Phosphorylation of Ser232Directly Regulates the Transcriptional Activity of the P Protein of Human Respiratory Syncytial Virus: Phosphorylation of Ser237May Play an Accessory Role

Abstract The phosphoprotein P of human respiratory syncytial virus (RSV) was expressed in eukaryotic cells in phosphorylated form. Site-directed mutagenesis of the recombinant protein established Ser 232 as the major site of phosphorylation in vivo. Phosphorylation of bacterially made P protein in vitro by purified casein kinase II (CKII) resulted in the phosphorylation of Ser 237 , whereas mainly Ser 232 was phosphorylated by a crude cell extract. The P kinase activity in the cell extract exhibited properties characteristic of CKII. While the Ser 232,237 to Ala double mutant was nearly completely defective for phosphorylation and transcription, phosphorylation at Ser 232 , through the use of appropriate P mutant or kinase, activated P protein. Phosphorylation of Ser 237 restored activity only to the extent it facilitated phosphorylation of Ser 232 . Phosphate groups of P protein in RSV-infected cells were highly stable; inhibitors of protein serine phosphatases had no effect on the intracellular turn-over of the phosphates. Highly purified viral polymerase L was transcriptionally active but devoid of P protein kinase activity. Thus, CKII-mediated phosphorylation of Ser 232 appears to be the primary regulator of P protein activity while phosphorylation of Ser 237 may be involved in a modulatory role under certain conditions.