Characterization of the Mutations Responsible for the Electrophoretic Mobility Differences in the NS Proteins of Vesicular Stomatitis Virus New Jersey Complementation Group E Mutants

Rae, Brendan P.; Elliott, Richard M.

doi:10.1099/0022-1317-67-12-2635

Cited by 21 publications

(10 citation statements)

References 41 publications

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“…The precise nature of this complex interaction remains to be understood. The acidic domain I, like the eukaryotic transcriptional activators, contains stretches of a-helical structures (18,26). These structures probably interact with the helical structures of the N-RNA template and transiently displace the RNA-bound N protein, allowing the L protein to contact the genome RNA.…”

Section: Discussionmentioning

confidence: 99%

NH2-terminal acidic region of the phosphoprotein of vesicular stomatitis virus can be functionally replaced by tubulin.

Chattopadhyay

Banerjee²

1988

Proc. Natl. Acad. Sci. U.S.A.

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The phosphoprotein (NS) of vesicular stomatitis virus is an indispensable subunit of the vinrion-associated RNA polymerase (L). NS consists of a highly acidic NH2-terminal domain and a basic COOH-terminal domain. Unlike the latter, the amino acid sequences of the NH2-terminal regions are highly dissimilar among different viral serotypes, although they share structural similarities. We have cloned an NS gene into the SP6 transcription vector and replaced the 5'-terminal 80% by a full-length gene for fi-tubulin, which contains an acidic COOH-terminal domain. Here we present evidence that the chimeric tubulin-NS protein is biologically active and that the acidic region in tubulin directly affects the transcription reaction. These observations indicate that NS probably functions as an activator protein in which the acidic domain stimulates transcription of the viral genes by interacting with the RNA polymerase as observed for eukaryotic cellular transcription activators.An RNA-dependent RNA polymerase packaged within the purified virion of vesicular stomatitis virus (VSV) transcribes the negative-strand genome RNA in vitro into five distinct mRNAs and a leader RNA (1, 2). This transcription is essential for initiation of infection when VSV infects cells. The RNA polymerase consists of two distinct polypeptide subunits: a large polypeptide, L, of 240 kDa and a small phosphoprotein, NS, of 29 kDa tightly associated with each other and also with the N protein-RNA template (N-RNA template). There are approximately 50 molecules of the L protein and 500 molecules of the NS protein per virion (3). The unique feature of the VSV transcription complex is that each of the reacting components can be separated free from the others and in vitro RNA synthesis can be restored effectively when L and NS proteins are added to the N-RNA template (4). By using the above transcription reconstitution system, it was shown (5) that the L protein, which acts catalytically in the initiation of RNA chains, fails to synthesize full-length RNA unless the NS protein, which acts stoichiometrically, is added to the transcription mixture. These results indicated that the L protein is the RNA polymerase whereas the NS protein is a regulatory protein that plays a role in the transcription process, possibly by interacting with the N-RNA template to facilitate access of L protein to the genome template for RNA synthesis (5,6). Phosphorylation ofthe NS protein seems to have important regulatory effect(s) in this process (1).To study the precise function of the NS protein in the transcription process, we (7) inserted a full-length NS gene (New Jersey serotype) into an SP6 transcription vector. This system enabled us to obtain NS mRNA in amounts that, when subsequently translated in vitro, resulted in the synthesis of biologically active protein. The latter activity was demonstrated by transcription reconstitution with purified L protein and the N-RNA template. Systematic deletion mapping studies (7) showed (i) that the COOH-terminal basic 27 amino...

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Section: Discussionmentioning

confidence: 99%

NH2-terminal acidic region of the phosphoprotein of vesicular stomatitis virus can be functionally replaced by tubulin.

Chattopadhyay

Banerjee²

1988

Proc. Natl. Acad. Sci. U.S.A.

