The herpes simplex virus type 1 (HSV-1) immediate-early (IE) regulatory protein infected-cell protein 0 (ICP0) is a strong and global transactivator of both viral and cellular genes. In a previous study, we reported that ICP0 is highly phosphorylated and contains at least seven distinct phosphorylation signals as determined by phosphotryptic peptide mapping (D. J. Davido et al., J. Virol. 76:1077-1088, 2002). Since phosphorylation affects the activities of many viral regulatory proteins, we sought to determine whether the phosphorylation of ICP0 affects its functions. To address this question, it was first necessary to identify the regions of ICP0 that are phosphorylated. For this purpose, ICP0 was partially purified, and phosphorylation sites were mapped by microcapillary high-pressure liquid chromatography tandem mass spectrometry. Three phosphorylated regions containing 11 putative phosphorylation sites, all within or adjacent to domains important for the transactivating activity of ICP0, were identified. The 11 sites were mutated to alanine as clusters in each of the three regions by site-directed mutagenesis, generating plasmids expressing mutant forms of ICP0: Phos 1 (four mutated sites), Phos 2 (three mutated sites), and Phos 3 (four mutated sites). One-dimensional phosphotryptic peptide analysis confirmed that the phosphorylation state of each Phos mutant form of ICP0 is altered relative to that of wild-type ICP0. In functional assays, the ICP0 phosphorylation site mutations affected the subcellular and subnuclear localization of ICP0, its ability to alter the staining pattern of the nuclear domain 10 (ND10)-associated protein PML, and/or its transactivating activity in Vero cells. Only mutations in Phos 1, however, impaired the ability of ICP0 to complement the replication of an ICP0 null mutant in Vero cells. This study thus suggests that phosphorylation is an important regulator of ICP0 function.Phosphorylation is a universal posttranslational modification that alters the activities of many viral regulatory proteins. Among the best examples of this effect is large T antigen of simian virus 40, a multifunctional 708-amino-acid nuclear phospho-and oncoprotein required for the replication of simian virus 40 (reviewed in references 61 and 76). Many phosphorylation sites have been identified on T antigen, and several lie adjacent to its nuclear localization signal and origin-binding domain, modulating simian virus 40 DNA replication (reviewed in reference 61). Specifically, mutation of these sites alters T antigen's origin-binding activity, hexamer-hexamer interaction, nuclear localization, and/or DNA replication activity, affecting both its biochemical and biological functions (11, 14, 41, 50-52, 54, 78).In herpes simplex virus type 1 (HSV-1) infection, the first genes to be expressed are the immediate-early (IE) genes (39). These genes encode infected-cell protein (ICPs) 0, 4, 22, 27, and 47, which collectively exhibit diverse regulatory and immunomodulatory functions. At least four of the five IE regul...

Herpes simplex virus type 1 (HSV-1) ICP0 directs the degradation of cellular proteins associated with nuclear structures called ND10, a function thought to be closely associated with its broad transactivating activity. Roscovitine (Rosco), an inhibitor of cyclin-dependent kinases (cdks), inhibits the replication of HSV-1, HSV-2, human cytomegalovirus, varicella-zoster virus, and human immunodeficiency virus type 1 by inhibiting specific steps or activities of viral regulatory proteins, indicating the broad and pleiotropic effects that cdks have on the replication of these viruses. We previously demonstrated that Rosco inhibits the transactivating activity of ICP0. In the present study, we asked whether Rosco also affects the ability of ICP0 to direct the degradation of ND10-associated proteins. For this purpose, WI-38 cells treated with cycloheximide (CHX) were mock infected or infected with wild-type HSV-1 or an ICP0؊ mutant (7134). After release from the CHX block, the infections were allowed to proceed for 2 h in the presence or absence of Rosco at a concentration known to inhibit ICP0's transactivating activity. The cells were then examined for the presence of ICP0 and selected ND10-associated antigens (promyelocytic leukemia antigen [PML], sp100, hDaxx, and NDP55) by immunofluorescence. Staining for the ND10-associated antigens was detected in <20% of KOS-infected cells in the presence or absence of Rosco, demonstrating that Rosco-sensitive kinases are not required for ICP0's ability to direct the dispersal or degradation of these antigens. In contrast, >90% of 7134-and mock-infected cells stained positive for all ND10-associated antigens in the presence or absence of Rosco. Similar results were obtained with a non-ND10-associated antigen, DNA-PK cs , a known target of ICP0-directed degradation. The results of the PML and DNA-PK cs immunofluorescence studies correlated with a decrease in the levels of these proteins as determined by Western blotting. Thus, the differential requirement for Rosco-sensitive cdk activities distinguishes ICP0's ability to direct the dispersal or degradation of cellular proteins from its transactivating activity.The molecular mechanisms responsible for the activation of herpes simplex virus (HSV) gene expression during productive infection and reactivation from latency are not well understood. Available evidence indicates, however, that the functions of viral regulatory proteins that activate viral-gene expression are themselves activated by cell cycle regulatory proteins. For example, two cellular proteins, HCF (36) and TAF250 (84), whose expression and activities are cell cycle associated, activate the functions of two viral regulatory proteins, VP16 (36) and ICP4 (12), respectively, and cyclin-dependent kinases (cdks), which drive the cell cycle, are required for the synthesis and activities of HSV immediate-early (IE) regulatory proteins (75-77). Notably, inhibitors of cdks have been shown to inhibit the replication of HSV type 1 (HSV-1) (74), HSV-2 (74), human cytomegaloviru...