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.     

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.     

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.     

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.     

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.     

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.     

Secretory protein with RING finger domain (SPRING) specific to Trypanosoma cruzi is directed, as a ubiquitin ligase related protein, to the nucleus of host cells

While some intracellular bacterial and viral proteins secreted into host cell possess ubiquitin ligase (E3) activity for their profit, it has not been reported whether intracellular parasites secrete such molecules. We identified a gene that encodes a protein containing a secretory signal peptide and a RING finger domain in the intracellular protozoan parasite, Trypanosoma cruzi . This gene was specific to T. cruzi and was designated spring ( secretory p rotein with RING finger domain). An in vitro ubiquitination assay showed that SPRING possessed E3 activity in a RING finger domain-dependent manner. SPRING could utilize human ubiquitin-activating enzymes (E2), UbcH5 and UbcH13. Although SPRING was found to be a secretory protein, the signal peptide-cleaved mature form of SPRING was localized in the nucleus of host cells, indicating that SPRING may function in the host cell nuclei. Yeast two-hybrid screening identified 52 putative SPRING interactors in HeLa cells, suggesting that SPRING affects the stability or function of a number of host proteins. Furthermore, a co-immunoprecipitation assay showed that breast cancer-associated protein 3 interacted with SPRING, as well as being ubiquitinated by SPRING in vitro . These findings are the first to show that this protozoan parasite secretes an ubiquitin ligase-related protein into host cells.     

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.     

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.     

NK lytic-associated molecule, involved in NK cytotoxic function, is an E3 ligase

NK lytic-associated molecule (NKLAM) is a protein involved in the cytolytic function of NK cells and CTLs. It has been localized to the cytolytic granules in NK cells and is up-regulated when cells are exposed to cytokines IL-2 or IFN-beta. We report in this study that NKLAM contains a cysteine-rich really interesting new gene (RING) in between RING-RING domain, and that this domain possesses strong homology to the RING domain of the known E3 ubiquitin ligase, Dorfin. To determine whether NKLAM functions as an E3 ligase, we performed coimmunoprecipitation binding assays with ubiquitin conjugates (Ubcs) UbcH7, UbcH8, and UbcH10. We demonstrated that both UbcH7 and UbcH8 bind to full-length NKLAM. We then performed a similar binding assay using endogenous NKLAM and UbcH8 expressed by human NK clone NK3.3 to show that the protein interaction occurs in vivo. Using the yeast two-hybrid system, we identified uridine kinase like-1 (URKL-1) protein as a substrate for NKLAM. We confirmed that NKLAM and URKL-1 interact in mammalian cells by using both immunoprecipitation and confocal microscopy. We demonstrated decreased protein expression and enhanced ubiquitination of URKL-1 in the presence of NKLAM. These data indicate that NKLAM is a RING finger protein that binds Ubcs and has as one of its substrates, URKL-1, thus defining this cytolytic protein as an E3 ubiquitin ligase.     

Features of the parkin/ariadne-like ubiquitin ligase, HHARI, that regulate its interaction with the ubiquitin-conjugating enzyme, Ubch7

We recently reported the identification of a RING finger-containing protein, HHARI (human homologue of Drosophila ariadne), which binds to the human ubiquitin-conjugating enzyme UbcH7 in vitro. We now demonstrate that HHARI interacts and co-localizes with UbcH7 in mammalian cells, particularly in the perinuclear region. We have further defined a minimal interaction region of HHARI comprising residues 186-254, identified individual amino acid residues essential for the interaction, and determined that the distance between the RING1 finger and IBR (in between RING fingers) domains is critical to maintaining binding. We have also established that the RING1 finger of HHARI cannot be substituted for by the highly homologous RING finger domains of either of the ubiquitin-protein ligase components c-CBL or Parkin, despite their similarity in structure and their independent capabilities to bind UbcH7. Furthermore, mutation of the RING1 finger domain of HHARI from a RING-HC to a RING-H2 type abolishes interaction with UbcH7. These studies demonstrate that very subtle changes to the domains that regulate recognition between highly conserved components of the ubiquitin pathway can dramatically affect their ability to interact.     

The poxvirus p28 virulence factor is an E3 ubiquitin ligase

A majority of the orthopoxviruses, including the variola virus that causes the dreaded smallpox disease, encode a highly conserved 28-kDa protein with a classic RING finger sequence motif (C(3)HC(4)) at their carboxyl-terminal domains. The RING domain of p28 has been shown to be a critical determinant of viral virulence for the ectromelia virus (mousepox virus) in a murine infection model (Senkevich, T. G., Koonin, E. V., and Buller, R. M. (1994) Virology 198, 118-128). Here, we demonstrate that the p28 proteins encoded by the ectromelia virus and the variola virus possess E3 ubiquitin ligase activity in biochemical assays as well as in cultured mammalian cells. Point mutations disrupting the RING finger domain of p28 completely abolish its E3 ligase activity. In addition, p28 functions cooperatively with Ubc4 and UbcH5c, the E2 conjugating enzymes involved in 26 S proteasome degradation of protein targets. Moreover, p28 catalyzes the formation of Lys-63-linked polyubiquitin chains in the presence of Ubc13/Uev1A, a heterodimeric E2 conjugating enzyme, indicating that p28 may regulate the biological activity of its cognate viral and/or host cell target(s) by Lys-63-linked ubiquitin multimers. We thus conclude that the poxvirus p28 virulence factor is a new member of the RING finger E3 ubiquitin ligase family and has a unique polyubiquitylation activity. We propose that the E3 ligase activity of the p28 virulence factor may be targeted for therapeutic intervention against infections by the variola virus and other poxviruses.     

The p53-inducible E3 ubiquitin ligase p53RFP induces p53-dependent apoptosis

Recently, it has been shown that really interesting new gene (RING)-in between ring finger (IBR)-RING domain-containing proteins, such as Parkin and Parc, are E3 ubiquitin ligases and are involved in regulation of apoptosis. In this report, we show that p53-inducible RING-finger protein (p53RFP), a p53-inducible E3 ubiquitin ligase, induces p53-dependent but caspase-independent apoptosis. p53RFP contains an N-terminal RING-IBR-RING domain and an uncharacterized, evolutionally highly conserved C-terminal domain. p53RFP interacts with E2 ubiquitin-conjugating enzymes UbcH7 and UbcH8 but not with UbcH5, and this interaction is mediated through the RING-IBR-RING domain of p53RFP. Interestingly, the conserved C-terminal domain of p53RFP is required and sufficient for p53RFP-mediated apoptosis, suggesting p53RFP-mediated apoptosis does not require its E3 ubiquitin ligase activity. Together with a recent report showing that p53RFP is involved in ubiquitination and degradation of p21, a p53 downstream protein promoting growth arrest and antagonizing apoptosis, our findings suggest that p53RFP is involved in switching a cell from p53-mediated growth arrest to apoptosis.     

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.     

Topors acts as a SUMO-1 E3 ligase for p53 in vitro and in vivo

Human Topors, which was originally identified as cellular binding partner of DNA topoisomerase I and of p53, has recently been shown to function as an ubiquitin E3 ligase for p53 in a manner dependent on its N'-terminally located RING finger. Here, we demonstrate that Topors also enhances the conjugation of the small ubiquitin-like modifier 1 (SUMO-1) to p53 in vivo and in a reconstituted in vitro system. The Topors SUMO-1 E3 ligase activity does not depend upon its RING finger motif. In HeLa cells, Topors induced p53 sumoylation was accompanied by an increase in endogenous p53 protein levels. Furthermore, Topors enhances the sumoylation of a variety of other, yet unidentified, cellular proteins.