Degradation of TRIM32 is induced by RTA for Kaposi's sarcoma-associated herpesvirus lytic replication.

TRIM32 is often aberrantly expressed in many types of cancers. Kaposi's sarcoma-associated herpesvirus (KSHV) is linked with several human malignancies, including Kaposi's sarcoma and primary effusion lymphomas (PELs). Increasing evidence has demonstrated the crucial role of KSHV lytic replication in viral tumorigenesis. However, the role of TRIM32 in herpesvirus lytic replication remains unclear. Here, we reveal that the expression of TRIM32 is upregulated by KSHV in latency, and reactivation of KSHV lytic replication leads to the inhibition of TRIM32 in PEL cells. Strikingly, RTA, the master regulator of lytic replication, interacts with TRIM32 and dramatically promotes TRIM32 for degradation via the proteasome systems. Inhibition of TRIM32 induces cell apoptosis and in turn inhibits the proliferation and colony formation of KSHV-infected PEL cells and facilitates the reactivation of KSHV lytic replication and virion production. Thus, our data imply that the degradation of TRIM32 is vital for the lytic activation of KSHV and is a potential therapeutic target for KSHV-associated cancers. IMPORTANCE: TRIM32 is associated with many cancers and viral infections; however, the role of TRIM32 in viral oncogenesis remains largely unknown. In this study, we found that the expression of TRIM32 is elevated by Kaposi's sarcoma-associated herpesvirus (KSHV) in latency, and RTA (the master regulator of lytic replication) induces TRIM32 for proteasome degradation upon viral lytic reactivation. This finding provides a potential therapeutic target for KSHV-associated cancers.

Broad-spectrum vaccine via combined immunization routes triggers potent immunity to SARS-CoV-2 and its variants.

IMPORTANCE: The development of broad-spectrum SARS-CoV-2 vaccines will reduce the global economic and public health stress from the COVID-19 pandemic. The use of conserved T-cell epitopes in combination with spike antigen that induce humoral and cellular immune responses simultaneously may be a promising strategy to further enhance the broad spectrum of COVID-19 vaccine candidates. Moreover, this research suggests that the combined vaccination strategies have the ability to induce both effective systemic and mucosal immunity, which may represent promising strategies for maximizing the protective efficacy of respiratory virus vaccines.

The efficacy of acupuncture for attention deficit hyperactivity disorder (ADHD): An overview of systematic reviews and meta-analyses.

BACKGROUND: Attention deficit hyperactivity disorder (ADHD) is one of the most common neurological and mental developmental disorders in children. Published systematic reviews (SRs) and meta-analyses (MAs) concerning the use of acupuncture for ADHD have compared the efficacy of acupuncture treatment to that of drug therapies. However, the quality of these articles has not been evaluated and the evidence varies widely. OBJECTIVE: To summarize and assess the efficacy of acupuncture for ADHD based on existing SRs and MAs. METHODS: A systematic search of the literature was conducted from inception until September 16 2021, using seven electronic databases. The AMSTAR-2 tool was used to evaluate the quality of SRs and MAs, and the GRADE system was used to assess the quality of evidence. RESULTS: There are a total of five SRs and MAs included in this overview. Using the AMSTAR-2, three articles were rated as having 'Low' quality, while two were rated as having of 'Critically Low' quality. The GRADE system was used to measure the quality of evidence for ten outcomes (five response rate outcomes, three Conners' Index of Hyperactivity (CIH) score outcomes, one Conners' rating scale score outcome, and one Chinese medicine syndrome outcome) across the five included MAs. Four of the ten outcomes demonstrated 'moderate' quality, four demonstrated 'low' quality, and two demonstrated 'very low' quality. The risk of bias and inconsistency accounted for most downgrading factors in the included reviews. CONCLUSION: It is still debatable whether acupuncture is efficacious in improving the CIH score and the Response rate. Considering the heterogeneity of clinical trials and the fact that this study did not search and evaluate the relevant data of each randomized controlled trial, large-sample and high-quality randomized controlled trials are still needed to draw reliable conclusions regarding acupuncture's role in treating ADHD. Due to the poor quality of existing available evidence, little inference can be drawn from the included studies.

Lactate-mediated Fascin protrusions promote cell adhesion and migration in cervical cancer.

