Stepwise phosphorylation of vesicular stomatitis virus P protein by virion-associated kinases and uncoupling of second step from in vitro transcription.
Transcription-competent cores of vesicular stomatitis virus (VSV) contain two tightly bound protein kinase activities capable of phosphorylating the viral P protein (Beckes and Perrault, Virology 184, 383-386, 1991). We examined here the specificity of these kinases for the P protein substrate and their activity during the in vitro transcription process. Conditions favoring the VSVK1 kinase activity resulted in phosphorylation of the P1 species predominantly whereas conditions favoring VSVK2, or transcription conditions, led to an increase in the proportion of the faster migrating P2 and P3 species. A minimum of 2 mol phosphate/mol P protein was incorporated in 1 hr under optimal transcription conditions. Pulse-chase experiments revealed that the VSVK2 activity converted phosphorylated P1 to P2/P3 species. Most or all of the sites modified by VSVK1 (serines only) mapped to the 78 amino acid-long N-terminal fragment of the P protein; additional serine acceptor sites of undetermined location were also phosphorylated under VSVK2 conditions. Pretreatment of virion cores with 5'-p-fluorosulfonylbenzoyl adenosine had little or no effect on P1 phosphorylation but inhibited P1 to P2/P3 conversion nearly completely, with no effect on subsequent transcription. Likewise, the addition of cell extracts had relatively little effect on P1 phosphorylation but strongly inhibited the appearance of P2/P3, without affecting concurrent transcription. We conclude that phosphorylation of the P protein during transcription in vitro is a two-step process carried out by two distinct kinase activities, but only the first step may be essential for viral mRNA synthesis.
Beckes
J D
JD
Department of Biology, San Diego State University, California 92182.
Perrault
J
J
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002213:Capsid / Q000737:chemistry / Q000378:metabolism*; D002478:Cells, Cultured; D006224:Cricetinae; D005033:Ethylmaleimide / Q000494:pharmacology; D015967:Gene Expression Regulation, Viral; D066298:In Vitro Techniques; D010449:Peptide Mapping; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D014158:Transcription, Genetic; D014721:Vesicular stomatitis Indiana virus / Q000201:enzymology / Q000378:metabolism*; D014758:Viral Core Proteins / Q000737:chemistry / Q000378:metabolism*; D017361:Viral Nonstructural Proteins
D010750:Phosphoproteins; D014758:Viral Core Proteins; D017361:Viral Nonstructural Proteins; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D005033:Ethylmaleimide