A germ-free humanized mouse model shows the contribution of resident microbiota to human-specific pathogen infection.

Germ-free (GF) mice, which are depleted of their resident microbiota, are the gold standard for exploring the role of the microbiome in health and disease; however, they are of limited value in the study of human-specific pathogens because they do not support their replication. Here, we develop GF mice systemically reconstituted with human immune cells and use them to evaluate the role of the resident microbiome in the acquisition, replication and pathogenesis of two human-specific pathogens, Epstein-Barr virus (EBV) and human immunodeficiency virus (HIV). Comparison with conventional (CV) humanized mice showed that resident microbiota enhance the establishment of EBV infection and EBV-induced tumorigenesis and increase mucosal HIV acquisition and replication. HIV RNA levels were higher in plasma and tissues of CV humanized mice compared with GF humanized mice. The frequency of CCR5+ CD4+ T cells throughout the intestine was also higher in CV humanized mice, indicating that resident microbiota govern levels of HIV target cells. Thus, resident microbiota promote the acquisition and pathogenesis of two clinically relevant human-specific pathogens.

HIV co-infection augments EBV-induced tumorigenesis in vivo.

In most individuals, EBV maintains a life-long asymptomatic latent infection. However, EBV can induce the formation of B cell lymphomas in immune suppressed individuals including people living with HIV (PLWH). Most individuals who acquire HIV are already infected with EBV as EBV infection is primarily acquired during childhood and adolescence. Although antiretroviral therapy (ART) has substantially reduced the incidence of AIDS-associated malignancies, EBV positive PLWH are at an increased risk of developing lymphomas compared to the general population. The direct effect of HIV co-infection on EBV replication and EBV-induced tumorigenesis has not been experimentally examined. Using a humanized mouse model of EBV infection, we demonstrate that HIV co-infection enhances systemic EBV replication and immune activation. Importantly, EBV-induced tumorigenesis was augmented in EBV/HIV co-infected mice. Collectively, these results demonstrate a direct effect of HIV co-infection on EBV pathogenesis and disease progression and will facilitate future studies to address why the incidence of certain types of EBV-associated malignancies are stable or increasing in ART treated PLWH.

Small molecule screening identifies inhibitors of the Epstein-Barr virus deubiquitinating enzyme, BPLF1.

Herpesviral deubiquitinating enzymes (DUBs) were discovered in 2005, are highly conserved across the family, and are proving to be increasingly important players in herpesviral infection. EBV's DUB, BPLF1, is known to regulate both cellular and viral target activities, yet remains largely unstudied. Our work has implicated BPLF1 in a wide range of processes including infectivity, viral DNA replication, and DNA repair. Additionally, knockout of BPLF1 delays and reduces human B-cell immortalization and lymphoma formation in humanized mice. These findings underscore the importance of BPLF1 in viral infectivity and pathogenesis and suggest that inhibition of EBV's DUB activity may offer a new approach to specific therapy for EBV infections. We set out to discover and characterize small molecule inhibitors of BPLF1 deubiquitinating activity through high-throughput screening. An initial small pilot screen resulted in discovery of 10 compounds yielding >80% decrease in BPLF1 DUB activity at a 10 µM concentration. Follow-up dose response curves of top hits identified several compounds with an IC50 in the low micromolar range. Four of these hits were tested for their ability to cleave ubiquitin chains as well as their effects on viral infectivity and cell viability. Further characterization of the top hit, commonly known as suramin was found to not be selective yet decreased viral infectivity by approximately 90% with no apparent effects on cell viability. Due to the conserved nature of Herpesviral deubiquitinating enzymes, identification of an inhibitor of BPLF1 may prove to be an effective and promising new avenue of therapy for EBV and other herpesviral family members.

Inhibition of UCH-L1 Deubiquitinating Activity with Two Forms of LDN-57444 Has Anti-Invasive Effects in Metastatic Carcinoma Cells.

