Reciprocal activities between herpes simplex virus type 1 regulatory protein ICP0, a ubiquitin E3 ligase, and ubiquitin-specific protease USP7

Herpes simplex virus type 1 (HSV-1) regulatory protein ICP0 stimulates lytic infection and the reactivation of quiescent viral genomes. These roles of ICP0 require its RING finger E3 ubiquitin ligase domain, which induces the degradation of several cellular proteins, including components of promyelocytic leukemia nuclear bodies and centromeres. ICP0 also interacts very strongly with the cellular ubiquitin-specific protease USP7 (also known as HAUSP). We have shown previously that ICP0 induces its own ubiquitination and degradation in a RING finger-dependent manner, and that its interaction with USP7 regulates this process. In the course of these studies we found and report here that ICP0 also targets USP7 for ubiquitination and proteasome-dependent degradation. The reciprocal activities of the two proteins reveal an intriguing situation that poses the question of the balance of the two processes during productive HSV-1 infection. Based on a thorough analysis of the properties of an HSV-1 mutant virus that expresses forms of ICP0 that are unable to bind to USP7, we conclude that USP7-mediated stabilization of ICP0 is dominant over ICP0-induced degradation of USP7 during productive HSV-1 infection. We propose that the biological significance of the ICP0-USP7 interaction may be most pronounced in natural infection situations, in which limited amounts of ICP0 are expressed.     

A RING finger ubiquitin ligase is protected from autocatalyzed ubiquitination and degradation by binding to ubiquitin-specific protease USP7

Herpes simplex virus type 1 immediate-early regulatory protein ICP0 stimulates lytic infection and reactivation from latency, processes that require the ubiquitin E3 ligase activity mediated by the RING finger domain in the N-terminal portion of the protein. ICP0 stimulates the production of polyubiquitin chains by the ubiquitin-conjugating enzymes UbcH5a and UbcH6 in vitro, and in infected and transfected cells it induces the proteasome-dependent degradation of a number of cellular proteins including PML, the major constituent protein of PML nuclear bodies. However, ICP0 binds strongly to the cellular ubiquitin-specific protease USP7, a member of a family of proteins that cleave polyubiquitin chains and/or ubiquitin precursors. The region of ICP0 that is required for its interaction with USP7 has been mapped, and mutations in this domain reduce the functionality of ICP0. These findings pose the question: why does ICP0 include domains that are associated with the potentially antagonistic functions of ubiquitin conjugation and deconjugation? Here we report that although neither protein affected the intrinsic activities of the other in vitro, USP7 protected ICP0 from autoubiquitination in vitro, and their interaction can greatly increase the stability of ICP0 in vivo. These results demonstrate that RING finger-mediated autoubiquitination of ICP0 is biologically relevant and can be regulated by interaction with USP7. This principle may extend to a number of cellular RING finger E3 ubiquitin ligase proteins that have analogous interactions with ubiquitin-specific cleavage enzymes.     

Functional characterization of residues required for the herpes simplex virus 1 E3 ubiquitin ligase ICP0 to interact with the cellular E2 ubiquitin-conjugating enzyme UBE2D1 (UbcH5a)

The viral ubiquitin ligase ICP0 is required for efficient initiation of herpes simplex virus 1 (HSV-1) lytic infection and productive reactivation of viral genomes from latency. ICP0 has been shown to target a number of specific cellular proteins for proteasome-dependent degradation during lytic infection, including the promyelocytic leukemia protein (PML) and its small ubiquitin-like modified (SUMO) isoforms. We have shown previously that ICP0 can catalyze the formation of unanchored polyubiquitin chains and mediate the ubiquitination of specific substrate proteins in vitro in the presence of two E2 ubiquitin-conjugating enzymes, namely, UBE2D1 (UbcH5a) and UBE2E1 (UbcH6), in a RING finger-dependent manner. Using homology modeling in conjunction with site-directed mutagenesis, we identify specific residues required for the interaction between the RING finger domain of ICP0 and UBE2D1, and we report that point mutations at these residues compromise the ability of ICP0 to induce the colocalization of conjugated ubiquitin and the degradation of PML and its SUMO-modified isoforms. Furthermore, we show that RING finger mutants that are unable to interact with UBE2D1 fail not only to complement the plaque-forming defect of an ICP0-null mutant virus but also to mediate the derepression of quiescent HSV-1 genomes in cell culture. These data demonstrate that the ability of ICP0 to interact with cellular E2 ubiquitin-conjugating enzymes is fundamentally important for its biological functions during HSV-1 infection.     