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“…The 39 base pair difference between these initiation sites appears to be too small to account for the 3K difference in size of NS3 (28K) and NS3A (25K). However, Rae & Elliott (1986) have shown that even a single base pair change in the 5' coding region of vesicular stomatitis virus NS protein resulted in a large difference in electrophoretic mobility in the corresponding proteins, as determined on SDS-PAGE. The apparent 3K size difference between NS3 and NS3A is therefore not necessarily a reflection of a large difference in the length of the peptide chain, but could reflect differences in the polarity and structure of the terminal regions of the two proteins.…”

Section: Discussionmentioning

confidence: 99%

In vitro Transcription and Translation of Bluetongue Virus mRNA

Dijk

Huismans

1988

Journal of General Virology

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SUMMARYFractionation of in vitro transcribed bluetongue virus (BTV) mRNA by agarose gel electrophoresis resulted in the separation of eight of the 10 species. The relative molar ratio of the mRNAs confirmed that mRNA 5 was transcribed more frequently than would be predicted from the size of the $5 genome segment, while mRNA 10 was transcribed less frequently. In vitro translation ofunfractionated BTV mRNAs resulted in the synthesis of the seven known structural proteins (P1 to P7) and two known nonstructural proteins (NSI and NS2). Two additional non-structural proteins (NS3 and NS3A) with Mr of 28K and 25K respectively were identified. The protein coding assignments for the medium-and small-sized double-stranded RNA genome segments ofBTV serotype 10 were found to correspond to those reported for BTV-1 and BTV-17. The peptide maps of NSI, NS2, NS3 and NS3A synthesized in vitro corresponded to those of their counterparts synthesized in infected cells. Protein NS3A appeared to be a truncated form of NS3, since its peptide map completely overlapped that of NS3. Proteins NS3 and NS3A were present in very small amounts in the soluble fraction of the cytoplasm of infected cells, and were synthesized in variable amounts in vitro, whereas the other nine viral proteins were synthesized in constant molar ratios. A difference in the relative molar ratios in which some of the BTV proteins were synthesized in vitro and in vivo was observed. In vivo, protein NS1 was translated in the largest amount but in vitro, NS2 was the most efficiently translated protein. Conversely, protein P6 was translated much more efficiently in vitro than in vivo.

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“…In addition, the weakly phosphorylated NS protein from treated cells displayed the same electrophoretic mobility as the phosphorylated form from untreated cells. As noted by other workers (Marnell & Summers, 1984;Rae & Elliott, 1986), the anomalous mobility of the NS phosphoprotein is caused mainly by numerous acidic amino acid residues, and hence non-stoichiometric binding of SDS, rather than by the phosphate groups.…”

Section: Effect On Phosphorylation Of the Ns Proteinmentioning

confidence: 50%

Inhibition of the Phosphorylation of the Regulatory Non-structural Protein of Vesicular Stomatitis Virus by an Antiviral Xanthate Compound

Müller‐Decker

Amtmann²,

Sauer³

1987

Journal of General Virology

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SUMMARYThe growth of vesicular stomatitis virus (VSV) can be inhibited by the antiviral compound tricycloodecane-9-yl-xanthogenate (D609). On analysing the antiviral mechanism we found no effect on the primary transcription of infecting VSV genomes. In contrast, the processes of replication and transcription during late stages of infection were inhibited. Despite the synthesis of all five virus-coded proteins (41 ~ to 56 ~ of the uninhibited control), as shown by labelling with [35S]methionine, the phosphorylation of the non-structural (NS) protein was reduced in the presence of the xanthate by a factor of at least 17. The pattern of phosphorylation of the bulk of cellular proteins remained unaltered under the same conditions. A relation between a possible loss of biological activity of the NS protein owing to the lack of phosphorylation and the decreased VSV RNA synthesis is suggested.

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Characterization of the Mutations Responsible for the Electrophoretic Mobility Differences in the NS Proteins of Vesicular Stomatitis Virus New Jersey Complementation Group E Mutants

Cited by 21 publications

References 41 publications

NH2-terminal acidic region of the phosphoprotein of vesicular stomatitis virus can be functionally replaced by tubulin.

NH2-terminal acidic region of the phosphoprotein of vesicular stomatitis virus can be functionally replaced by tubulin.

In vitro Transcription and Translation of Bluetongue Virus mRNA

Inhibition of the Phosphorylation of the Regulatory Non-structural Protein of Vesicular Stomatitis Virus by an Antiviral Xanthate Compound

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