Temporal regulation of herpes simplex virus type 2 VP22 expression and phosphorylation

The VP22 protein of herpes simplex virus type 2 (HSV-2) is a major componen t of the virion tegument. Previous work with HSV-1 indicated that VP22 is p hosphorylated during infection, and phosphorylation may play a role in modu lating VP22 localization in infected cells. It is not clear, however, when phosphorylation occurs in infected cells or how it is regulated. Less is kn own about the synthesis and phosphorylation of HSV-2 VP22. To study the com plete biosynthetic history of HSV-2 VP22, we generated a monoclonal antibod y to the carboxy terminus of VP22. Using immunoprecipitation and Western bl ot analyses, we show that HSV-2 VP22 can be found in three distinct isoform s in infected cells, two of which are phosphorylated. Like HSV-1 VP22, HSV- 2 VP22 is synthesized ca. 4 h after infection, and the isoform later incorp orated into virions is hypophosphorylated. In addition, we demonstrate for the first time (i) that newly synthesized VP22 is phosphorylated rapidly af ter synthesis, (ii) that this phosphorylation occurs in a virus-dependent m anner, (iii) that the HSV-2 kinase UL13 is capable of inducing phosphorylat ion of VP22 in the absence of other viral proteins, (iv) that phosphorylate d VP22 is very stable in infected cells, (v) that phosphorylated isoforms o f VP22 are gradually dephosphorylated late in infection to produce the viri on tegument form, and (vi) that this dephosphorylation occurs independently of viral DNA replication or virion assembly. These results indicate that H SV-2 VP22 is a stable protein that undergoes highly regulated, virus-depend ent phosphorylation events in infected cells.