The herpes simplex virus type 1 (HSV-1) regulatory protein ICP0 interacts with and Ubiquitinates p53

Herpes simplex virus type 1 regulatory protein ICP0 contains a zinc-binding RING finger and has been shown to induce the proteasome-dependent degradation of a number of cellular proteins in a RING finger-dependent manner during infection. This domain of ICP0 is also required to induce the formation of unanchored polyubiquitin chains in vitro in the presence of ubiquitin-conjugating enzymes UbcH5a and UbcH6. These data indicate that ICP0 has the potential to act as a RING finger ubiquitin ubiquitin-protein isopeptide ligase (E3) and to induce the degradation of certain cellular proteins through ubiquitination and proteasome-mediated degradation. Here we demonstrate that ICP0 is a genuine RING finger ubiquitin E3 ligase that can interact with and mediate the ubiquitination of the major oncoprotein p53 both in vitro and in vivo. Ubiquitination of p53 requires ICP0 to have an intact RING finger domain and occurs independently of its ability to bind to the ubiquitin-specific protease USP7.     

Replication of ICP0-null mutant herpes simplex virus type 1 is restricted by both PML and Sp100

Herpes simplex virus type 1 (HSV-1) mutants that fail to express the viral immediate-early protein ICP0 have a pronounced defect in viral gene expression and plaque formation in limited-passage human fibroblasts. ICP0 is a RING finger E3 ubiquitin ligase that induces the degradation of several cellular proteins. PML, the organizer of cellular nuclear substructures known as PML nuclear bodies or ND10, is one of the most notable proteins that is targeted by ICP0. Depletion of PML from human fibroblasts increases ICP0-null mutant HSV-1 gene expression, but not to wild-type levels. In this study, we report that depletion of Sp100, another major ND10 protein, results in a similar increase in ICP0-null mutant gene expression and that simultaneous depletion of both proteins complements the mutant virus to a greater degree. Although chromatin assembly and modification undoubtedly play major roles in the regulation of HSV-1 infection, we found that inhibition of histone deacetylase activity with trichostatin A was unable to complement the defect of ICP0-null mutant HSV-1 in either normal or PML-depleted human fibroblasts. These data lend further weight to the hypothesis that ND10 play an important role in the regulation of HSV-1 gene expression.     

PML residue lysine 160 is required for the degradation of PML induced by herpes simplex virus type 1 regulatory protein ICP0

During the early stages of herpes simplex virus type 1 (HSV-1) infection, viral immediate-early regulatory protein ICP0 localizes to and disrupts cellular nuclear structures known as PML nuclear bodies or ND10. These activities correlate with the functions of ICP0 in stimulating lytic infection and reactivating quiescent HSV-1. The disruption of ND10 occurs because ICP0 induces the loss of the SUMO-1-modified forms of PML and the subsequent proteasome-mediated degradation of the PML protein. The functions of ICP0 are largely dependent on the integrity of its zinc-binding RING finger domain. Many RING finger proteins have been found to act as ubiquitin E3 ligase enzymes, stimulating the production of conjugated polyubiquitin chains in the presence of ubiquitin, the ubiquitin-activating enzyme E1, and the appropriate E2 ubiquitin-conjugating enzyme. Substrate proteins that become polyubiquitinated are then subject to degradation by proteasomes. We have previously shown that purified full-length ICP0 acts as an efficient E3 ligase in vitro, producing high-molecular-weight polyubiquitin chains in a RING finger-dependent but substrate-independent manner. In this paper we report on investigations into the factors governing the degradation of PML induced by ICP0 in a variety of in vivo and in vitro assays. We found that ICP0 expression increases the levels of ubiquitinated PML in transfected cells. However, ICP0 does not interact with or directly ubiquitinate either unmodified PML or SUMO-1-modified PML in vitro, suggesting either that additional factors are required for the ICP0-mediated ubiquitination of PML in vivo or that PML degradation is an indirect consequence of some other activity of ICP0 at ND10. Using a transfection-based approach and a family of deletion and point mutations of PML, we found that efficient ICP0-induced PML degradation requires sequences within the C-terminal part of PML and lysine residue 160, one of the principal targets for SUMO-1 modification of the protein.     

