Ubiquitin-specific Protease USP7 Research Articles

Human testis–specific Y-encoded protein-like protein 5 is a histone H3/H4-specific chaperone that facilitates histone deposition in vitro

DNA and core histones are hierarchically packaged into a complex organization called chromatin. The nucleosome assembly protein (NAP) family of histone chaperones is involved in the deposition of histone complexes H2A/H2B and H3/H4 onto DNA and prevents nonspecific aggregation of histones. Testis-specific Y-encoded protein (TSPY)–like protein 5 (TSPYL5) is a member of the TSPY-like protein family, which has been previously reported to interact with ubiquitin-specific protease USP7 and regulate cell proliferation and is thus implicated in various cancers, but its interaction with chromatin has not been investigated. In this study, we characterized the chromatin association of TSPYL5 and found that it preferentially binds histone H3/H4 via its C-terminal NAP-like domain both in vitro and ex vivo. We identified the critical residues involved in the TSPYL5–H3/H4 interaction and further quantified the binding affinity of TSPYL5 toward H3/H4 using biolayer interferometry. We then determined the binding stoichiometry of the TSPYL5–H3/H4 complex in vitro using a chemical cross-linking assay and size-exclusion chromatography coupled with multiangle laser light scattering. Our results indicate that a TSPYL5 dimer binds to either two histone H3/H4 dimers or a single tetramer. We further demonstrated that TSPYL5 has a specific affinity toward longer DNA fragments and that the same histone-binding residues are also critically involved in its DNA binding. Finally, employing histone deposition and supercoiling assays, we confirmed that TSPYL5 is a histone chaperone responsible for histone H3/H4 deposition and nucleosome assembly. We conclude that TSPYL5 is likely a new member of the NAP histone chaperone family.

Proteins of the ubiquitin system in the Shoc2 ‐ ERK1/2 signaling axis and Noonan‐like syndrome with loose anagen hair (NSLAH) RASopathy

The ERK1/2 signaling pathway is critical in organismal development and tissue morphogenesis. Deregulation of this pathway leads to congenital abnormalities with severe developmental dysmorphisms, i.e. RASopathies. The core ERK1/2 cascade relies on scaffold proteins such as Shoc2 to guide and fine‐tune its signals. Mutations in shoc2 lead to the development of the pathology termed Noonan‐like Syndrome with Loose Anagen Hair (NSLAH) RASopathy. However, the mechanisms underlying the functions of Shoc2 and its contributions to disease progression remain unclear.We found that Shoc2 assembles an elegant multi‐component complex that incorporates several proteins of the ubiquitin system. To fine‐tune the amplitude of ERK1/2 signal transmitted via the complex, Shoc2 tethers the E3 ligase HUWE1, the (AAA+) ATPases, PSMC5 and VCP/p97, and the deubiquitinating enzyme, USP7. All of these enzymes are integral to the intricate feedback mechanism.Our recent studies demonstrated that ERK1/2 pathway activation triggers the interaction of Shoc2 with the ubiquitin‐specific protease USP7. We identified that in the Shoc2 module, USP7 functions as a molecular “switch” that controls the E3 ligase HUWE1 and the HUWE1‐induced regulatory feedback loop. We also found that disruption of Shoc2‐USP7 binding leads to aberrant activation of the Shoc2‐ERK1/2 axis. The zebrafish vertebrate model was then used to show that Shoc2 congenital mutations affecting Shoc2 interaction with USP7 lead to aberrant Shoc2 ubiquitination and signal transmission. Thus, our studies reveal a role for USP7 in the pathogenic mechanisms underlying NSLAH extending our understanding of how ubiquitin‐specific proteases regulate intracellular signaling.In summary, our studies are the first to demonstrate that the Shoc2 scaffold employs multi‐protein enzymatic machinery to govern the amplitude of Shoc2‐ERK1/2 signals. We also uncover novel molecular mechanisms underlying the pathogenesis of Noonan‐like syndrome with loose anagen hair. Overall, these studies significantly advance our understanding of the mechanisms by which non‐enzymatic scaffolds regulate the specificity and dynamics of the ERK1/2 signaling networks.

