Casein kinase 2-mediated phosphorylation of respiratory syncytial virus phosphoprotein P is essential for the transcription elongation activity of the viral polymerase; Phosphorylation by casein kinase 1 occurs mainly at Ser(215) and is without effect

The major site of in vitro phosphorylation by casein kinase 2 (CK2) was the conserved Ser(232) in the P proteins of human, bovine, and ovine strains o f respiratory syncytial virus (RSV). Enzymatic removal of this phosphate gr oup from the P protein instantly halted transcription elongation in vitro. Transcription reconstituted in the absence of P protein or in the presence of phosphate-free P protein produced abortive initiation products but no fu ll-length transcripts. A recombinant P protein in which Ser(232) was mutate d to Asp exhibited about half of the transcriptional activity of I-he wild- type phosphorylated protein, suggesting that the negative charge of the pho sphate groups is an important contributor to P protein function. Use of a t emperature-sensitive CK2 mutant yeast revealed that in yeast, phosphorylati on of recombinant P by non-CK2 kinase(s) occurs mainly at Ser(215). In vitr o, P protein could, be phosphorylated by purified CK1 at Ser(215) but this phosphorylation did not result in transcriptionally active P protein. A tri ple mutant P protein in which Ser(215), Ser(232), and Ser(237) were all mut ated to AIa was completely defective in phosphorylation in vitro as well as ex vivo. The xanthate compound D609 inhibited CK2 but not CK1 in vitro and had a very modest effect on P protein phosphorylation and RSV yield ex viv o. Together, these results suggest a role for CK2-mediated phosphorylation of the P protein in the promoter clearance and elongation properties of the viral RNA-dependent RNA polymerase.