ICP0 induces the accumulation of colocalizing conjugated ubiquitin

Herpes simplex virus type 1 (HSV-1) immediate-early protein ICP0 is a general activator of viral gene expression which stimulates the initiation of lytic infection and reactivation from quiescence and latency. The importance of ICP0 to the biology of HSV-1 infection has stimulated interest in its mode of action. Previous studies have reported its interactions with other viral regulatory molecules, with the translation apparatus, with cyclin D3, and with a ubiquitin-specific protease. It has been demonstrated that ICP0 is able to induce the proteasome-dependent degradation of a number of cellular proteins, including components of centromeres and small nuclear substructures known as ND10 or PML nuclear bodies. ICP0 has a RING finger zinc-binding domain which is essential for its functions. In view of several recent examples of other RING finger proteins which modulate the stability of specific target proteins by acting as components of E3 ubiquitin ligase complexes, this study has explored whether ICP0 might operate via a similar mechanism. Evidence that the foci of accumulated ICP0 in transfected and infected cells contain enhanced levels of conjugated ubiquitin is presented. This effect was dependent on the RING finger region of ICP0, and comparison of the properties of a number of ICP0 mutants revealed an excellent correlation between previously established functions of ICP0 and its ability to induce concentrations of colocalizing conjugated ubiquitin. These results strongly support the hypothesis that a major factor in the mechanism by which ICP0 influences virus infection is its ability to induce the degradation of specific cellular targets by interaction with the ubiquitin-proteasome pathway.     

Impact of the interaction between herpes simplex virus type 1 regulatory protein ICP0 and ubiquitin-specific protease USP7 on activation of adeno-associated virus type 2 rep gene expression

Expression of the herpes simplex virus type 1 (HSV-1) regulatory protein ICP0 in transfected cells reactivates rep gene expression from integrated adeno-associated virus (AAV) type 2 genomes via a mechanism that requires both its RING finger and USP7 interaction domains. In this study, we found that the rep reactivation defect of USP7-binding-negative ICP0 mutants can be overcome by further deletion of sequences in the C-terminal domain of ICP0, indicating that binding of USP7 to ICP0 is not directly required. Unlike the case in transfected cells, only the RING finger domain of ICP0 was essential for rep gene reactivation during HSV-1 infection. However, mutants unable to bind to USP7 activate HSV-1 gene expression and reactivate rep gene expression with reduced efficiencies. These results further elucidate the role of ICP0 as a helper factor for AAV replication and illustrate that care is required when extrapolating from the properties of ICP0 in transfection assays to events occurring during HSV-1 infection.     

PML contributes to a cellular mechanism of repression of herpes simplex virus type 1 infection that is inactivated by ICP0

Promyelocytic leukemia (PML) nuclear bodies (also known as ND10) are nuclear substructures that contain several proteins, including PML itself, Sp100, and hDaxx. PML has been implicated in many cellular processes, and ND10 are frequently associated with the replicating genomes of DNA viruses. During herpes simplex virus type 1 (HSV-1) infection, the viral regulatory protein ICP0 localizes to ND10 and induces the degradation of PML, thereby disrupting ND10 and dispersing their constituent proteins. ICP0-null mutant viruses are defective in PML degradation and ND10 disruption, and concomitantly they initiate productive infection very inefficiently. Although these data are consistent with a repressive role for PML and/or ND10 during HSV-1 infection, evidence in support of this hypothesis has been inconclusive. By use of short interfering RNA technology, we demonstrate that depletion of PML increases both gene expression and plaque formation by an ICP0-negative HSV-1 mutant, while having no effect on wild-type HSV-1. We conclude that PML contributes to a cellular antiviral repression mechanism that is countered by the activity of ICP0.     

Alphaherpesvirus proteins related to herpes simplex virus type 1 ICP0 induce the formation of colocalizing, conjugated ubiquitin

Herpes simplex virus type 1 immediate early protein ICP0 influences virus infection by inducing the degradation of specific cellular proteins via a mechanism requiring its RING finger and the ubiquitin-proteasome pathway. Many RING finger proteins, by virtue of their RING finger domain, interact with E2 ubiquitin-conjugating enzymes and act as a component of an E3 ubiquitin ligase. We have recently shown that ICP0 induces the accumulation of colocalizing, conjugated ubiquitin, suggesting that ICP0 can act as or contribute to an E3 ubiquitin ligase. In this report we demonstrate that the ICP0-related RING finger proteins encoded by other alphaherpesviruses also induce colocalizing, conjugated ubiquitin, thereby suggesting that they act by similar biochemical mechanisms.     

