Sánchez A, De B P, Banerjee A K
J Gen Virol. 1985 May;66 ( Pt 5):1025-36. doi: 10.1099/0022-1317-66-5-1025.
The structural proteins L and NS of vesicular stomatitis virus were obtained from purified viral ribonucleoprotein complex followed by phosphocellulose column chromatography and assayed for protein kinase activity using [gamma-32P]ATP as the phosphate donor. The fractions containing purified L protein phosphorylated NS protein in vitro. 8-Azido-ATP, a photoreactive analogue of ATP, was also used as the phosphate donor for phosphorylation of NS protein by the L protein. In the presence of ultraviolet light, only L protein was specifically cross-linked with 8-azido-[gamma-32P]ATP. In the absence of u.v. light 8-azido ATP did no inhibit RNA transcription in a reconstituted reaction or substitute ATP for RNA synthesis in vitro. The above results, taken together, suggest that 8-azido-ATP was bound to the kinase site and phosphorylation of NS protein was mediated by the L protein. Exogenous phosphate acceptor proteins such as phosvitin and casein were also phosphorylated by the L protein fraction. However, addition of an excess of phosvitin failed to compete with the phosphorylation of NS by L, indicating that the protein kinase activity possessed higher affinity for NS. The phosphorylation of NS was strongly inhibited by photoreaction of L protein with 8-azido-ATP with concomitant inhibition of transcription in vitro. These results suggest that phosphorylation of NS protein by L may have a role in the regulation of the virus genome transcription in vitro.
水泡性口炎病毒的结构蛋白L和NS是从纯化的病毒核糖核蛋白复合物中获得的，随后经过磷酸纤维素柱层析，并以[γ-32P]ATP作为磷酸盐供体来检测蛋白激酶活性。含有纯化L蛋白的组分在体外使NS蛋白磷酸化。8-叠氮基-ATP是ATP的一种光反应类似物，也被用作L蛋白使NS蛋白磷酸化的磷酸盐供体。在紫外线存在的情况下，只有L蛋白与8-叠氮基-[γ-32P]ATP特异性交联。在没有紫外线的情况下，8-叠氮基ATP在重组反应中不抑制RNA转录，也不能在体外替代ATP进行RNA合成。综合上述结果表明，8-叠氮基-ATP与激酶位点结合，NS蛋白的磷酸化由L蛋白介导。外源性磷酸盐受体蛋白如卵黄高磷蛋白和酪蛋白也被L蛋白组分磷酸化。然而，加入过量的卵黄高磷蛋白并不能与L对NS的磷酸化竞争，这表明蛋白激酶活性对NS具有更高的亲和力。L蛋白与8-叠氮基-ATP的光反应强烈抑制了NS的磷酸化，并同时抑制了体外转录。这些结果表明，L对NS蛋白的磷酸化可能在体外病毒基因组转录的调控中起作用。