The role of USP7 in the Shoc2-ERK1/2 signaling axis and Noonan-like syndrome with loose anagen hair.

The ERK1/2 (also known as MAPK3 and MAPK1, respectively) signaling pathway is critical in organismal development and tissue morphogenesis. Deregulation of this pathway leads to congenital abnormalities with severe developmental dysmorphisms. The core ERK1/2 cascade relies on scaffold proteins, such as Shoc2 to guide and fine-tune its signals. Mutations in SHOC2 lead to the development of the pathology termed Noonan-like Syndrome with Loose Anagen Hair (NSLAH). However, the mechanisms underlying the functions of Shoc2 and its contributions to disease progression remain unclear. Here, we show that ERK1/2 pathway activation triggers the interaction of Shoc2 with the ubiquitin-specific protease USP7. We reveal that, in the Shoc2 module, USP7 functions as a molecular 'switch' that controls the E3 ligase HUWE1 and the HUWE1-induced regulatory feedback loop. We also demonstrate that disruption of Shoc2-USP7 binding leads to aberrant activation of the Shoc2-ERK1/2 axis. Importantly, our studies reveal a possible role for USP7 in the pathogenic mechanisms underlying NSLAH, thereby extending our understanding of how ubiquitin-specific proteases regulate intracellular signaling.

Bimodal regulation of the PRC2 complex by USP7 underlies tumorigenesis.

Although overexpression of EZH2, a catalytic subunit of the polycomb repressive complex 2 (PRC2), is an eminent feature of various cancers, the regulation of its abundance and function remains insufficiently understood. We report here that the PRC2 complex is physically associated with ubiquitin-specific protease USP7 in cancer cells where USP7 acts to deubiquitinate and stabilize EZH2. Interestingly, we found that USP7-catalyzed H2BK120ub1 deubiquitination is a prerequisite for chromatin loading of PRC2 thus H3K27 trimethylation, and this process is not affected by H2AK119 ubiquitination catalyzed by PRC1. Genome-wide analysis of the transcriptional targets of the USP7/PRC2 complex identified a cohort of genes including FOXO1 that are involved in cell growth and proliferation. We demonstrated that the USP7/PRC2 complex drives cancer cell proliferation and tumorigenesis in vitro and in vivo. We showed that the expression of both USP7 and EZH2 elevates during tumor progression, corresponding to a diminished FOXO1 expression, and the level of the expression of USP7 and EZH2 strongly correlates with histological grades and prognosis of tumor patients. These results reveal a dual role for USP7 in the regulation of the abundance and function of EZH2, supporting the pursuit of USP7 as a therapeutic target for cancer intervention.

Deubiquitinase USP7 regulates Drosophila aging through ubiquitination and autophagy.

Ubiquitination-mediated protein degradation is the selective degradation of diverse forms of damaged proteins that are tagged with ubiquitin, while deubiquitinating enzymes reverse ubiquitination-mediated protein degradation by removing the ubiquitin chain from the target protein. The interactions of ubiquitinating and deubiquitinating enzymes are required to maintain protein homeostasis. The ubiquitin-specific protease USP7 is a deubiquitinating enzyme that indirectly plays a role in repairing DNA damage and development. However, the mechanism of its participation in aging has not been fully explored. Regarding this issue, we found that USP7 was necessary to maintain the normal lifespan of Drosophila melanogaster, and knockdown of dusp7 shortened the lifespan and reduced the ability of Drosophila to cope with starvation, oxidative stress and heat stress. Furthermore, we showed that the ability of USP7 to regulate aging depends on the autophagy and ubiquitin signaling pathways. Furthermore, 2,5-dimethyl-celecoxib (DMC), a derivative of celecoxib, can partially restore the shortened lifespan and aberrant phenotypes caused by dusp7 knockdown. Our results suggest that USP7 is an important factor involved in the regulation of aging, and related components in this regulatory pathway may become new targets for anti-aging treatments.