Background: Lactate is associated with the poor prognosis of many human malignancies. Cervical cancer, one of main causes of women mortality worldwide, is aggressive and absent of effective pharmacological treatment, and its underlying mechanisms of progression remain elusive. Methods: The regulation of ß-catenin to fascin protrusion formation upon acidic lactate (Lactic acid [LA]) stimulation was evaluated through in ß-catenin or fascin deficiency cell line models by immunofluorescence assays, and subcellular fractionation. The effect of ß-catenin and fascin relocation by LA and its antagonist were evaluated by immunohistochemistry assay in patient tissues and mouse tumor xenograft model. Trypsin digestion, Transwell assay, cell proliferation in vitro was performed to explore the role of LA in the cell growth, adhesion and migration. Results: Low concentration of LA significantly promotes cytoskeleton remodeling via `protrusion formation to increase cell adhesion and migration. Mechanistically, upon LA stimulation, ß-catenin diffuses from the cytoplasmic membrane into the nucleus, which in turn induces fascin nuclear-cytoplasm redistribution to the protrusion compartment. Moreover, the antagonist of LA sufficiently blocks the LA-mediated ß-catenin nuclear import, fascin nuclear export, and the growth and invasion of cervical cancer cells in vitro and in vivo using a murine xenograft model. Conclusions: This study uncovers ß-catenin-fascin axis as a key signal in response to extracellular lactate and indicates that antagonist of LA may serve as a potential clinical intervention for cancer development.

Aberrant expression of GOLM1 protects ALK+ anaplastic large cell lymphoma from apoptosis by enhancing BCL-XL stability.

Golgi membrane protein 1 (GOLM1) is aberrantly expressed in many types of solid tumors and contributes to cancer development; however, its role in hematopoietic and lymphoid neoplasms remains unknown. Here, we report that GOLM1 was significantly upregulated in anaplastic large cell lymphoma (ALCL), particularly in anaplastic lymphoma kinase-positive (ALK+) ALCL. Mechanistically, the expression of GOLM1 was induced by nucleophosmin-ALK in both ALK-transformed T cells and ALCL cell lines through AKT/mTOR pathway. Knockdown of GOLM1 expression led to a reduction in the growth and viability of ALCL cells with increased spontaneous apoptosis, whereas ectopic expression of GOLM1 protected ALCL cells from apoptosis induced by staurosporine treatment. Moreover, GOLM1 directly interacted with B-cell lymphoma-extra large protein (a crucial anti-apoptosis regulator) and significantly prolonged its stability. Introduction of GOLM1 promoted ALK+ ALCL cells colony formation in vitro and tumor growth in a murine xenograft model. Taken together, our findings demonstrate, to our knowledge, for the first time that GOLM1 plays a critical role in suppressing apoptosis and promoting the progression of ALK+ ALCL and provide evidence that GOLM1 is a potential biomarker and therapeutic target in ALK-induced hematological malignancies.

Identification of hACE2-interacting sites in SARS-CoV-2 spike receptor binding domain for antiviral drugs screening.

The key structure of the interface between the spike protein of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and human angiotensin-converting enzyme 2 (hACE2) acts as an essential switch for cell entry by the virus and drugs targets. However, this is largely unknown. Here, we tested three peptides of spike receptor binding domain (RBD) and found that peptide 391-465 aa is the major hACE2-interacting sites in SARS-CoV-2 spike RBD. We then identified essential amino acid residues (403R, 449Y, 454R) of peptide 391-465 aa that were critical for the interaction between the RBD and hACE2. Additionally, a pseudotyped virus containing SARS-CoV-2 spike with individual mutation (R454G, Y449F, R403G, N439I, or N440I) was determined to have very low infectivity compared with the pseudotyped virus containing the wildtype (WT) spike from reference strain Wuhan 1, respectively. Furthermore, we showed the key amino acids had the potential to drug screening. For example, molecular docking (Docking) and infection assay showed that Cephalosporin derivatives can bind with the key amino acids to efficiently block infection of the pseudoviruses with wild type spike or new variants. Moreover, Cefixime inhibited live SARS-CoV-2 infection. These results also provide a novel model for drug screening and support further clinical evaluation and development of Cephalosporin derivatives as novel, safe, and cost-effective drugs for prevention/treatment of SARS-CoV-2.

Neutralization mechanism of a human antibody with pan-coronavirus reactivity including SARS-CoV-2.

