Static and dynamic protein phosphorylation in the Vaccinia virion

To the best of our knowledge, two phosphorylation sites have been reported previously, among 11 known Vaccinia virus phosphoproteins. Here, via phosphopeptide mass spectrometry, up to 189 phosphorylation sites were identified among 48 proteins in preparations of purified Vaccinia mature virus (MV). 8.5% of phospho-residues were pTyr. Viral phosphoproteins were found in diverse functional classes, including structural proteins, membrane proteins and RNA polymerase subunits. Among the nine identified membrane phosphoproteins, the sites in just one, namely A14L, were deduced to be internal with respect to the accompanying membrane. Examination of sites in known substrates of the Vaccinia-encoded protein kinase VPK2, indicated VPK2 to be a proline-dependent kinase. The MV phosphoproteome was enriched in potential substrates of cellular kinases belonging to the CDK2/CDK3, CK2, and p38 groups. Quantitative mass spectrometry identified several sites that became phosphorylated during intravirion kinase activation in vitro, each showing one of two distinct pH-dependency profiles.