USP7 regulates ALS-associated proteotoxicity and quality control through the NEDD4L–SMAD pathway

An imbalance in cellular homeostasis occurring as a result of protein misfolding and aggregation contributes to the pathogeneses of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Here, we report the identification of a ubiquitin-specific protease, USP7, as a regulatory switch in a protein quality-control system that defends against proteotoxicity. A genome-wide screen in a Caenorhabditis elegans model of SOD1-linked ALS identified the USP7 ortholog as a suppressor of proteotoxicity in the nervous system. The actions of USP7 orthologs on misfolded proteins were found to be conserved in Drosophila and mammalian cells. USP7 acts on protein quality control through the SMAD2 transcription modulator of the transforming growth factor β pathway, which activates autophagy and enhances the clearance of misfolded proteins. USP7 deubiquitinates the E3 ubiquitin ligase NEDD4L, which mediates the degradation of SMAD2. Inhibition of USP7 protected against proteotoxicity in mammalian neurons, and SMAD2 was found to be dysregulated in the nervous systems of ALS patients. These findings reveal a regulatory pathway of protein quality control that is implicated in the proteotoxicity-associated neurodegenerative diseases.

The deubiquitinase USP7 stabilizes Maf proteins to promote myeloma cell survival

The Maf proteins, including c-Maf, MafA, and MafB, are critical transcription factors in myelomagenesis. Previous studies demonstrated that Maf proteins are processed by the ubiquitin-proteasome pathway, but the mechanisms remain elusive. This study applied MS to identify MafB ubiquitination-associated proteins and found that the ubiquitin-specific protease USP7 was present in the MafB interactome. Moreover, USP7 also interacted with c-Maf and MafA and blocked their polyubiquitination and degradation. Consistently, knockdown of USP7 resulted in Maf protein degradation along with increased polyubiquitination levels. The action of USP7 thus promoted Maf transcriptional activity as evidenced by luciferase assays and by the up-regulation of the expression of Maf-modulated genes. Furthermore, USP7 was up-regulated in myeloma cells, and it was negatively associated with the survival of myeloma patients. USP7 promoted myeloma cell survival, and when it was inhibited by its specific inhibitor P5091, myeloma cell lines underwent apoptosis. These results therefore demonstrated that USP7 is a deubiquitinase of Maf proteins and promotes MM cell survival in association with Maf stability. Given the significance of USP7 and Maf proteins in myeloma genesis, targeting the USP7/Maf axle is a potential strategy to the precision therapy of MM.

A feedforward circuit shaped by ECT2 and USP7 contributes to breast carcinogenesis.

Rationale: A number of guanine nucleotide exchange factors (GEFs) including epithelial cell transforming factor ECT2 are believed to drive carcinogenesis through activating distinct oncogenic GTPases. Yet, whether GEF-independent activity of ECT2 also plays a role in tumorigenesis remains unclear.Methods: Immunohistochemical (IHC) staining, colony formation and xenograft assays were used to examine the role of ECT2 in breast carcinogenesis. Co-immunoprecipitation, immunofluorescent stainings, in vivo deubiquitination and in vitro deubiquitination experiments were performed to examine the physical and functional interaction between ECT2 and ubiquitin-specific protease USP7. High-throughput RNA sequencing, quantitative reverse transcription-PCR and Western blotting were employed to investigate the biological significance of the interplay between ECT2 and USP7.Results: We report that ECT2 plays a tumor-promoting role in breast cancer, and GEF activity-deficient ECT2 is able to alleviate ECT2 depletion associated growth defects in breast cancer cells. Mechanistically, we demonstrated that ECT2 physically interacts with ubiquitin-specific protease USP7 and functionally facilitates USP7 intermolecular self-association, -deubiquitination and -stabilization in a GEF activity-independent manner. USP7 in turn, deubiquitinates and stabilizes ECT2, resulting in a feedforward regulatory circuit that ultimately sustains the expression of oncogenic protein MDM2.Conclusion: Our study uncovers a GEF-independent role of ECT2 in promoting survival of breast cancer cells, provides a molecular insight for the reciprocal regulation of ECT2 and USP7, and supports the pursuit of ECT2/USP7 as potential targets for breast cancer intervention.