Frequent outbreaks of coronaviruses underscore the need for antivirals and vaccines that can counter a broad range of coronavirus types. We isolated a human antibody named 76E1 from a COVID-19 convalescent patient, and report that it has broad-range neutralizing activity against multiple α- and ß-coronaviruses, including the SARS-CoV-2 variants. 76E1 also binds its epitope in peptides from γ- and δ-coronaviruses. 76E1 cross-protects against SARS-CoV-2 and HCoV-OC43 infection in both prophylactic and therapeutic murine animal models. Structural and functional studies revealed that 76E1 targets a unique epitope within the spike protein that comprises the highly conserved S2' site and the fusion peptide. The epitope that 76E1 binds is partially buried in the structure of the SARS-CoV-2 spike trimer in the prefusion state, but is exposed when the spike protein binds to ACE2. This observation suggests that 76E1 binds to the epitope at an intermediate state of the spike trimer during the transition from the prefusion to the postfusion state, thereby blocking membrane fusion and viral entry. We hope that the identification of this crucial epitope, which can be recognized by 76E1, will guide epitope-based design of next-generation pan-coronavirus vaccines and antivirals.

Comprehensive role of SARS-CoV-2 spike glycoprotein in regulating host signaling pathway.

Since the outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, global public health and the economy have suffered unprecedented damage. Based on the increasing related literature, the characteristics and pathogenic mechanisms of the virus, and epidemiological and clinical features of the disease are being rapidly discovered. The spike glycoprotein (S protein), as a key antigen of SARS-CoV-2 for developing vaccines, antibodies, and drug targets, has been shown to play an important role in viral entry, tissue tropism, and pathogenesis. In this review, we summarize the molecular mechanisms of interaction between S protein and host factors, especially receptor-mediated viral modulation of host signaling pathways, and highlight the progression of potential therapeutic targets, prophylactic and therapeutic agents for prevention and treatment of SARS-CoV-2 infection.

A System Based on Novel Parainfluenza Virus PIV5-L for Efficient Gene Delivery of B-Lymphoma Cells.

Aggressive B-cell lymphoma is one of the most common types of blood malignancy. Robust delivery of genes of interest into target cells, long-term gene expression, and minimal risk of secondary effects are highly desirable for translational medicine including gene therapy and studies on gene function. However, efficient gene delivery into viral or nonviral B-lymphoma cells remains a challenge. Here, we report a strategy for inducing foreign gene expression in B-lymphoma cells by using a vector based on the novel parainfluenza virus PIV5-L (a strain isolated from B cells) that enabled us to study and control the function of a gene product within B-lymphoma cells. Using enhanced green fluorescent protein (eGFP) as a reporter, we successfully rescued PIV5-L and established a one-step system to generate PIV5-L virus-like particles (L-VLPs) with efficient delivery into a broad spectrum of susceptible B-lymphoma cell lines, including Epstein-Barr virus (EBV)- or Kaposi's sarcoma-associated herpesvirus (KSHV)-transformed B-lymphoblastoid cells. Similar to lentiviral vector, the L-VLP highly expressed exogenous genes and remained stable for long periods without obvious negative effects on cell viability. Taken together, these data demonstrate that the PIV5-L-based system provides a potential new strategy for the delivery of desirable genes and the treatment of cancer. IMPORTANCE B-cell lymphoma is a common aggressive neoplastic disorder of lymphocytes. Delivery of genes of interest into B cells, particularly virus-mediated B-lymphoma cells, is still a challenge. In this study, we report that a system (L-VLP) based on the parainfluenza virus PIV5-L strain isolated from B cells had highly expressed exogenous genes and remained stable without obvious cell toxicity, which provides a potential new strategy for gene delivery and treatment of B-cell cancer.

Rapid establishment of murine gastrointestinal organoids using mechanical isolation method.

Gastrointestinal (GI) diseases, including pathological dysplasia, inflammation, neoplasia and injury, suffer millions of patients globally per year. Organoids, three-dimensional cell mass structures supported by biomaterials in dishes, are currently used as a research model for diseases of the small intestine. However, the traditional enzymatic-digestion method for establishing small-intestinal organoids (EnzyOs) is time consuming and often loses the bulk of crypts, a more efficient and reliable method needs to be developed. In this study, using mouse GI organoids as a model, we formulated a rapid mechanical isolation method that could efficiently isolate and culture villi-crypts into small intestinal organoids (MechOs). Primary duodenum organoids generated by MechOs displayed three types of morphology: spheroid, semi-budding and budding, while EnzyOs produced much less budding. Moreover, primary duodenum organoids from MechOs could be subcultured and presented similar gene expression profiles of small intestine specific markers as that from EnzyOs. Importantly, the MechOs method could also be used to generate other types of organoids derived from the stomach, jejunum-ileum, sigmoid-rectum and bile cysts. Taken together, the results show that MechOs could efficiently and economically generate digestive system organoids, providing a potential basis of epithelial organoids for the clinical treatment of gastroenterological diseases.