Normally ubiquitin C-terminal hydrolase L1 (UCH-L1) is expressed in the central nervous and reproductive systems of adults, but its de novo expression has been detected in many human cancers. There is a growing body of evidence that UCH-L1 de-ubiquitinating (DUB) activity plays a major pro-metastatic role in certain carcinomas. Here we tested anti-metastatic effects of the small-molecule inhibitor of UCH-L1 DUB activity, LDN-57444, in cell lines from advanced oral squamous cell carcinoma (OSCC) as well as invasive nasopharyngeal (NP) cell lines expressing the major pro-metastatic gene product of Epstein-Barr virus (EBV) tumor virus, LMP1. To overcome the limited aqueous solubility of LDN-57444 we developed a nanoparticle formulation of LDN-57444 by incorporation of the compound in polyoxazoline micellear nanoparticles (LDN-POx). LDN-POx nanoparticles were equal in effects as the native compound in vitro. Our results demonstrate that inhibition of UCH-L1 DUB activity with LDN or LDN-POx inhibits secretion of exosomes and reduces levels of the pro-metastatic factor in exosomal fractions. Both forms of UCH-L1 DUB inhibitor suppress motility of metastatic squamous carcinoma cells as well as nasopharyngeal cells expressing EBV pro-metastatic Latent membrane protein 1 (LMP1) in physiological assays. Moreover, treatment with LDN and LDN-POx resulted in reduced levels of pro-metastatic markers, a decrease of carcinoma cell adhesion, as well as inhibition of extra-cellular vesicle (ECV)-mediated transfer of viral invasive factor LMP1. We suggest that soluble inhibitors of UCH-L1 such as LDN-POx offer potential forms of treatment for invasive carcinomas including EBV-positive malignancies.

Epstein-Barr Virus Latent Membrane Protein-1 Induces the Expression of SUMO-1 and SUMO-2/3 in LMP1-positive Lymphomas and Cells.

Epstein-Barr Virus latent membrane protein-1 (LMP1) interacts with the SUMO-conjugating enzyme Ubc9, which induces protein sumoylation and may contribute to LMP1-mediated oncogenesis. After analyzing human lymphoma tissues and EBV-positive cell lines, we now document a strong correlation between LMP1 and sumo-1/2/3 or SUMO-1/2/3 levels, and show that LMP1-induced sumo expression requires the activation of NF-κB signaling through CTAR1 and CTAR2. Together, these results point to a second mechanism by which LMP1 dysregulates sumoylation processes and adds EBV-associated lymphomas to the list of malignancies associated with increased SUMO expression.

Prevalence of Epstein­Barr Virus Genotypes in Pakistani Lymphoma Patients

The Epstein-Barr virus (EBV) is a herpesvirus infecting more than 90% of the human population. The tropism of EBV for B lymphocytes is evidenced in its association with many lymphoproliferative disorders. Different types of EBV (EBV-1 and EBV-2), classified on the basis of EBV nuclear antigen-2 (EBNA-2) genotyping, have been reported in benign and malignant pathologies, but there is almost no information about their frequency in the Pakistani population. The aim of this study was to determine the frequency and distribution of EBNA-2-based EBV genotypes in lymphoma patients. Genomic DNA was extracted from formalin-fixed paraffin embedded (FFPE) tissue samples obtained from 73 EBV-DNA-positive lymphoma patients. The ß-globin gene was amplified to assess the presence and quality of cellular DNA from all samples. EBER-1 DNA was detected by PCR to confirm EBV presence in tissue samples. EBNA-1 mRNA relative quantification done by quantitative PCR substantiated EBNA-1 mRNA overexpression in 43.8% of EBV-positive cases in comparison to EBV-positive control cell line. EBNA-2 genotyping was done by nested PCR. Among typable samples, EBV-1 was found in 90.7% of samples while EBV-2 was present in 9.3% cases. These results show that EBV-1 was the most prevalent type in the lymphoma population of Pakistan. This epidemiology of EBV in Pakistani lymphoma patients represents an important first step in using EBV for prognosis and monitoring treatment response.

Antiviral Drugs for EBV.

