HIERARCHICAL 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 c omplex (P:L) reportedly requires phosphorylation of the N-terminal dom ain of P by CKII. Two constitutively phosphorylated sites have been im plicated in this activation for both VSV Indiana and New Jersey seroty pe P proteins. We show here that, in contrast to New Jersey, the India na P protein is constitutively phosphorylated on three sites in vivo. The evidence rests on assessing the phosphorylation status of transfec ted P gene constructs containing all possible combinations of Ala subs titutions 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 subst itution and displayed no significant phosphorylation, while the S64A m utant 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 a nd thought to be carried out by an L-protein-associated kinase. All of our transfected mutant P proteins readily converted to P2 in the pres ence or absence of L cotransfection, and the latter had no significant effect on P phosphorylation. We conclude that VSV Indiana P protein d iffers in significant ways from New Jersey P. It is hierarchically and constitutively phosphorylated on a cluster of three sites, not two, s uggesting that an additional kinase may be involved. Moreover, Indiana P1 to P2 conversion is independent of prior constitutive phosphorylat ion and does not require the presence of L protein. (C) 1995 Academic Press, Inc.