Temperature-Sensitive Mutants of Complementation Group E of Vesicular Stomatitis Virus New Jersey Serotype Possess Altered NS Polypeptides

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Abstract
In vesicular stomatitis virus New Jersey serotype, polyacrylamide gel electrophoresis was unable to distinguish the polypeptides of the temperature-sensitive (ts) mutants of complementation groups A, B, C and F from those of the wild-type virus. The NS polypeptide of the representative mutant of group E ts E1, had a significantly greater electrophoretic mobility than that of the wild-type virus NS polypeptide. The electrophoretic mobilities of the NS polypeptides of the 3 mutants of complementation group E varied, being greatest in the case of ts E1, slightly less for ts E2 and only a little greater than that of wild-type virus NS polypeptide in the case of ts E3. Since the NS polypeptides of the revertant clones ts E1/R1 and ts E3/R1 have mobilities identical to that of wild-type NS polypeptide, the observed altered mobilities of the group E mutants are almost certainly the direct result of the ts mutations in the E locus. The electrophoretic mobilities of the intracellular NS polypeptides of the group E mutants were indistinguishable from those of their virion NS polypeptides. The electrophoretic mobilities of the NS polypeptides of the group E mutants synthesized in vitro using mRNA synthesized in vitro by TNP [transcribing nucleoprotein] were identical to those of the NS polypeptides of their purified virions. The NS polypeptides of all three mutants were labeled with 32Pi to approximately the same extent as wild-type virus NS polypeptide, indicating that gross differences in phosphorylation of this polypeptide are unlikely to account for the altered mobilities. A model is proposed in which the NS polypeptide consists of at least 3 loops held in this configuration by hydrophobic or ionic forces or both and stabilized by phosphodiester bridges. If a mutation affects one of the amino acids to which the phosphate is covalently linked, the phosphodiester bridge cannot be formed, and, as a result, in the presence of sodium dodecyl sulfate the affected loop opens and the NS polypeptide migrates further into the gel. Such a configuration may also explain the multifunctional nature of the NS polypeptide.