In vitro phosphorylation of NS protein by the L protein of vesicular stomatitis virus.
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.
Sánchez
A
A
De
B P
BP
Banerjee
A K
AK
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D001386:Azides / Q000378:metabolism; D010766:Phosphorylation; D010774:Phosvitin / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D012367:RNA, Viral / Q000096:biosynthesis; D012324:RNA-Dependent RNA Polymerase; D013379:Substrate Specificity; D014158:Transcription, Genetic; D014466:Ultraviolet Rays; D014721:Vesicular stomatitis Indiana virus / Q000201:enzymology* / Q000235:genetics; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins / Q000302:isolation & purification / Q000378:metabolism*
D001386:Azides; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; C017040:8-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D010774:Phosvitin; D011494:Protein Kinases; C040530:L protein, vesicular stomatitis virus; D012324:RNA-Dependent RNA Polymerase