Herpes simplex virus type 1 immediate-early protein ICP0 and is isolated RING finger domain act as ubiquitin E3 ligases in vitro.

Proteasome-dependent degradation of ubiquitinated proteins plays a key role in many important cellular processes. Ubiquitination requires the E1 ubiquitin activating enzyme, an E2 ubiquitin conjugating enzyme, and frequently a substrate-specific ubiquitin protein ligase (E3). One class of E3 ubiquitin ligases has been shown to contain a common zinc-binding RING finger motif. We have previously shown that herpes simplex virus type 1 ICP0, itself a RING finger protein, induces the proteasome-dependent degradation of several cellular proteins and induces the accumulation of colocalizing conjugated ubiquitin in vivo. We now report that both full-length ICP0 and its isolated RING finger domain induce the accumulation of polyubiquitin chains in vitro in the presence of E1 and the E2 enzymes UbcH5a and UbcH6. Mutations within the RING finger region that abolish the in vitro ubiquitination activity also cause severe reductions in ICP0 activity in other assays. We conclude that ICP0 has the potential to act as an E3 ubiquitin ligase during viral infection and to target specific cellular proteins for destruction by the 26S proteasome.     

A viral ubiquitin ligase has substrate preferential SUMO targeted ubiquitin ligase activity that counteracts intrinsic antiviral defence

Intrinsic antiviral resistance represents the first line of intracellular defence against virus infection. During herpes simplex virus type-1 (HSV-1) infection this response can lead to the repression of viral gene expression but is counteracted by the viral ubiquitin ligase ICP0. Here we address the mechanisms by which ICP0 overcomes this antiviral response. We report that ICP0 induces the widespread proteasome-dependent degradation of SUMO-conjugated proteins during infection and has properties related to those of cellular SUMO-targeted ubiquitin ligases (STUbLs). Mutation of putative SUMO interaction motifs within ICP0 not only affects its ability to degrade SUMO conjugates, but also its capacity to stimulate HSV-1 lytic infection and reactivation from quiescence. We demonstrate that in the absence of this viral countermeasure the SUMO conjugation pathway plays an important role in mediating intrinsic antiviral resistance and the repression of HSV-1 infection. Using PML as a model substrate, we found that whilst ICP0 preferentially targets SUMO-modified isoforms of PML for degradation, it also induces the degradation of PML isoform I in a SUMO modification-independent manner. PML was degraded by ICP0 more rapidly than the bulk of SUMO-modified proteins in general, implying that the identity of a SUMO-modified protein, as well as the presence of SUMO modification, is involved in ICP0 targeting. We conclude that ICP0 has dual targeting mechanisms involving both SUMO- and substrate-dependent targeting specificities in order to counteract intrinsic antiviral resistance to HSV-1 infection.     

The Viral Ubiquitin Ligase ICP0 Is neither Sufficient nor Necessary for Degradation of the Cellular DNA Sensor IFI16 during Herpes Simplex Virus 1 Infection

The cellular protein IFI16 colocalizes with the herpes simplex virus 1 (HSV-1) ubiquitin ligase ICP0 at early times of infection and is degraded as infection progresses. Here, we report that the factors governing the degradation of IFI16 and its colocalization with ICP0 are distinct from those of promyelocytic leukemia protein (PML), a well-characterized ICP0 substrate. Unlike PML, IFI16 colocalization with ICP0 was dependent on the ICP0 RING finger and did not occur when proteasome activity was inhibited. Expression of ICP0 in the absence of infection did not destabilize IFI16, the degradation occurred efficiently in the absence of ICP0 if infection was progressing efficiently, and IFI16 was relatively stable in wild-type (wt) HSV-1-infected U2OS cells. Therefore, IFI16 stability appears to be regulated by cellular factors in response to active HSV-1 infection rather than directly by ICP0. Because IFI16 is a DNA sensor that becomes associated with viral genomes during the early stages of infection, we investigated its role in the recruitment of PML nuclear body (PML NB) components to viral genomes. Recruitment of PML and hDaxx was less efficient in a proportion of IFI16-depleted cells, and this correlated with improved replication efficiency of ICP0-null mutant HSV-1. Because the absence of interferon regulatory factor 3 (IRF3) does not increase the plaque formation efficiency of ICP0-null mutant HSV-1, we speculate that IFI16 contributes to cell-mediated restriction of HSV-1 in a manner that is separable from its roles in IRF3-mediated interferon induction, but that may be linked to the PML NB response to viral infection.     

