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.     

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.     

Depletion of intracellular zinc inhibits the ubiquitin ligase activity of viral regulatory protein ICP0 and restricts herpes simplex virus 1 replication in cell culture

The viral ubiquitin ligase ICP0 stimulates the onset of HSV-1 lytic infection and productive reactivation of viral genomes from latency. In order to mediate these processes, it requires its C3HC4 RING finger domain, a tertiary structural fold that is coordinated by the binding of two zinc (Zn(2+)) atoms. Here we formally demonstrate that Zn(2+) binding and intracellular Zn(2+) levels are critical for ICP0's biochemical activity and that depletion of intracellular Zn(2+) severely attenuates HSV-1 replication.     

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 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.     

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.     

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.     

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.     

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.     

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.     

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.     

Insights into Ubiquitination from the Unique Clamp-like Binding of the RING E3 AO7 to the E2 UbcH5B

RING proteins constitute the largest class of E3 ubiquitin ligases. Unlike most RINGs, AO7 (RNF25) binds the E2 ubiquitin-conjugating enzyme, UbcH5B (UBE2D2), with strikingly high affinity. We have defined, by co-crystallization, the distinctive means by which AO7 binds UbcH5B. AO7 contains a structurally unique UbcH5B binding region (U5BR) that is connected by an 11-amino acid linker to its RING domain, forming a clamp surrounding the E2. The U5BR interacts extensively with a region of UbcH5B that is distinct from both the active site and the RING-interacting region, referred to as the backside of the E2. An apparent paradox is that the high-affinity binding of the AO7 clamp to UbcH5B, which is dependent on the U5BR, decreases the rate of ubiquitination. We establish that this is a consequence of blocking the stimulatory, non-covalent, binding of ubiquitin to the backside of UbcH5B. Interestingly, when non-covalent backside ubiquitin binding cannot occur, the AO7 clamp now enhances the rate of ubiquitination. The high-affinity binding of the AO7 clamp to UbcH5B has also allowed for the co-crystallization of previously described and functionally important RING mutants at the RING-E2 interface. We show that mutations having marked effects on function only minimally affect the intermolecular interactions between the AO7 RING and UbcH5B, establishing a high degree of complexity in activation through the RING-E2 interface.     

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 ubiquitin-conjugating enzymes UbcH7 and UbcH8 interact with RING finger/IBR motif-containing domains of HHARI and H7-AP1.

Ubiquitinylation of proteins appears to be mediated by the specific interplay between ubiquitin-conjugating enzymes (E2s) and ubiquitin-protein ligases (E3s). However, cognate E3s and/or substrate proteins have been identified for only a few E2s. To identify proteins that can interact with the human E2 UbcH7, a yeast two-hybrid screen was performed. Two proteins were identified and termed human homologue of Drosophila ariadne (HHARI) and UbcH7-associated protein (H7-AP1). Both proteins, which are widely expressed, are characterized by the presence of RING finger and in between RING fingers (IBR) domains. No other overt structural similarity was observed between the two proteins. In vitro binding studies revealed that an N-terminal RING finger motif (HHARI) and the IBR domain (HHARI and H7-AP1) are involved in the interaction of these proteins with UbcH7. Furthermore, binding of these two proteins to UbcH7 is specific insofar that both HHARI and H7-AP1 can bind to the closely related E2, UbcH8, but not to the unrelated E2s UbcH5 and UbcH1. Although it is not clear at present whether HHARI and H7-AP1 serve, for instance, as substrates for UbcH7 or represent proteins with E3 activity, our data suggests that a subset of RING finger/IBR proteins are functionally linked to the ubiquitin/proteasome pathway.     

UBE1L2, a novel E1 enzyme specific for ubiquitin

UBE1 is known as the human ubiquitin-activating enzyme (E1), which activates ubiquitin in an ATP-dependent manner. Here, we identified a novel human ubiquitin-activating enzyme referred to as UBE1L2, which also shows specificity for ubiquitin. The UBE1L2 sequence displays a 40% identity to UBE1 and also contains an ATP-binding domain and an active site cysteine conserved among E1 family proteins. UBE1L2 forms a covalent link with ubiquitin in vitro and in vivo, which is sensitive to reducing conditions. In an in vitro polyubiquitylation assay, recombinant UBE1L2 could activate ubiquitin and transfer it onto the ubiquitin-conjugating enzyme UbcH5b. Ubiquitin activated by UBE1L2 could be used for ubiquitylation of p53 by MDM2 and supported the autoubiquitylation of the E3 ubiquitin ligases HectH9 and E6-AP. The UBE1L2 mRNA is most abundantly expressed in the testis, suggesting an organ-specific regulation of ubiquitin activation.     

Functional interactions between ubiquitin E2 enzymes and TRIM proteins

The TRIM (tripartite motif) family of proteins is characterized by the presence of the tripartite motif module, composed of a RING domain, one or two B-box domains and a coiled-coil region. TRIM proteins are involved in many cellular processes and represent the largest subfamily of RING-containing putative ubiquitin E3 ligases. Whereas their role as E3 ubiquitin ligases has been presumed, and in several cases established, little is known about their specific interactions with the ubiquitin-conjugating E2 enzymes or UBE2s. In the present paper, we report a thorough screening of interactions between the TRIM and UBE2 families. We found a general preference of the TRIM proteins for the D and E classes of UBE2 enzymes, but we also revealed very specific interactions between TRIM9 and UBE2G2, and TRIM32 and UBE2V1/2. Furthermore, we demonstrated that the TRIM E3 activity is only manifest with the UBE2 with which they interact. For most specific interactions, we could also observe subcellular co-localization of the TRIM involved and its cognate UBE2 enzyme, suggesting that the specific selection of TRIM-UBE2 pairs has physiological relevance. Our findings represent the basis for future studies on the specific reactions catalysed by the TRIM E3 ligases to determine the fate of their targets.