Phosphorylation of simian cytomegalovirus assembly protein precursor (pAPNG.5) and proteinase precursor (pAPNG1): Multiple attachment sites identified, including two adjacent serines in a casein kinase II consensus sequence

The assembly protein precursor (pAP) of cytomegalovirus (CMV), and its homo logs in other herpesviruses, functions at several key steps during the proc ess of capsid formation, This protein, and the genetically related maturati onal proteinase, is distinguished from the other capsid proteins by posttra nslational modifications, including phosphorylation, The objective of this study was to identify sites at which pAP is phosphorylated so that the func tional significance of this modification and the enzyme(s) responsible for it can be determined, In the work reported here, we used peptide mapping, m ass spectrometry, and site-directed mutagenesis to identify two sets of pAP phosphorylation sites. One is a casein kinase II (CKII) consensus sequence that contains two adjacent serines, both of which are phosphorylated, The other site(s) is in a different domain of the protein, is phosphorylated le ss frequently than the CKII site, does not require preceding CKII-site phos phorylation, and causes an electrophoretic mobility shift when phosphorylat ed, Transfection/expression assays for proteolytic activity showed no gross effect of CKII-site phosphorylation on the enzymatic activity of the prote inase or on the substrate behavior of pAP, Evidence is presented that both the CKII sites and the secondary sites are phosphorylated in virus-infected cells and plasmid-transfected cells, indicating that these modifications c an be made by a cellular enzyme(s), Apparent compartmental differences in p hosphorylation of the CKII-site (cytoplasmic) and secondary-site (nuclear) serines suggest the involvement of more that one enzyme in these modificati ons.