USP7 Deubiquitinates and Stabilizes SIRT1

The NAD+ -dependent protein deacetylase silent information regulator 1 (SIRT1) targets multiple proteins for deacetylation, and it has been implicated in a variety of cellular pathways and human diseases. However, it remains unclear how the abundance of SIRT1 is regulated. Here, by mass spectrometry analysis of SIRT1-containing protein complexes, we revealed that SIRT1 is physically associated with the ubiquitin-specific protease USP7. Importantly, we found that USP7 cleaves K48-linked polyubiquitin chains of SIRT1 and promotes SIRT1 stabilization. Accordingly, we demonstrated that treatment of cells with an enzymatic inhibitor of USP7 led to a decreased level of SIRT1 expression and accumulation of SIRT1 polyubiquitination. Collectively, our findings indicate that USP7 is a critical regulator of SIRT1 and provide a new pathway for the maintenance of SIRT1 abundance in cells. Anat Rec, 303:1337-1345, 2020. © 2019 American Association for Anatomy.

Usp7 regulates Hippo pathway through deubiquitinating the transcriptional coactivator Yorkie

The Hippo pathway plays an important role in organ development and adult tissue homeostasis, and its deregulation has been implicated in many cancers. The Hippo signaling relies on a core kinase cascade culminating in phosphorylation of the transcription coactivator Yorkie (Yki). Although Yki is the key effector of Hippo pathway, the regulation of its protein stability is still unclear. Here, we show that Hippo pathway attenuates the binding of a ubiquitin-specific protease Usp7 to Yki, which regulates Hippo signaling through deubiquitinating Yki. Furthermore, the mammalian homolog of Usp7, HAUSP plays a conserved role in regulating Hippo pathway by modulating Yap ubiquitination and degradation. Finally, we find that the expression of HAUSP is positively correlated with that of Yap, both showing upregulated levels in clinical hepatocellular carcinoma (HCC) specimens. In summary, our findings demonstrate that Yki/Yap is stabilized by Usp7/HAUSP, and provide HAUSP as a potential therapeutic target for HCC.

Ubiquitin-specific protease 7 sustains DNA damage response and promotes cervical carcinogenesis.

Central to the recognition, signaling, and repair of DNA double-strand breaks (DSBs) are the MRE11-RAD50-NBS1 (MRN) complex and mediator of DNA damage checkpoint protein 1 (MDC1), the interplay of which is essential for initiation and amplification of the DNA damage response (DDR). The intrinsic rule governing the regulation of the function of this molecular machinery remains to be investigated. We report here that the ubiquitin-specific protease USP7 was physically associated with the MRN-MDC1 complex and that the MRN-MDC1 complex acted as a platform for USP7 to efficiently deubiquitinate and stabilize MDC1, thereby sustaining the DDR. Accordingly, depletion of USP7 impaired the engagement of the MRN-MDC1 complex and the consequent recruitment of the downstream factors p53-binding protein 1 (53BP1) and breast cancer protein 1 (BRCA1) at DNA lesions. Significantly, USP7 was overexpressed in cervical cancer, and the level of its expression positively correlated with that of MDC1 and worse survival rates for patients with cervical cancer. We demonstrate that USP7-mediated MDC1 stabilization promoted cervical cancer cell survival and conferred cellular resistance to genotoxic insults. Together, our study reveals a role for USP7 in regulating the function of the MRN-MDC1 complex and activity of the DDR, supporting the pursuit of USP7 as a potential therapeutic target for MDC1-proficient cancers.

Dual-utility NLS drives RNF169-dependent DNA damage responses

Loading of p53-binding protein 1 (53BP1) and receptor-associated protein 80 (RAP80) at DNA double-strand breaks (DSBs) drives cell cycle checkpoint activation but is counterproductive to high-fidelity DNA repair. ring finger protein 169 (RNF169) maintains the balance by limiting the deposition of DNA damage mediator proteins at the damaged chromatin. We report here that this attribute is accomplished, in part, by a predicted nuclear localization signal (NLS) that not only shuttles RNF169 into the nucleus but also promotes its stability by mediating a direct interaction with the ubiquitin-specific protease USP7. Guided by the crystal structure of USP7 in complex with the RNF169 NLS, we uncoupled USP7 binding from its nuclear import function and showed that perturbing the USP7-RNF169 complex destabilized RNF169, compromised high-fidelity DSB repair, and hypersensitized cells to poly (ADP-ribose) polymerase inhibition. Finally, expression of USP7 and RNF169 positively correlated in breast cancer specimens. Collectively, our findings uncover an NLS-mediated bipartite mechanism that supports the nuclear function of a DSB response protein.