Broad neutralization of SARS-CoV-2 variants by an inhalable bispecific single-domain antibody.

The effectiveness of SARS-CoV-2 vaccines and therapeutic antibodies have been limited by the continuous emergence of viral variants and by the restricted diffusion of antibodies from circulation into the sites of respiratory virus infection. Here, we report the identification of two highly conserved regions on the Omicron variant receptor-binding domain recognized by broadly neutralizing antibodies. Furthermore, we generated a bispecific single-domain antibody that was able to simultaneously and synergistically bind these two regions on a single Omicron variant receptor-binding domain as revealed by cryo-EM structures. We demonstrated that this bispecific antibody can be effectively delivered to lung via inhalation administration and exhibits exquisite neutralization breadth and therapeutic efficacy in mouse models of SARS-CoV-2 infections. Importantly, this study also deciphered an uncommon and highly conserved cryptic epitope within the spike trimeric interface that may have implications for the design of broadly protective SARS-CoV-2 vaccines and therapeutics.

Receptome profiling identifies KREMEN1 and ASGR1 as alternative functional receptors of SARS-CoV-2.

Host cellular receptors play key roles in the determination of virus tropism and pathogenesis. However, little is known about SARS-CoV-2 host receptors with the exception of ACE2. Furthermore, ACE2 alone cannot explain the multi-organ tropism of SARS-CoV-2 nor the clinical differences between SARS-CoV-2 and SARS-CoV, suggesting the involvement of other receptor(s). Here, we performed genomic receptor profiling to screen 5054 human membrane proteins individually for interaction with the SARS-CoV-2 capsid spike (S) protein. Twelve proteins, including ACE2, ASGR1, and KREMEN1, were identified with diverse S-binding affinities and patterns. ASGR1 or KREMEN1 is sufficient for the entry of SARS-CoV-2 but not SARS-CoV in vitro and in vivo. SARS-CoV-2 utilizes distinct ACE2/ASGR1/KREMEN1 (ASK) receptor combinations to enter different cell types, and the expression of ASK together displays a markedly stronger correlation with virus susceptibility than that of any individual receptor at both the cell and tissue levels. The cocktail of ASK-related neutralizing antibodies provides the most substantial blockage of SARS-CoV-2 infection in human lung organoids when compared to individual antibodies. Our study revealed an interacting host receptome of SARS-CoV-2, and identified ASGR1 and KREMEN1 as alternative functional receptors that play essential roles in ACE2-independent virus entry, providing insight into SARS-CoV-2 tropism and pathogenesis, as well as a community resource and potential therapeutic strategies for further COVID-19 investigations.

Proteomic Profiling Identifies Kaposi's Sarcoma-Associated Herpesvirus (KSHV)-Encoded LANASIM-Associated Proteins in Hypoxia.

Hypoxia signaling is a key regulator in the development and progression of many types of human malignancies, including viral cancers. The latency-associated nuclear antigen (LANA), encoded by Kaposi's sarcoma-associated herpesvirus (KSHV) during latency, is a multifunctional protein that plays an essential role in viral episome maintenance and lytic gene silencing for inducing tumorigenesis. Although our previous studies have shown that LANA contains a SUMO-interacting motif (LANASIM), and hypoxia reduces SUMOylated KAP1 association with LANASIM, the physiological proteomic network of LANASIM-associated cellular proteins in response to hypoxia is still unclear. In this study, we individually established cell lines stably expressing wild-type LANA (LANAWT) and its SIM-deleted mutant (LANAdSIM) and treated them with or without hypoxia, followed by coimmunoprecipitation and mass spectrometry analysis to systemically identify the hypoxia-responsive profile of LANASIM-associated cellular proteins. We found that in hypoxia, the number of cellular proteins associated with LANAWT instead of LANAdSIM was dramatically increased. Functional network analysis revealed that two major pathways, which included cytoskeleton organization and DNA/RNA binding and processing pathways, were significantly enriched for 28 LANASIM-associated proteins in response to hypoxia. HNRNPU was one of the proteins consistently identified that interacted with LANASIM in different proteomic screening systems and responded to hypoxia. This study provides a proteomic profile of LANASIM-associated proteins in hypoxia and facilitates our understanding of the role of the collaboration between viral infection and the hypoxia response in inducing viral persistence and tumorigenesis. IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) has been reported to be involved in the regulation of host proteins in response to hypoxic stress. LANA, one of the key latent proteins, contains a SUMO-interacting motif (LANASIM) and reduces the association with SUMOylated KAP1 upon hypoxic treatment. However, the physiological systematic network of LANASIM-associated cellular proteins in hypoxia is still unclear. Here, we revealed two major pathways, which included cytoskeleton organization and DNA/RNA binding and processing pathways, that were significantly enriched for 28 LANASIM-associated proteins in hypoxia. This discovery not only provides a proteomic profile of LANASIM-associated proteins in hypoxia but also facilitates our understanding of the collaboration between viral infection and hypoxic stress in inducing viral persistence and tumorigenesis.

