title: Display of disparate transcription phenotype by the phosphorylation negative P protein mutants of vesicular stomatitis virus, Indiana serotype, expressed in E. coli and eucaryotic cells.
abstract: The phosphoprotein (P) of vesicular stomatitis virus (VSV) is a subunit of the RNA polymerase (L) that transcribes the negative strand genome RNA into mRNAs both in vitro and in vivo. We have recently shown that the P protein of VSV, New Jersey serotype (PNJ), expressed in E. coli, is biologically inactive unless phosphorylated at specific serine residues by cellular casein kinase II (CKII). In the present work, we are studying the role of phosphorylation in the activation of the P protein of Indiana serotype (PIND), which is highly nonhomologous in amino acid sequence yet structurally similar to its New Jersey counterpart. Despite the fact that E. coli-expressed PIND required phosphorylation by CKII for activation, the phosphorylation negative P protein mutants generated by altering the phosphate acceptors S and T to alanine, surprisingly, showed transcription activity similar to wild-type in vitro. Alteration of S and T residues to phenylalanine, similarly, supported substantial transcription activity (approx. 60% of wild-type), whereas substitution with arginine residue abrogated transcription (approx. 5% of wild-type). In contrast, the same mutants, when expressed in eucaryotic cells, exhibited greatly reduced transcription activity in vitro. This disparate display of transcription phenotype by the PIND mutants expressed in bacteria and eucaryotic cells suggests that these mutants are unique in assuming different secondary structure or conformation when synthesized in two different cellular milieu. The findings that, unless phosphorylated by CKII, the bacterially expressed unphosphorylated (P0) form of PIND, as well as the phosphorylation negative mutants expressed in eucaryotic cells, demonstrates transcription negative phenotype indicate that, like PNJ, phosphorylation of PIND is essential for its activity.
authors.affiliation: Department of Molecular Biology, Cleveland Clinic Foundation, OH 44195, USA.
authors.forename: M
authors.initials: M
authors.lastname: Mathur
authors.forename: T
authors.initials: T
authors.lastname: Das
authors.forename: J L
authors.initials: JL
authors.lastname: Chen
authors.forename: D
authors.initials: D
authors.lastname: Chattopadhyay
authors.forename: A K
authors.initials: AK
authors.lastname: Banerjee
mesh_terms: D000818:Animals; D019556:COS Cells; D047390:Casein Kinase II; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D004926:Escherichia coli; D005057:Eukaryotic Cells; D005822:Genetic Vectors; D007196:Indiana; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D010750:Phosphoproteins; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D012703:Serotyping; D014158:Transcription, Genetic; D014721:Vesicular stomatitis Indiana virus / Q000378:metabolism*; D018116:Vesiculovirus; D015678:Viral Structural Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*
chemical_list: C081250:P protein, Vesicular stomatitis virus; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D015678:Viral Structural Proteins; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases