378:metabolism; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism; D054243:Vesicular Stomatitis / Q000821:virology*; D014721:Vesicular stomatitis Indiana virus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015678:Viral Structural Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
chemical_list: C081250:P protein, Vesicular stomatitis virus; D010750:Phosphoproteins; D012367:RNA, Viral; D015678:Viral Structural Proteins; D014443:Tyrosine; D012694:Serine