PHOSPHORYLATION OF SER(232) DIRECTLY REGULATES THE TRANSCRIPTIONAL ACTIVITY OF THE P-PROTEIN OF HUMAN RESPIRATORY SYNCYTIAL VIRUS - PHOSPHORYLATION OF SER(237) MAY PLAY AN ACCESSORY ROLE

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 turnover 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. (C) 1995 academic Press, Inc.