A non-ACE2 competing human single-domain antibody confers broad neutralization against SARS-CoV-2 and circulating variants.

The current COVID-19 pandemic has heavily burdened the global public health system and may keep simmering for years. The frequent emergence of immune escape variants have spurred the search for prophylactic vaccines and therapeutic antibodies that confer broad protection against SARS-CoV-2 variants. Here we show that the bivalency of an affinity maturated fully human single-domain antibody (n3113.1-Fc) exhibits exquisite neutralizing potency against SARS-CoV-2 pseudovirus, and confers effective prophylactic and therapeutic protection against authentic SARS-CoV-2 in the host cell receptor angiotensin-converting enzyme 2 (ACE2) humanized mice. The crystal structure of n3113 in complex with the receptor-binding domain (RBD) of SARS-CoV-2, combined with the cryo-EM structures of n3113 and spike ecto-domain, reveals that n3113 binds to the side surface of up-state RBD with no competition with ACE2. The binding of n3113 to this novel epitope stabilizes spike in up-state conformations but inhibits SARS-CoV-2 S mediated membrane fusion, expanding our recognition of neutralization by antibodies against SARS-CoV-2. Binding assay and pseudovirus neutralization assay show no evasion of recently prevalent SARS-CoV-2 lineages, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), and Delta (B.1.617.2) for n3113.1-Fc with Y58L mutation, demonstrating the potential of n3113.1-Fc (Y58L) as a promising candidate for clinical development to treat COVID-19.

Yeast-produced RBD-based recombinant protein vaccines elicit broadly neutralizing antibodies and durable protective immunity against SARS-CoV-2 infection.

Massive production of efficacious SARS-CoV-2 vaccines is essential for controlling the ongoing COVID-19 pandemic. We report here the preclinical development of yeast-produced receptor-binding domain (RBD)-based recombinant protein SARS-CoV-2 vaccines. We found that monomeric RBD of SARS-CoV-2 could be efficiently produced as a secreted protein from transformed Pichia pastoris (P. pastoris) yeast. Yeast-derived RBD-monomer possessed functional conformation and was able to elicit protective level of neutralizing antibodies in mice. We further designed and expressed a genetically linked dimeric RBD protein in yeast. The engineered dimeric RBD was more potent than the monomeric RBD in inducing long-lasting neutralizing antibodies. Mice immunized with either monomeric RBD or dimeric RBD were effectively protected from live SARS-CoV-2 virus challenge even at 18 weeks after the last vaccine dose. Importantly, we found that the antisera raised against the RBD of a single SARS-CoV-2 prototype strain could effectively neutralize the two predominant circulating variants B.1.1.7 and B.1.351, implying broad-spectrum protective potential of the RBD-based vaccines. Our data demonstrate that yeast-derived RBD-based recombinant SARS-CoV-2 vaccines are feasible and efficacious, opening up a new avenue for rapid and cost-effective production of SARS-CoV-2 vaccines to achieve global immunization.

Delayed Antiviral Immune Responses in Severe Acute Respiratory Syndrome Coronavirus Infected Pregnant Mice.

