Single serine phosphorylation within the acidic domain of Chandipura virus p protein regulates the transcription in vitro

Abstract

Bacterially expressed unphosphorylated P protein of Chandipura Virus was found to be efficiently phosphorylatedin vitroby casein kinase II (CKII). The phosphorylated form of the P protein supported the transcriptionin vitrobut the unphosphorylated form could not. Kinetic data suggests that CKII incorporates one molecule of phosphate. Western blotting with monoclonal antibody against phosphoserine and phosphoaminoacid analysis confirmed that the phosphate accepting residue was serine. Comparison with P protein of other viruses and tryptic digest of the phosphorylated protein predicted the ser⁶²was the probable site for phosphorylation. This was further confirmed by substituting ser⁶²with alanine by site-directed mutagenesis. CKII was unable to phosphorylate the mutated P protein which in turn could not support the transcriptionin vitro.The phosphorylated P protein eluted from the gel filtration at the position of its dimer in contrast to the unphosphorylated protein which eluted as monomer.