Site-specific phosphorylation regulates the transcriptive activity of vesicular stomatitis virus NS protein.
In vitro transcription by vesicular stomatitis virus nucleocapsids is inhibited by enzymatic dephosphorylation of the NS protein. We provide evidence that specific, partial dephosphorylation of NS molecules is the only detectable change in nucleocapsids treated with bacterial alkaline phosphatase under conditions that prevent the action of adventitious protease. Dephosphorylation appeared to affect only the rate of transcription; there were no changes in sedimentation rates of transcripts. To identify the sites of phosphorylation required for NS activity in transcription, we examined phosphopeptides produced by chymotrypsin digestion of the two electrophoretic classes of NS molecules found in virions and infected cells. The electrophoretically slower class, NS1, abundant in the intracellular soluble pool, has a lower activity in transcription; it contained six chymotryptic phosphopeptides. Five of these peptides contained both phosphoserine and phosphothreonine, indicating that this peptide cluster represents at least 11 separate sites of phosphorylation. In the electrophoretically faster nucleocapsid-associated NS2 class of molecules, which support a higher rate of transcription, another group of eight phosphopeptides was superimposed on this pattern. Two of these peptides contained both phosphoserine and phosphothreonine, so this cluster of peptides represents at least 10 additional phosphorylation sites. These sites were especially sensitive to dephosphorylation by bacterial alkaline phosphatase. One or more of them appears to be responsible for the higher transcription rates medicated by NS2 molecules.
D000469:Alkaline Phosphatase / Q000494:pharmacology; D007700:Kinetics; D010750:Phosphoproteins / Q000032:analysis / Q000502:physiology*; D010766:Phosphorylation; D011487:Protein Conformation; D014158:Transcription, Genetic; D014721:Vesicular stomatitis Indiana virus / Q000235:genetics* / Q000378:metabolism; D014764:Viral Proteins / Q000032:analysis / Q000502:physiology*
D010750:Phosphoproteins; D014764:Viral Proteins; D000469:Alkaline Phosphatase