Sex differences in immune responses had been reported to correlate with different symptoms and mortality in the disease course of coronavirus disease 2019 (COVID-19). However, whether severe acute respiratory syndrome coronavirus (SARS-CoV-2) infection interferes with females' fertility and causes different symptoms among pregnant and non-pregnant females remains unknown. Here, we examined the differences in viral loads, SARS-CoV-2-specific antibody titers, proinflammatory cytokines, and levels of T cell activation after SARS-CoV-2 sub-lethal infection between pregnant and non-pregnant human Angiotensin-Converting Enzyme II (ACE2) transgenic mouse models. Both mice showed elevated levels of viral loads in the lung at 4 days post-infection (dpi). However, viral loads in the pregnant group remained elevated at 7 dpi while decreased in the non-pregnant group. Consistent with viral loads, increased production of proinflammatory cytokines was detected from the pregnant group, and the IgM or SARS-CoV-2-specific IgG antibody in serum of pregnant mice featured delayed elevation compared with non-pregnant mice. Moreover, by accessing kinetics of activation marker expression of peripheral T cells after infection, a lower level of CD8+ T cell activation was observed in pregnant mice, further demonstrating the difference of immune-response between pregnant and non-pregnant mice. Although vertical transmission did not occur as SARS-CoV-2 RNA was absent in the uterus and fetus from the infected pregnant mice, a lower pregnancy rate was observed when the mice were infected before embryo implantation after mating, indicating that SARS-CoV-2 infection may interfere with mice's fertility at a specific time window. In summary, pregnant mice bear a weaker ability to eliminate the SARS-CoV-2 virus than non-pregnant mice, which was correlated with lower levels of antibody production and T cell activation.

Lactate Induces Production of the tRNAHis Half to Promote B-lymphoblastic Cell Proliferation.

High plasma lactate is emerging as a critical regulator in development and progression of many human malignancies. Small RNAs derived from cleavage of mature tRNAs have been implicated in many cellular stresses, but the detailed mechanisms that respond to lactic acid (LA; acidic lactate) are not well defined. Here, using an Epstein-Barr virus (EBV)-immortalized B lymphoblastic cell line (LCL) as a model, we report that LA induces cleavage of mature tRNA at the anticodon loop, particularly production of three 5'-tRNA halves (5'-HisGUG, 5'-ValAAC, and 5'-GlyGCC), along with increased expression of RNA polymerase III and angiogenin (ANG). Of these, only the 5'-HisGUG half binds to the chromatin regulator argonaute-2 (AGO2) instead of the AGO1 protein for stability. Notably, the levels of ANG and 5'-HisGUG half expression in peripheral blood mononuclear cells from B cell lymphoma patients are tightly correlated with lactate dehydrogenase (LDH; a lactate indicator) in plasma. Silencing production of the 5'-HisGUG half by small interfering RNA or inhibition of ANG significantly reduces colony formation and growth of LA-induced tumor cells in vitro and in vivo using a murine xenograft model. Overall, our findings identify a novel molecular therapeutic target for the diagnosis and treatment of B cell lymphoma.

STUB1 is targeted by the SUMO-interacting motif of EBNA1 to maintain Epstein-Barr Virus latency.

Latent Epstein-Barr virus (EBV) infection is strongly associated with several malignancies, including B-cell lymphomas and epithelial tumors. EBNA1 is a key antigen expressed in all EBV-associated tumors during latency that is required for maintenance of the EBV episome DNA and the regulation of viral gene transcription. However, the mechanism utilized by EBV to maintain latent infection at the levels of posttranslational regulation remains largely unclear. Here, we report that EBNA1 contains two SUMO-interacting motifs (SIM2 and SIM3), and mutation of SIM2, but not SIM3, dramatically disrupts the EBNA1 dimerization, while SIM3 contributes to the polySUMO2 modification of EBNA1 at lysine 477 in vitro. Proteomic and immunoprecipitation analyses further reveal that the SIM3 motif is required for the EBNA1-mediated inhibitory effects on SUMO2-modified STUB1, SUMO2-mediated degradation of USP7, and SUMO1-modified KAP1. Deletion of the EBNASIM motif leads to functional loss of both EBNA1-mediated viral episome maintenance and lytic gene silencing. Importantly, hypoxic stress induces the SUMO2 modification of EBNA1, and in turn the dissociation of EBNA1 with STUB1, KAP1 and USP7 to increase the SUMO1 modification of both STUB1 and KAP1 for reactivation of lytic replication. Therefore, the EBNA1SIM motif plays an essential role in EBV latency and is a potential therapeutic target against EBV-associated cancers.