Herpes simplex virus 1 mutant in which the ICP0 HUL-1 E3 ubiquitin ligase site is disrupted stabilizes cdc34 but degrades D-type cyclins and exhibits diminished neurotoxicity

Herpes simplex virus type 1 (HSV-1) infected cell protein 0 (ICP0) is a multifunctional protein that functions as a promiscuous transactivator and promotes the degradation of multiple cellular proteins. In vitro studies indicated that it encodes two physically separated functional E3 ubiquitin ligase domains. One, designated herpesvirus ubiquitin ligase 1 (HUL-1), maps to a region encoded by exon 3 and is contained between residues 543 and 680. Deletion of amino acids 621 to 625 abolishes this activity. The second, designated HUL-2, maps to the RING finger domain present in ICP0 encoded by exon 2. Earlier studies have shown that ICP0 stabilizes cyclins D1 and D3, and several lines of investigation led to the hypothesis that this function of ICP0 is the consequence of degradation of the E2 enzyme cdc34, known to be involved in the proteasome-dependent degradation of D-type cyclins. Consistent with this hypothesis, we have previously shown that cdc34 physically interacts with ICP0 at or near aspartate 199 and at amino acids 621 to 625 and that the former site is required for effective ubiquitylation and degradation of cdc34. Furthermore, the ICP0 HUL-1 domain promotes the polyubiquitination of cdc34 in vitro. If the mechanism by which D-type cyclins are salvaged in wild-type-infected cells is dependent on polyubiquitination and consequent destruction of cdc34, than the mutant virus R6701, which was constructed for these studies and lacks ICP0 residues 621 to 625, should destabilize the D cyclins and preclude the degradation of cdc34. We report that ICP0 residues 621 to 625 are essential for degradation of cdc34 in infected cells and for the ICP0-mediated stabilization of D-type cyclins, that a mutation that specifically disrupted the ring finger domain of the HUL-2 site had no effect on the degradation of cdc34 in infected cells, and that deletion of ICP0 residues 621 to 625 decreased the replicative capacity of the virus in growth-arrested but not in dividing cells and resulted in diminished pathogenicity on intracerebral inoculation of mice. We conclude that the ICP0 HUL-1 domain acts in infected cells to degrade cdc34 and that this function requires the interaction of cdc34 with sequences in exons 2 and 3 but does not involve the HUL-2 RING finger E3 domain.     

Stabilization of the E3 ubiquitin ligase Nrdp1 by the deubiquitinating enzyme USP8

Nrdp1 is a RING finger-containing E3 ubiquitin ligase that physically interacts with and regulates steady-state cellular levels of the ErbB3 and ErbB4 receptor tyrosine kinases and has been implicated in the degradation of the inhibitor-of-apoptosis protein BRUCE. Here we demonstrate that the Nrdp1 protein undergoes efficient proteasome-dependent degradation and that mutations in its RING finger domain that disrupt ubiquitin ligase activity enhance stability. These observations suggest that Nrdp1 self-ubiquitination and stability could play an important role in regulating the activity of this protein. Using affinity chromatography, we identified the deubiquitinating enzyme USP8 (also called Ubpy) as a protein that physically interacts with Nrdp1. Nrdp1 and USP8 could be coimmunoprecipitated, and in transfected cells USP8 specifically bound to Nrdp1 but not cbl, a RING finger E3 ligase involved in ligand-stimulated epidermal growth factor receptor down-regulation. The USP8 rhodanese and catalytic domains mediated Nrdp1 binding. USP8 markedly enhanced the stability of Nrdp1, and a point mutant that disrupts USP8 catalytic activity destabilized endogenous Nrdp1. Our results indicate that Nrdp1 is a specific target for the USP8 deubiquitinating enzyme and are consistent with a model where USP8 augments Nrdp1 activity by mediating its stabilization.     

