Human Parainfluenza Virus Type 3 Phosphoprotein: Identification of Serine 333 as the Major Site for PKC ζ Phosphorylation

The human parainfluenza virus type 3P protein is an RNA polymerase subunit involved in both transcription and replication during the life cycle of the virus. Our laboratory has recently shown that the P protein is phosphorylated both in vitro and in vivo by the cellular protein kinase C (PKC) isoform ζ and that this phosphorylation is essential for viral replication. To identify the site(s) of phosphorylation, we have used CNBr cleavage, phosphoamino acid analysis, and two-dimensional tryptic peptide mapping of the in vitro and in vivo phosphorylated P protein. We demonstrate that when bacterially expressed unphosphorylated P is labeled in vitro with either commercial PKC or purified recombinant PKC ζ, P protein has one major phosphorylation site. By site-directed mutagenesis of PKC consensus sites in the P protein, the primary phosphorylation site is found to be Ser 333. The same site appeared to be modified when vital P protein was phosphorylated in vitro by the PKC packaged within the virion and in the P protein of progeny virion labeled in vivo.