Temporal Regulation of Herpes Simplex Virus Type 2 VP22 Expression and Phosphorylation

The tegument of the herpesvirus virion is an amorphous structure that lies between the capsid and the envelope. The function of this subvirion structure appears to be twofold: to link the capsid of the newly forming viral particle with the envelope and to introduce regulatory proteins into a newly infected cell. The capacity of herpes simplex virus type 1 (HSV-1) and HSV-2 tegument proteins to regulate viral gene expression and to subvert cellular functions has been the focus of considerable study. For example, it is known that the tegument proteins VP16 and vhs transactivate viral α genes and degrade host and viral mRNAs, respectively (14, 15, 22). These and other tegument proteins help to create a cellular environment that is advantageous to virus replication. VP22 is an abundant HSV tegument protein that possesses the remarkable property of intercellular spread (5). VP22 also can associate with and stabilize microtubules (7), interact with the transactivating protein VP16 (6), and associate with chromatin (13, 23). Although the function(s) of VP22 has not been established, its properties suggest that VP22 must play an important role in the HSV life cycle. Posttranslational modification is a common mechanism to regulate protein localization and function. VP22 is a 300-amino-acid protein that has been shown in both HSV-1 and HSV-2 to be posttranslationally modified by phosphorylation (1, 8, 13, 23). Phosphorylation of HSV-1 VP22 has been implicated in directing VP22 to distinct subcellular regions, with the hypophosphorylated form localizing to the cytoplasm and phosphorylated forms localizing to the nucleus (24). HSV-1 VP22 appears to be phosphorylated early in infection, whereas at later times during infection the majority of VP22 is hypophosphorylated (24). Considerable evidence from in vitro kinase assays has implicated casein kinase II (CKII) in the phosphorylation of HSV-1 VP22 (8), and other data suggest that a virus-associated kinase may also be involved (9). A viral kinase, UL13, has been implicated in inducing phosphorylation of HSV-1 VP22 based on observations that a protein of ca. 38 kDa is phosphorylated in in vitro kinase assays using lysates of cells infected with wild-type virus, but not when the lysates derive from cells infected with UL13− virus (2, 20). However, lack of an observable mobility difference in VP22 from transfected and infected cells led to the conclusion that UL13 does not play a major role in phosphorylation of VP22 (8). Whether UL13 is itself capable of phosphorylating VP22 is unknown. It is also not clear whether phosphorylation of VP22 is regulated, although this seems likely since the fastest-mobility form accumulates late in infection (24) and hypophosphorylated VP22 is preferentially incorporated into extracellular virions (8, 17). HSV-2 VP22 is highly homologous to HSV-1 VP22, with 65% identity at the amino acid level. The homology shared by these two proteins suggests that they may act in similar ways. Despite the potential importance of VP22 in human disease caused by HSV-2, information about HSV-2 VP22 is limited. It is known to exist in one phosphorylated and one hypophosphorylated form and to be ADP-ribosylated (1). We generated a monoclonal antibody (MAb) to VP22 that is not sensitive to phosphorylation to more fully characterize the phosphorylation pattern of HSV-2 VP22 in the context of the infected cell. Better separation of the HSV-2 VP22 isoforms on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) compared to those of HSV-1 facilitated examination of their phosphorylation pattern. Based on its similarity to HSV-1 VP22, we used HSV-2 VP22 as a model to investigate whether VP22 is differentially phosphorylated at various stages of the HSV-2 infectious cycle and whether this phosphorylation is regulated, possibly to control VP22 localization or function.