Usp7 promotes medulloblastoma cell survival and metastasis by activating Shh pathway

The ubiquitin-specific protease Usp7 plays roles in multiple cellular processes through deubiquitinating and stabilizing numerous substrates, including P53, Pten and Gli. Aberrant Usp7 activity has been implicated in many disorders and tumorigenesis, making it as a potential target for therapeutic intervention. Although it is clear that Usp7 is involved in many types of cancer, its role in regulating medulloblastoma (MB) is still unknown. In this study, we show that knockdown of Usp7 inhibits the proliferation and migration of MB cells, while Usp7 overexpression exerts an opposite effect. Furthermore, we establish Usp7 knockout MB cell line using the CRISPR/Cas9 system and further confirm that Usp7 knockout also blocks MB cell proliferation and metastasis. In addition, we reveal that knockdown of Usp7 compromises Shh pathway activity and decrease Gli protein levels, while P53 level and P53 target gene expression have no obvious changes. Finally, we find that Usp7 inhibitors apparently inhibit MB cell viability and migration. Taken together, our findings suggest that Usp7 is important for MB cell proliferation and metastasis by activating Shh pathway, and is a putative therapeutic target for MBs.

STIP is a critical nuclear scaffolding protein linking USP7 to p53-Mdm2 pathway regulation.

The ubiquitin-specific protease USP7 stabilizes both Mdm2 and p53 by removing ubiquitins, hence playing an important enzymatic role in the p53-Mdm2 pathway. However, it is poorly understood how USP7 executes its dual-stabilization effect on Mdm2 and p53 in cellular context. Here, we report that STIP is a novel macromolecular scaffold that links USP7 to the p53-Mdm2 pathway. STIP and a fraction of USP7 interact and constitutively colocalize in nucleoplasma. Overexpression of STIP stabilizes Mdm2 and p53, whereas downregulation of STIP decreases Mdm2 and p53 levels. The effect of STIP on Mdm2 and p53 depends on USP7 function as a deubiquitinating enzyme. Furthermore, we demonstrate that STIP mediates the assembly of two separate ternary protein complexes in vivo as STIP-USP7-Mdm2 and STIP-USP7-p53, which facilitates USP7-mediated stabilization of Mdm2 and p53. Collectively, these results pinpoint a new molecular function of STIP and reveal a novel mechanism whereby USP7 executes its dual-stabilization effect on Mdm2 and p53 via STIP scaffolding.

Deubiquitination of Ci/Gli by Usp7/HAUSP Regulates Hedgehog Signaling

Hedgehog (Hh) signaling plays essential roles in animal development and tissue homeostasis, and its misregulation causes congenital diseases and cancers. Regulation of the ubiquitin/proteasome-mediated proteolysis of Ci/Gli transcription factors is central to Hh signaling, but whether deubiquitinase is involved in this process remains unknown. Here, we show that Hh stimulates the binding of a ubiquitin-specific protease Usp7 to Ci, which positively regulates Hh signaling activity through inhibiting Ci ubiquitination and degradation mediated by both Slimb-Cul1 and Hib-Cul3 E3 ligases. Furthermore, we find that Usp7 forms a complex with GMP-synthetase (GMPS) to promote Hh pathway activity. Finally, we show that the mammalian counterpart of Usp7, HAUSP, positively regulates Hh signaling by modulating Gli ubiquitination and stability. Our findings reveal a conserved mechanism by which Ci/Gli is stabilized by a deubiquitination enzyme and identify Usp7/HUASP as a critical regulator of Hh signaling and potential therapeutic target for Hh-related cancers.