Epstein⁻Barr virus (EBV) infects up to 95% of the adult human population, with primary infection typically occurring during childhood and usually asymptomatic. However, EBV can cause infectious mononucleosis in approximately 35⁻50% cases when infection occurs during adolescence and early adulthood. Epstein⁻Barr virus is also associated with several B-cell malignancies including Burkitt lymphoma, Hodgkin lymphoma, and post-transplant lymphoproliferative disease. A number of antiviral drugs have proven to be effective inhibitors of EBV replication, yet have resulted in limited success clinically, and none of them has been approved for treatment of EBV infections.

The Translesion Polymerase Pol η Is Required for Efficient Epstein-Barr Virus Infectivity and Is Regulated by the Viral Deubiquitinating Enzyme BPLF1.

Epstein-Barr virus (EBV) infection and lytic replication are known to induce a cellular DNA damage response. We previously showed that the virally encoded BPLF1 protein interacts with and regulates several members of the translesion synthesis (TLS) pathway, a DNA damage tolerance pathway, and that these cellular factors enhance viral infectivity. BPLF1 is a late lytic cycle gene, but the protein is also packaged in the viral tegument, indicating that BPLF1 may function both early and late during infection. The BPLF1 protein expresses deubiquitinating activity that is strictly conserved across the Herpesviridae; mutation of the active site cysteine results in a loss of enzymatic activity. Infection with an EBV BPLF1 knockout virus results in decreased EBV infectivity. Polymerase eta (Pol η), a specialized DNA repair polymerase, functions in TLS and allows for DNA replication complexes to bypass lesions in DNA. Here we report that BPLF1 interacts with Pol η and that Pol η protein levels are increased in the presence of functional BPLF1. BPLF1 promotes a nuclear relocalization of Pol η molecules which are focus-like in appearance, consistent with the localization observed when Pol η is recruited to sites of DNA damage. Knockdown of Pol η resulted in decreased production of infectious virus, and further, Pol η was found to bind to EBV DNA, suggesting that it may allow for bypass of damaged viral DNA during its replication. The results suggest a mechanism by which EBV recruits cellular repair factors, such as Pol η, to sites of viral DNA damage via BPLF1, thereby allowing for efficient viral DNA replication.IMPORTANCE Epstein-Barr virus is the causative agent of infectious mononucleosis and infects approximately 90% of the world's population. It causes lymphomas in individuals with acquired and innate immune disorders and is strongly associated with Hodgkin's lymphoma, Burkitt's lymphoma, diffuse large B-cell lymphomas, nasopharyngeal carcinoma (NPC), and lymphomas that develop in organ transplant recipients. Cellular DNA damage is a major determinant in the establishment of oncogenic processes and is well studied, but there are few studies of endogenous repair of viral DNA. This work evaluates how EBV's BPLF1 protein and its conserved deubiquitinating activity regulate the cellular DNA repair enzyme polymerase eta and recruit it to potential sites of viral damage and replication, resulting in enhanced production of infectious virus. These findings help to establish how EBV enlists and manipulates cellular DNA repair factors during the viral lytic cycle, contributing to efficient infectious virion production.

Expression of interferon regulatory factor 7 correlates with the expression of Epstein-Barr Virus latent membrane protein 1 and cervical lymph node metastasis in nasopharyngeal cancer.

Interferon regulatory factor 7 (IRF7) has oncogenic properties in several malignancies such as Epstein-Barr virus (EBV)-associated lymphoma. However, there is no evidence whether IRF7 is associated with the oncogenesis of nasopharyngeal cancer (NPC), the pathogenesis of which is closely associated with EBV. Herein, we report that expression of IRF7 was increased in normal nasopharyngeal cells that expressed the EBV principal oncoprotein, latent membrane protein 1 (LMP1). In addition, IRF7 was mainly expressed in the nucleus in both normal nasopharyngeal cells and nasopharyngeal cancer cells that expresses LMP1. On immunohistochemical analysis, IRF7 was predominantly localized in the nucleus in biopsy samples of NPC tissues. In total, IRF7 expression was detected with 36 of 49 specimens of these tissues. Furthermore, the expression score of IRF7 correlated with the expression score of LMP1. Moreover, the expression score of IRF7 is associated with cervical lymph-node metastasis, which reflects the highly metastatic nature of this cancer. Taken together, our results suggest that expression of IRF7 is one of the metastatic effectors of LMP1 signalling in EBV-associated NPC.

