Supplementary Materials Supplemental material supp_88_3_1461__index. phosphorylated P proteins by mutagenesis. Nevertheless,

Supplementary Materials Supplemental material supp_88_3_1461__index. phosphorylated P proteins by mutagenesis. Nevertheless, there’s been no organized biochemical seek out phosphorylation sites on genuine, 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. INTRODUCTION Nonsegmented, negative-strand RNA viruses encode their own RNA-dependent RNA polymerase (RdRp) to carry out the RNA synthetic activities of mRNA transcription and genome replication. The RdRp is composed of two multifunctional Suvorexant supplier proteins, the large (L) protein and the phosphoprotein (P) (1). The L protein is the main catalytic subunit, harboring the RdRp, capping, methyltransferase, and polyadenylation activities (2,C6). The P protein is the cofactor, essential for the formation of an active Suvorexant supplier polymerase complex. The template for RNA synthesis is the negative-sense RNA genome encapsidated by oligomers of the viral nucleocapsid (N) protein (7, 8). The L protein gains Suvorexant supplier access to the N-RNA template via the P protein, which interacts with the L protein and the oligomers of N protein simultaneously (9,C12). The P protein also functions to maintain N protein in a soluble, encapsidation-competent form by acting as an N-specific chaperone (13,C15). The vesicular stomatitis virus (VSV) P protein is divided into three domains, an N-terminal domain (PNTD), a central domain (PCD), and a C-terminal domain (PCTD) (Fig. 1) (10, 16,C18). The PNTD has been shown to bind to both the L and N proteins. Upon binding to L protein, it causes a conformational rearrangement of L protein and increases the processivity of the polymerase (12, 19). The PNTD also binds free N protein (N), acting as a chaperone protein (13, 14, 17). The PCTD contacts two adjacent N monomers of the N-RNA template and, along with PNTD binding of L, serves as a bridge between the polymerase and RNA template (9, 12). The PCD is a homodimerization domain (16). Recent data suggest that structured regions of P are separated by intrinsically disordered regions (IDRs) (18, 20). Open in a separate window FIG 1 (A) Schematic representation of the organization of the VSV P protein domain in light of recent structural studies (16,C18). The P protein has been divided into three domains, an N-terminal domain (PNTD), an autonomously folded central domain (PCD), and a negatively charged C-terminal domain (PCTD). Reported phosphorylation sites and surrounding residues are demonstrated Previously. (B) Protein profile of VSV-infected cells. BHK 21 cells either mock contaminated or contaminated with VSV had been metabolically tagged with [35S]methionine-cysteine. Cell lysates had been solved by low-bis SDS-PAGE (discover Materials and Strategies). (C) P proteins was isolated from VSV-infected BHK 21 cells by immunoaffinity purification (discover Materials and Strategies). N proteins was copurified with P as demonstrated. (D) The identities from the P and N proteins were determined by Western blotting analyses. The values between the blots are molecular sizes in kilodaltons. The VSV P protein becomes phosphorylated during the viral life cycle. Recombinant P protein isolated from and thus devoid of any phosphorylation was unable to support transcription, indicating that P CPB2 protein phosphorylation is important for viral RNA synthesis (21, 22). P protein isolated by DEAE cellulose column chromatography, either from virions or from infected cells, was reported to have different phosphorylated forms (23). These differentially phosphorylated forms varied in the ability to support transcription (23, 24). Doublet bands of P proteins were observed when resolved through urea-SDS-polyacrylamide gels, and these two forms of the P protein were further divided into subspecies when subjected to isoelectric focusing (23, 25). The specific sites of phosphorylation of the VSV P protein were analyzed initially by chemical and enzymatic cleavage, which indicated that most of the phosphate acceptor sites were in the PNTD (26, 27). Hsu and Kingsbury (28) localized these phosphorylation sites between amino acids 35 and 75 of P protein of the VSV Indiana (PInd) serotype. Residues Ser60, Thr62, and Ser64 in this region were subsequently identified as phosphorylation sites in PInd by incubating exogenous casein kinase II with bacterium-expressed mutant P protein; however, phosphorylation at residue 62 varied with the type of assay and the enzyme source (29). A separate study reported constitutive phosphorylation at all three sites (30). These phosphorylation sites Suvorexant supplier in the PNTD were reported to be important for the homodimerization of the protein and its interaction with L (31,C34)..