HUMAN PARAINFLUENZA VIRUS TYPE-3 PHOSPHOPROTEIN - IDENTIFICATION OF SERINE-333 AS THE MAJOR SITE FOR PKC-ZETA PHOSPHORYLATION

The human parainfluenza virus type 3 P protein is an RNA polymerase su bunit involved in both transcription and replication during the life c ycle of the virus. Our laboratory has recently shown that the P protei n is phosphorylated both in vitro and in vivo by the cellular protein kinase C (PKC) isoform zeta and that this phosphorylation is essential for viral replication. To identify the site(s) of phosphorylation, we have used CNBr cleavage, phosphoamino acid analysis, acid two-dimensi onal tryptic peptide mapping of the in vitro and in vivo phosphorylate d P protein. We demonstrate that when bacterially expressed unphosphor ylated P is labeled in vitro with either commercial PKC or purified re combinant PKC zeta, P protein has one major phosphorylation site. By s ite-directed mutagenesis of PKC consensus sites in the P protein, the primary phosphorylation site is found to be Ser 333. The same site app eared to be modified when viral P protein was phosphorylated in vitro by the PKC packaged within the virion and in the P protein of progeny Virion labeled in vivo. (C) 1995 Academic Press, Inc.