INTRACELLULAR PHOSPHORYLATION OF THE SENDAI VIRUS P-PROTEIN

Phosphorylation status of the Sendai virus P protein was examined duri ng virus infection and compared with cell-free phosphorylation. P prot ein from Sendai virus-infected (VI) and P/C gene-transfected (PT) mamm alian cells and from purified virions (PV) was phosphorylated at only serine residues. In contrast, cell-free phosphorylation of the P prote in with virion-associated protein kinase (VAPK) occurred at both threo nine and serine. Tryptic phosphopeptide maps of the P protein from VI, PT, and PV showed that the phosphorylation was primarily localized on one peptide (TP1), while VAPK phosphorylated the P protein on several peptides. There was no change in the steady-state phosphopeptide map of the P protein during virus replication, indicating that the TP1 is constitutively phosphorylated. Inhibition of cellular phosphatases (PP 1 and PP2A) by okadaic acid (OA) in virus-infected cells caused a sixf old increase in the P protein phosphorylation, solely at serine residu es. The phosphopeptide map of the OA-P protein revealed that phosphory lation occurred on several peptides, but the OA-P map was significantl y different from the VAPK-P map. However, additional phosphorylation o f the P protein did not block its association with nucleocapsids. Thes e results suggest that the Sendai virus P protein is constitutively ph osphorylated primarily at one locus but has the potential for phosphor ylation at additional sites. Further, our results do not show any corr elations between the intracellular and cell-free phosphorylation of th e P protein and, therefore, question the validity of cell-free phospho rylations. (C) 1995 Academic Press, Inc.