Identification of TRIM27 as a novel degradation target of herpes simplex virus 1 ICP0.

The herpes simplex virus 1 (HSV-1) immediate early protein ICP0 performs many functions during infection, including transactivation of viral gene expression, suppression of innate immune responses, and modification and eviction of histones from viral chromatin. Although these functions of ICP0 have been characterized, the detailed mechanisms underlying ICP0's complex role during infection warrant further investigation. We thus undertook an unbiased proteomic approach to identifying viral and cellular proteins that interact with ICP0 in the infected cell. Cellular candidates resulting from our analysis included the ubiquitin-specific protease USP7, the transcriptional repressor TRIM27, DNA repair proteins NBN and MRE11A, regulators of apoptosis, including BIRC6, and the proteasome. We also identified two HSV-1 early proteins involved in nucleotide metabolism, UL39 and UL50, as novel candidate interactors of ICP0. Because TRIM27 was the most statistically significant cellular candidate, we investigated the relationship between TRIM27 and ICP0. We observed rapid, ICP0-dependent loss of TRIM27 during HSV-1 infection. TRIM27 protein levels were restored by disrupting the RING domain of ICP0 or by inhibiting the proteasome, arguing that TRIM27 is a novel degradation target of ICP0. A mutant ICP0 lacking E3 ligase activity interacted with endogenous TRIM27 during infection as demonstrated by reciprocal coimmunoprecipitation and supported by immunofluorescence data. Surprisingly, ICP0-null mutant virus yields decreased upon TRIM27 depletion, arguing that TRIM27 has a positive effect on infection despite being targeted for degradation. These results illustrate a complex interaction between TRIM27 and viral infection with potential positive or negative effects of TRIM27 on HSV under different infection conditions. During productive infection, a virus must simultaneously redirect multiple cellular pathways to replicate itself while evading detection by the host's defenses. To orchestrate such complex regulation, viruses, including herpes simplex virus 1 (HSV-1), rely on multifunctional proteins such as the E3 ubiquitin ligase ICP0. This protein regulates various cellular pathways concurrently by targeting a diverse set of cellular factors for degradation. While some of these targets have been previously identified and characterized, we undertook a proteomic screen to identify additional targets of this activity to further characterize ICP0's role during infection. We describe a set of candidate interacting proteins of ICP0 identified through this approach and our characterization of the most statistically significant result, the cellular transcriptional repressor TRIM27. We present TRIM27 as a novel degradation target of ICP0 and describe the relationship of these two proteins during infection.

A Role for USP7 in DNA Replication

The minichromosome maintenance (MCM) complex, which plays multiple important roles in DNA replication, is loaded onto chromatin following mitosis, remains on chromatin until the completion of DNA synthesis, and then is unloaded by a poorly defined mechanism that involves the MCM binding protein (MCM-BP). Here we show that MCM-BP directly interacts with the ubiquitin-specific protease USP7, that this interaction occurs predominantly on chromatin, and that MCM-BP can tether USP7 to MCM proteins. Detailed biochemical and structure analyses of the USP7-MCM-BP interaction showed that the (155)PSTS(158) MCM-BP sequence mediates critical interactions with the TRAF domain binding pocket of USP7. Analysis of the effects of USP7 knockout on DNA replication revealed that lack of USP7 results in slowed progression through late S phase without globally affecting the fork rate or origin usage. Lack of USP7 also resulted in increased levels of MCM proteins on chromatin, and investigation of the cause of this increase revealed a defect in the dissociation of MCM proteins from chromatin in mid- to late S phase. This role of USP7 mirrors the previously described role for MCM-BP in MCM complex unloading and suggests that USP7 works with MCM-BP to unload MCM complexes from chromatin at the end of S phase.

