Role of Cellular Casein Kinase II in the Function of the Phosphoprotein (P) Subunit of RNA Polymerase of Vesicular Stomatitis Virus

Abstract

The phosphorylation of the P protein of vesicular stomatitis virus by cellular casein kinase II (CKII) is essential for its activity in viral transcription. Recent in vitro studies have demonstrated that CKII converts the inactive unphosphorylated form of P (P0) to an active phosphorylated form P1, after phosphorylation at two serine residues, Ser-59 and Ser-61. To gain insight into the role of CKII-mediated phosphorylation in the structure and function of the P protein, we have carried out circular dichroism (CD) and biochemical analyses of both P0 and P1. The results of CD analyses reveal that phosphorylation of P0 to P1 significantly increases the predicted ␣-helical structure of the P1 protein from 27 to 48%. The phosphorylation defective double serine mutant (P59/ 61), which is transcriptionally inactive, possesses a secondary structure similar to that of P0. P1, at a protein concentration of 50 g/ml, elutes from a gel filtration column apparently as a dimer, whereas both P0 and the double serine mutant elute as a monomer at the same concentration. Interestingly, unlike wild-type P1 protein, the P mutants in which either Ser-59 or Ser-61 is altered to alanine required a high concentration of CKII for optimal phosphorylation. We demonstrate here that phosphorylation of either Ser-59 or Ser-61 is necessary and sufficient to transactivate L polymerase although alteration of one serine residue significantly decreases its affinity for CKII. We have also shown that P1 binds to the N-RNA template more efficiently than P0 and the formation of P1 is a prerequisite for the subsequent phosphorylation by L protein-associated kinase. In addition, mutant P59/61 acts as a transdominant negative mutant when used in a transcription reconstitution assay in the presence of wild-type P protein.The RNA-dependent RNA polymerase of vesicular stomatitis virus (VSV) 1 consists of two proteins: the large protein L (241 kDa) and the phosphoprotein P (29 kDa). Together, these proteins are needed to transcribe the linear, single-stranded viral RNA genome of negative polarity, which is tightly wrapped with the nucleocapsid N protein (N-RNA template) (1, 3). Genetic and biochemical studies have suggested that the L protein encodes all the basic transcription activities, whereas the P protein appears to be an RNA virus transcription factor (1, 2, 7) with properties similar to many well studied eucaryotic transcription factors/activators (31). The P protein contains ␣-helical coiled structure and is highly acidic, with Asp and Glu residues constituting one-third of the first 100 amino acid residues in the N-terminal half (domain I) of the polypeptide (17, 18). The acidic domain is also phosphorylated by cellular protein kinase (14, 15). The possible contribution of the Nterminal acidic domain I in the function of P protein seems to transactivate the L protein for transcription similar to those observed for eucaryotic acidic transactivators (30 -32). The Cterminal end, on the other hand, serves as the binding site for the L protein (domain ...

N-Terminal Phosphorylation of Phosphoprotein of Vesicular Stomatitis Virus Is Required for Preventing Nucleoprotein from Binding to Cellular RNAs and for Functional Template Formation

“…Previous studies indicated that N-terminal phosphorylation is required for the self-association of P, which facilitates its interaction with L as well as the N-RNA template and its subsequent transcription activity (14,15,30). Here we provided direct evidence that N-terminal phosphorylation of P was not required for its self-association via three panels of experiments: a yeast twohybrid assay, in vivo coimmunoprecipitation, and an in vitro GST pulldown test.…”

Phosphoprotein of the Rinderpest Virus Forms a Tetramer through a Coiled Coil Region Important for Biological Function

“…Notably the study with VSV P also suggests the presence of a fraction of oligomeric population in the absence of any phosphorylation, and phosphorylation is only involved in the shift of equilibrium toward the multimer formation. Further at high concentration, the unphosphorylated VSV P exists predominately as a multimer (16). Considering these results, it can be generalized that P proteins of mononegaloviruses exist as a multimer, possibly as a tetramer, whose oligomeric form is independent of any phosphorylation.…”

“…Also VSV P when used in high concentrations is able to bring about transcriptional activity without requiring any phosphorylation (43). This activity of VSV P could be caused by the presence of some oligomers at high concentrations (16). From these studies, it appears that oligomerization and not the phosphorylation of P protein of mononegalovirales is essential for its transcription and replication function.…”

Phosphorylation of Vesicular Stomatitis Virus Phosphoprotein P Is Indispensable for Virus Growth

“…The P protein of VSV has a modular structure. In vitro studies as well as studies with transfected cells using DI RNAs or minigenomic RNAs strongly suggest that the phosphorylation of different structural domains of the P protein regulates its multiple functions in viral genome transcription and replication (2,3,7,8,16,17,26,44,53). In the present study, we addressed the role of P protein phosphorylation in the life cycle of VSV.…”

“…By mutational and biochemical studies, the phosphate acceptor sites were mapped to Ser-59 and Ser-61 in P NJ and Ser-60, Thr-62, and Ser-64 in P I (6,53,54). Further studies suggested that the phosphorylation of these residues results in multimerization of the P protein, which in turn facilitates its binding to the L protein for transcription (7,16,17). Using a plasmid-based reverse genetics system (46), we demonstrated that the phosphorylation of these residues is critical for the function of the P protein in transcription but not in replication (44).…”