SUMO pathway dependent recruitment of cellular repressors to herpes simplex virus type 1 genomes

Components of promyelocytic leukaemia (PML) nuclear bodies (ND10) are recruited to sites associated with herpes simplex virus type 1 (HSV-1) genomes soon after they enter the nucleus. This cellular response is linked to intrinsic antiviral resistance and is counteracted by viral regulatory protein ICP0. We report that the SUMO interaction motifs of PML, Sp100 and hDaxx are required for recruitment of these repressive proteins to HSV-1 induced foci, which also contain SUMO conjugates and PIAS2β, a SUMO E3 ligase. SUMO modification of PML and elements of its tripartite motif (TRIM) are also required for recruitment in cells lacking endogenous PML. Mutants of PML isoform I and hDaxx that are not recruited to virus induced foci are unable to reproduce the repression of ICP0 null mutant HSV-1 infection mediated by their wild type counterparts. We conclude that recruitment of ND10 components to sites associated with HSV-1 genomes reflects a cellular defence against invading pathogen DNA that is regulated through the SUMO modification pathway.     

The Replication Defect of ICP0-Null Mutant Herpes Simplex Virus 1 Can Be Largely Complemented by the Combined Activities of Human Cytomegalovirus Proteins IE1 and pp71

Herpes simplex virus 1 (HSV-1) immediate-early protein ICP0 is required for efficient lytic infection and productive reactivation from latency and induces derepression of quiescent viral genomes. Despite being unrelated at the sequence level, ICP0 and human cytomegalovirus proteins IE1 and pp71 share some functional similarities in their abilities to counteract antiviral restriction mediated by components of cellular nuclear structures known as ND10. To investigate the extent to which IE1 and pp71 might substitute for ICP0, cell lines were developed that express either IE1 or pp71, or both together, in an inducible manner. We found that pp71 dissociated the hDaxx-ATRX complex and inhibited accumulation of these proteins at sites juxtaposed to HSV-1 genomes but had no effect on the promyelocytic leukemia protein (PML) or Sp100. IE1 caused loss of the small ubiquitin-like modifier (SUMO)-conjugated forms of PML and Sp100 and inhibited the recruitment of these proteins to HSV-1 genome foci but had little effect on hDaxx or ATRX in these assays. Both IE1 and pp71 stimulated ICP0-null mutant plaque formation, but neither to the extent achieved by ICP0. The combination of IE1 and pp71, however, inhibited recruitment of all ND10 proteins to viral genome foci, stimulated ICP0-null mutant HSV-1 plaque formation to near wild-type levels, and efficiently induced derepression of quiescent HSV-1 genomes. These results suggest that ND10-related intrinsic resistance results from the additive effects of several ND10 components and that the effects of IE1 and pp71 on subsets of these components combine to mirror the overall activities of ICP0.     

Herpes simplex virus type 1 ICP0 phosphorylation mutants impair the E3 ubiquitin ligase activity of ICP0 in a cell type-dependent manner

Herpes simplex virus type 1 (HSV-1) infected cell protein 0 (ICP0) is a 110-kDa nuclear phosphoprotein that is required for both the efficient initiation of lytic infection and the reactivation of quiescent viral genomes from latency. The ability of ICP0 to act as a potent viral transactivator is mediated by its N-terminal zinc-binding RING finger domain. This domain confers E3 ubiquitin ligase activity to ICP0 and is required for the proteasome-dependent degradation of a number of cellular proteins during infection, including the major nuclear domain 10 (ND10) constituent protein promyelocytic leukemia. In previous work we mapped three phosphorylation regions within ICP0, two of which directly affected its transactivation capabilities in transient transfection assays (Davido et al., J. Virol. 79:1232-1243, 2005). Because ICP0 is a phosphoprotein, we initially sought to test the hypothesis that phosphorylation regulates the E3 ubiquitin ligase activity of ICP0. Although none of the mutations affected ICP0 E3 ligase activity in vitro, transient transfection analysis indicated that mutations within one or more of the phosphorylated regions impaired the ability of ICP0 to form foci with colocalizing conjugated ubiquitin and to disrupt ND10. Mutations within one of the regions also affected ICP0 stability, and all of these phenomena occurred in a cell type-dependent manner. In the context of viral infection, only one ICP0 phosphorylation mutant (P1) showed a significant defect in viral replication and enhanced protein stability compared to all the other viruses tested. This study suggests that specific cellular environments and context of expression (transfection versus infection) differentially regulate several activities of ICP0 related to its E3 ubiquitin ligase activity via phosphorylation.