Ubiquitination-Deubiquitination by the TRIM27-USP7 Complex Regulates Tumor Necrosis Factor Alpha-Induced Apoptosis

Tumor necrosis factor alpha (TNF-α) plays a role in apoptosis and proliferation in multiple types of cells, and defects in TNF-α-induced apoptosis are associated with various autoimmune diseases. Here, we show that TRIM27, a tripartite motif (TRIM) protein containing RING finger, B-box, and coiled-coil domains, positively regulates TNF-α-induced apoptosis. Trim27-deficient mice are resistant to TNF-α-d-galactosamine-induced hepatocyte apoptosis. Trim27-deficient mouse embryonic fibroblasts (MEFs) are also resistant to TNF-α-cycloheximide-induced apoptosis. TRIM27 forms a complex with and ubiquitinates the ubiquitin-specific protease USP7, which deubiquitinates receptor-interacting protein 1 (RIP1), resulting in the positive regulation of TNF-α-induced apoptosis. Our findings indicate that the ubiquitination-deubiquitination cascade mediated by the TRIM27-USP7 complex plays an important role in TNF-α-induced apoptosis.

The Ubiquitin-Specific Protease USP7 Modulates the Replication of Kaposi's Sarcoma-Associated Herpesvirus Latent Episomal DNA

Kaposi's sarcoma herpesvirus (KSHV) belongs to the gamma-2 Herpesviridae and is associated with three neoplastic disorders: Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease (MCD). The viral latency-associated nuclear antigen 1 (LANA) is expressed in all latently KSHV-infected cells and is involved in viral latent replication and maintenance of the viral genome. We show that LANA interacts with the ubiquitin-specific protease USP7 through its N-terminal TRAF (tumor necrosis factor [TNF] receptor-associated factor) domain. This interaction involves a short sequence (amino acids [aa] 971 to 986) within the C-terminal domain of LANA with strong similarities to the USP7 binding site of the Epstein-Barr virus (EBV) EBNA-1 protein. A LANA mutant with a deletion of the identified USP7 binding site showed an enhanced ability to replicate a plasmid containing the KSHV latent origin of replication but was comparable to the wild-type LANA (LANA WT) with regard to the regulation of viral and cellular promoters. Furthermore, the LANA homologues of two other gamma-2 herpesviruses, MHV68 and RRV, also recruit USP7. Our findings suggest that recruitment of USP7 to LANA could play a role in the regulation of viral latent replication. The recruitment of USP7, and its role in herpesvirus latent replication, previously described for the latent EBNA-1 protein of the gamma-1 herpesvirus (lymphocryptovirus) EBV (M. N. Holowaty et al., J. Biol. Chem. 278:29987-29994, 2003), may thereby be a conserved feature among gammaherpesvirus latent origin binding proteins.

Proteomic Profiling of the Human Cytomegalovirus UL35 Gene Products Reveals a Role for UL35 in the DNA Repair Response

Human cytomegalovirus infections involve the extensive modification of host cell pathways, including cell cycle control, the regulation of the DNA damage response, and averting promyelocytic leukemia (PML)-mediated antiviral responses. The UL35 gene from human cytomegalovirus is important for viral gene expression and efficient replication and encodes two proteins, UL35 and UL35a, whose mechanism of action is not well understood. Here, affinity purification coupled with mass spectrometry was used to identify previously unknown human cellular targets of UL35 and UL35a. We demonstrate that both viral proteins interact with the ubiquitin-specific protease USP7, and that UL35 expression can alter USP7 subcellular localization. In addition, UL35 (but not UL35a) was found to associate with three components of the Cul4(DCAF1) E3 ubiquitin ligase complex (DCAF1, DDB1, and DDA1) previously shown to be targeted by the HIV-1 Vpr protein. The coimmunoprecipitation and immunofluorescence microscopy of DCAF1 mutants revealed that the C-terminal region of DCAF1 is required for association with UL35 and mediates the dramatic relocalization of DCAF1 to UL35 nuclear bodies, which also contain conjugated ubiquitin. As previously reported for the Vpr-DCAF1 interaction, UL35 (but not UL35a) expression resulted in the accumulation of cells in the G(2) phase of the cell cycle, which is typical of a DNA damage response, and activated the G(2) checkpoint in a DCAF1-dependent manner. In addition, UL35 (but not UL35a) induced γ-H2AX and 53BP1 foci, indicating the activation of DNA damage and repair responses. Therefore, the identified interactions suggest that UL35 can contribute to viral replication through the manipulation of host responses.