Viral Carcinogenesis Beyond Malignant Transformation: EBV in the Progression of Human Cancers.

Cancer progression begins when malignant cells colonize adjacent sites, and it is characterized by increasing tumor heterogeneity, invasion and dissemination of cancer cells. Clinically, progression is the most relevant stage in the natural history of cancers. A given virus is usually regarded as oncogenic because of its ability to induce malignant transformation of cells. Nonetheless, oncogenic viruses may also be important for the progression of infection-associated cancers. Recently this hypothesis has been addressed because of studies on the contribution of the Epstein-Barr virus (EBV) to the aggressiveness of nasopharyngeal carcinoma (NPC). Several EBV products modulate cancer progression phenomena, such as the epithelial-mesenchymal transition, cell motility, invasiveness, angiogenesis, and metastasis. In this regard, there are compelling data about the effects of EBV latent membrane proteins (LMPs) and EBV nuclear antigens (EBNAs), as well as nontranslated viral RNAs, such as the EBV-encoded small nonpolyadenylated RNAs (EBERs) and viral microRNAs, notably EBV miR-BARTs. The available data on the mechanisms and players involved in the contribution of EBV infection to the aggressiveness of NPC are discussed in this review. Overall, this conceptual framework may be valuable for the understanding of the contribution of some infectious agents in the progression of cancers.

UCH-L1 in DLBCL: marker or target?

In this issue of Blood, Bedekovics et al have demonstrated that a multifunctional molecule of the ubiquitin system ubiquitin C-terminal hydrolase L1 (UCH-L1) is induced in diffuse large B-cell lymphomas (DLBCLs), and that levels of this molecule are higher in germinal center (GC) B-cell DLBCL (GCB-DLBCL) compared with activated B-cell DLBCL (ABC-DLBCL) and predict poor outcomes.

Knockout of Epstein-Barr virus BPLF1 retards B-cell transformation and lymphoma formation in humanized mice.

UNLABELLED: BPLF1 of Epstein-Barr virus (EBV) is classified as a late lytic cycle protein but is also found in the viral tegument, suggesting its potential involvement at both initial and late stages of viral infection. BPLF1 possesses both deubiquitinating and deneddylating activity located in its N-terminal domain and is involved in processes that affect viral infectivity, viral DNA replication, DNA repair, and immune evasion. A recently constructed EBV BPLF1-knockout (KO) virus was used in conjunction with a humanized mouse model that can be infected with EBV, enabling the first characterization of BPLF1 function in vivo. Results demonstrate that the BPLF1-knockout virus is approximately 90% less infectious than wild-type (WT) virus. Transformation of human B cells, a hallmark of EBV infection, was delayed and reduced with BPLF1-knockout virus. Humanized mice infected with EBV BPLF1-knockout virus showed less weight loss and survived longer than mice infected with equivalent infectious units of WT virus. Additionally, splenic tumors formed in 100% of mice infected with WT EBV but in only 25% of mice infected with BPLF1-KO virus. Morphological features of spleens containing tumors were similar to those in EBV-induced posttransplant lymphoproliferative disease (PTLD) and were almost identical to cases seen in human diffuse large B-cell lymphoma. The presence of EBV genomes was detected in all mice that developed tumors. The results implicate BPLF1 in human B-cell transformation and tumor formation in humanized mice. IMPORTANCE: Epstein-Barr virus infects approximately 90% of the world's population and is the causative agent of infectious mononucleosis. EBV also causes aggressive lymphomas in individuals with acquired and innate immune disorders and is strongly associated with diffuse large B-cell lymphomas, classical Hodgkin lymphoma, Burkitt lymphoma, and nasopharyngeal carcinoma (NPC). Typically, EBV initially infects epithelial cells in the oropharynx, followed by a lifelong persistent latent infection in B-cells, which may develop into lymphomas in immunocompromised individuals. This work is the first of its kind in evaluating the effects of EBV's BPLF1 in terms of pathogenesis and lymphomagenesis in humanized mice and implicates BPLF1 in B-cell transformation and tumor development. Currently, there is no efficacious treatment for EBV, and therapeutic targeting of BPLF1 may lead to a new path to treatment for immunocompromised individuals or transplant recipients infected with EBV.

