CONSTITUTIVE PHOSPHORYLATION OF THE VESICULAR STOMATITIS-VIRUS-P PROTEIN MODULATES POLYMERASE COMPLEX-FORMATION BUT IS NOT ESSENTIAL FOR TRANSCRIPTION OR REPLICATION

As a subunit of both the P-L polymerase complex and the P-N assembly c omplex, the vesicular stomatitis virus (VSV P protein plays a pivotal role in transcription and replication of the viral genome, Constitutiv e phosphorylation of this protein is currently thought to be essential . for formation of the P-L complex. We recently identified the three r elevant phosphate acceptor sites in the VSV Indiana serotype P protein (R. L. Jackson, D. Spadafora, and J. Perrault, Virology 214:189-197, 1995). We now report the effects of substituting Ata at these acceptor sites on transcription reconstitution in vitro and replication of def ective interfering virus (DI) templates in vivo. The singly substitute d S60A, T62A, and S64A mutants and the doubly substituted S60A/T62A an d T62A/S64A mutants, all of which retain some constitutive phosphoryla tion, were nearly as active as the wild type in both assays, Surprisin gly, the nonphosphorylated S60A/S64A protein was also active in transc ription (greater than or equal to 28%) and replication (greater than o r equal to 50%) under optimal conditions, However, this mutant was muc h less active in in vitro transcription (less than or equal to 5% of w ild type) at low P concentrations (<27 nM). In addition, S60A/S64A req uired higher concentrations of L protein than did the wild type for op timal DI replication in vivo. Df replication efficiency and intracellu lar accumulation of L, P, and N proteins in the transfected system wer e very similar to those in VSV-infected cells. We conclude that P prot ein constitutive phosphorylation is not essential for VSV RNA synthesi s per se but likely plays an important role in vivo In facilitating P- multimerization acid possibly P-L complex formation.