Hierarchal constitutive phosphorylation of the vesicular stomatitis virus P protein and lack of effect on P1 to P2 conversion.
In vitro reconstitution of a transcriptionally active VSV polymerase complex (P:L) reportedly requires phosphorylation of the N-terminal domain of P by CKII. Two constitutively phosphorylated sites have been implicated in this activation for both VSV Indiana and New Jersey serotype P proteins. We show here that, in contrast to New Jersey, the Indiana P protein is constitutively phosphorylated on three sites in vivo. The evidence rests on assessing the phosphorylation status of transfected P gene constructs containing all possible combinations of Ala substitutions at Ser60, Thr62, and Ser64. All mutants containing the T62A substitution showed a reduced level of phosphorylation and yielded no P-Thr. Surprisingly the S60A/S64A mutant behaved like the triple substitution and displayed no significant phosphorylation, while the S64A mutant yielded no P-Thr. Phosphorylation of Thr62 therefore depended on prior modification of Ser64. We also tested the ability of our mutant P proteins to convert to the more highly phosphorylated P2 species, a modification essential for transcription in the New Jersey serotype and thought to be carried out by an L-protein-associated kinase. All of our transfected mutant P proteins readily converted to P2 in the presence or absence of L cotransfection, and the latter had no significant effect on P phosphorylation. We conclude that VSV Indiana P protein differs in significant ways from New Jersey P. It is hierarchically and constitutively phosphorylated on a cluster of three sites, not two, suggesting that an additional kinase may be involved. Moreover, Indiana P1 to P2 conversion is independent of prior constitutive phosphorylation and does not require the presence of L protein.
Molecular Biology Institute, San Diego State University, California 92182, USA.
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Jackson
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Spadafora
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Perrault
D000409:Alanine / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D009838:Oligodeoxyribonucleotides; D010710:Phosphates / Q000378:metabolism; D010750:Phosphoproteins; D010766:Phosphorylation; D012324:RNA-Dependent RNA Polymerase; D012694:Serine / Q000378:metabolism; D013912:Threonine / Q000378:metabolism; D014162:Transfection; D014721:Vesicular stomatitis Indiana virus / Q000378:metabolism*; D018116:Vesiculovirus; D014764:Viral Proteins / Q000378:metabolism; D015678:Viral Structural Proteins / Q000235:genetics / Q000378:metabolism*
D009838:Oligodeoxyribonucleotides; C081250:P protein, Vesicular stomatitis virus; D010710:Phosphates; D010750:Phosphoproteins; D014764:Viral Proteins; D015678:Viral Structural Proteins; D013912:Threonine; D012694:Serine; C040530:L protein, vesicular stomatitis virus; D012324:RNA-Dependent RNA Polymerase; D012321:DNA-Directed RNA Polymerases; D000409:Alanine