LMP1-Induced Sumoylation Influences the Maintenance of Epstein-Barr Virus Latency through KAP1.

UNLABELLED: As a herpesvirus, Epstein-Barr virus (EBV) establishes a latent infection that can periodically undergo reactivation, resulting in lytic replication and the production of new infectious virus. Latent membrane protein-1 (LMP1), the principal viral oncoprotein, is a latency-associated protein implicated in regulating viral reactivation and the maintenance of latency. We recently found that LMP1 hijacks the SUMO-conjugating enzyme Ubc9 via its C-terminal activating region-3 (CTAR3) and induces the sumoylation of cellular proteins. Because protein sumoylation can promote transcriptional repression, we hypothesized that LMP1-induced protein sumoylation induces the repression of EBV lytic promoters and helps maintain the viral genome in its latent state. We now show that with inhibition of LMP1-induced protein sumoylation, the latent state becomes less stable or leakier in EBV-transformed lymphoblastoid cell lines. The cells are also more sensitive to viral reactivation induced by irradiation, which results in the increased production and release of infectious virus, as well as increased susceptibility to ganciclovir treatment. We have identified a target of LMP1-mediated sumoylation that contributes to the maintenance of latency in this context: KRAB-associated protein-1 (KAP1). LMP1 CTAR3-mediated sumoylation regulates the function of KAP1. KAP1 also binds to EBV OriLyt and immediate early promoters in a CTAR3-dependent manner, and inhibition of sumoylation processes abrogates the binding of KAP1 to these promoters. These data provide an additional line of evidence that supports our findings that CTAR3 is a distinct functioning regulatory region of LMP1 and confirm that LMP1-induced sumoylation may help stabilize the maintenance of EBV latency. IMPORTANCE: Epstein-Barr virus (EBV) latent membrane protein-1 (LMP1) plays an important role in the maintenance of viral latency. Previously, we documented that LMP1 targets cellular proteins to be modified by a ubiquitin-like protein (SUMO). We have now identified a function for this LMP1-induced modification of cellular proteins in the maintenance of EBV latency. Because latently infected cells have to undergo viral reactivation in order to be vulnerable to antiviral drugs, these findings identify a new way to increase the rate of EBV reactivation, which increases cell susceptibility to antiviral therapies.

Viruses and human cancers: a long road of discovery of molecular paradigms.

About a fifth of all human cancers worldwide are caused by infectious agents. In 12% of cancers, seven different viruses have been causally linked to human oncogenesis: Epstein-Barr virus, hepatitis B virus, human papillomavirus, human T-cell lymphotropic virus, hepatitis C virus, Kaposi's sarcoma herpesvirus, and Merkel cell polyomavirus. Here, we review the many molecular mechanisms of oncogenesis that have been discovered over the decades of study of these viruses. We discuss how viruses can act at different stages in the complex multistep process of carcinogenesis. Early events include their involvement in mutagenic events associated with tumor initiation such as viral integration and insertional mutagenesis as well as viral promotion of DNA damage. Also involved in tumor progression is the dysregulation of cellular processes by viral proteins, and we describe how this has been investigated by studies in cell culture and in experimental animals and by molecular cellular approaches. Also important are the molecular mechanisms whereby viruses interact with the immune system and the immune evasion strategies that have evolved.

The Rad6/18 ubiquitin complex interacts with the Epstein-Barr virus deubiquitinating enzyme, BPLF1, and contributes to virus infectivity.

