Identification of a domain within the phosphoprotein of vesicular stomatitis virus that is essential for transcription in vitro.

Abstract

A full-length cDNA copy of the phosphoprotein (NS) mRNA of vesicular stomatitis virus (New Jersey serotype) was inserted into pGEM4 vector downstream of the promoter for bacteriophage SP6 RNA polymerase. Transcription of the cDNA in vitro resulted in the synthesis of NS mRNA, which was subsequently translated into NS protein in a cell-free rabbit reticulocyte system. The biological activity of the expressed NS protein was demonstrated by in vitro synthesis of mRNA by transcription-reconstitution with purified viral L protein and N-RNA template. Deletion mapping of the NS gene dermed a specific domain between amino acid residues 213 and 247, which was essential for in vitro transcription. Removal of the COOH-terminal 21 amino acids, on the other hand, did not have a significant effect on transcription. This domain appears to be involved in efficient binding of NS protein to the N protein-RNA template.The phosphoprotein (NS) of vesicular stomatitis virus (VSV) is an essential polypeptide component of the RNA polymerase complex that is responsible for viral genome RNA transcription both in vitro and in vivo (1, 2). Within the purified virion, the NS protein (29 kDa) is complexed with the larger polymerase component L protein (241 kDa). This complex is tightly associated with the nucleocapsid protein N (49 kDa)-bound genome RNA template to form the transcribing ribonucleoprotein (RNP) complex (3). The RNP complex, in the presence of four ribonucleoside triphosphates, efficiently transcribes the genome RNA into a 47-base leader RNA (representing the 3' end of the genome RNA) followed sequentially by the synthesis of five 5' capped and 3'-polyadenylylated mRNA species coding for, in order, N-NS-M-G-L (4). A unique feature of the transcribing RNP is that the L and the NS proteins are readily dissociable from the complex, rendering the remaining N-RNA complex transcriptionally inactive (1). Effective reconstitution of in vitro RNA synthesis occurs only when both L and NS proteins are added to the N-RNA template (1). Thus, the complex containing the L and the NS protein constitutes the active RNA polymerase complex, whereas the N protein by its tight and specific interaction maintains the genome RNA template in a transcriptionally competent form.Although the phenomenon of in vitro transcription reconstitution has been known for some time, the precise roles played by the three constituent polypeptides of the RNP complex in the transcription process in vitro have remained obscure. Recently, it has been shown that the purified L protein interacts with the N-RNA complex and initiates transcription but can only synthesize short uncapped oligonucleotides (5). On the other hand, addition of purified NS protein along with the L protein effectively elongates the RNA chains to form matured capped and poly(A)-containing mRNAs. The requirements of the L and the NS proteins for RNA synthesis appeared to be catalytic and stoichiometric, respectively (5). The role of NS protein is particularly interesting because it is a phosph...

Role of Intermolecular Interactions of Vesicular Stomatitis Virus Nucleoprotein in RNA Encapsidation

“…The binding site for P has been mapped to the C-lobe of the N protein (12,17,19,34). In all the mutants generated in this study, the P binding site was not affected, as demonstrated by the copurification of the mutant N proteins with P, in similarity to the results seen with respect to the copurification of the wt N with P. However, we observed that mutant N (⌬347-352) protein free of P was present during size exclusion chromatography after copurification with P by use of a nickel-affinity column.…”

Altered ATP Function of a Vesicular Stomatitis Virus Mutant Detected by Kinetic Analysis of the Transcriptase Using Phosphorylated Ribavirin

“…There is evidence that the kinetic properties of ATP are related to phosphorylation events essential for transcription; a number of these have been reported for VSV. Phosphorylation of the NS protein is essential for in vitro transcription (Gill et al, 1986;Chattopadhyay & Banerjee, 1987). Specifically, two serine residues within domain II of the NS protein must be phosphorylated by the kinase associated with the L protein (Sfinchez et al, 1985).…”

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

“…1A) of both the Indiana (P I ) and New Jersey (P NJ ) serotypes of VSV (20,47). The amino-terminal domain I (residues 1 to approximately 150) is highly acidic and phosphorylated.…”