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 of respiratory syncytial virus (RSV). Enzymatic removal of this phosphate group 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 full-length transcripts. A recombinant P protein in which Ser(232) was mutated to Asp exhibited about half of the transcriptional activity of the wild-type phosphorylated protein, suggesting that the negative charge of the phosphate groups is an important contributor to P protein function. Use of a temperature-sensitive CK2 mutant yeast revealed that in yeast, phosphorylation of recombinant P by non-CK2 kinase(s) occurs mainly at Ser(215). In vitro, P protein could be phosphorylated by purified CK1 at Ser(215) but this phosphorylation did not result in transcriptionally active P protein. A triple mutant P protein in which Ser(215), Ser(232), and Ser(237) were all mutated to Ala 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 vivo. 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.
Dupuy
L C
LC
Department of Biochemistry and Molecular Biology, College of Medicine, University of South Alabama, Mobile, Alabama, USA.
Dobson
S
S
Bitko
V
V
Barik
S
S
D000818:Animals; D047390:Casein Kinase II; D047388:Casein Kinases; D002417:Cattle; D006801:Humans; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D012136:Respiratory Syncytial Viruses / Q000235:genetics / Q000378:metabolism*; D012694:Serine; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D010750:Phosphoproteins; D014764:Viral Proteins; D012694:Serine; D011494:Protein Kinases; D047390:Casein Kinase II; D047388:Casein Kinases; D017346:Protein Serine-Threonine Kinases