UNLABELLED: Epstein-Barr virus (EBV) encodes BPLF1, a lytic cycle protein with deubiquitinating activity that is contained in its N-terminal domain and conserved across the Herpesviridae. EBV replication is associated with cellular DNA replication and repair factors, and initiation of EBV lytic replication induces a DNA damage response, which can be regulated at least in part by BPLF1. The cellular DNA repair pathway, translesion synthesis (TLS), is disrupted by BPLF1, which deubiquitinates the DNA processivity factor, PCNA, and inhibits the recruitment of the TLS polymerase, polymerase eta (Pol eta), after damage to DNA by UV irradiation. Here we showed that the E3 ubiquitin ligase, which activates TLS repair by monoubiquitination of PCNA, is also affected by BPLF1 deubiquitinating activity. First, BPLF1 interacts directly with Rad18, and overexpression of BPLF1 results in increased levels of the Rad18 protein, suggesting that it stabilizes Rad18. Next, expression of functionally active BPLF1 caused relocalization of Rad18 into nuclear foci, which is consistent with sites of cellular DNA replication that occur during S phase. Also, levels of Rad18 remain constant during lytic reactivation of wild-type virus, but reactivation of BPLF1 knockout virus resulted in decreased levels of Rad18. Finally, the contribution of Rad18 levels to infectious virus production was examined with small interfering RNA (siRNA) targeting Rad18. Results demonstrated that reducing levels of Rad18 decreased production of infectious virus, and infectious titers of BPLF1 knockout virus were partially restored by overexpression of Rad18. Thus, BPLF1 interacts with and maintains Rad18 at high levels during lytic replication, which assists in production of infectious virus. IMPORTANCE: Characterization of EBV BPLF1's deubiquitinating activity and identification of its targets and subsequent functional effects remain little studied. All members of the Herpesviridae contain BPLF1 homologs with conserved enzymatic activity, and findings discovered with EBV BPLF1 are likely applicable to other members of the family. Discovery of new targets of BPLF1 will point to cellular pathways and viral processes regulated by the enzymatic activity of the EBV-encoded deubiquitinating enzyme. Here we determined the importance of the cellular ubiquitin ligase Rad18 in these processes and how it is affected by BPLF1. Our findings demonstrate that EBV can co-opt Rad18 as a novel accessory factor in the production of infectious virus.

KSHV LANA and EBV LMP1 induce the expression of UCH-L1 following viral transformation.

Ubiquitin C-terminal Hydrolase L1 (UCH-L1) has oncogenic properties and is highly expressed during malignancies. We recently documented that Epstein-Barr virus (EBV) infection induces uch-l1 expression. Here we show that Kaposi's Sarcoma-associated herpesvirus (KSHV) infection induced UCH-L1 expression, via cooperation of KSHV Latency-Associated Nuclear Antigen (LANA) and RBP-Jκ and activation of the uch-l1 promoter. UCH-L1 expression was also increased in Primary Effusion Lymphoma (PEL) cells co-infected with KSHV and EBV compared with PEL cells infected only with KSHV, suggesting EBV augments the effect of LANA on uch-l1. EBV latent membrane protein 1 (LMP1) is one of the few EBV products expressed in PEL cells. Results showed that LMP1 was sufficient to induce uch-l1 expression, and co-expression of LMP1 and LANA had an additive effect on uch-l1 expression. These results indicate that viral latency products of both human γ-herpesviruses contribute to uch-l1 expression, which may contribute to the progression of lymphoid malignancies.

Maribavir inhibits Epstein-Barr virus transcription through the EBV protein kinase.

Maribavir (MBV) inhibits Epstein-Barr virus (EBV) replication and the enzymatic activity of the viral protein kinase BGLF4. MBV also inhibits expression of multiple EBV transcripts during EBV lytic infection. Here we demonstrate, with the use of a BGLF4 knockout virus, that effects of MBV on transcription take place primarily through inhibition of BGLF4. MBV inhibits viral genome copy numbers and infectivity to levels similar to and exceeding levels produced by BGLF4 knockout virus.

Upregulation of special AT-rich-binding protein 1 by Epstein-Barr virus latent membrane protein 1 in human nasopharyngeal cells and nasopharyngeal cancer.

A global regulator of chromatin remodelling and gene expression, special AT-rich-binding protein 1 (SATB1) has been implicated in promotion of growth and metastasis of a number of cancers. Here, we demonstrate that the principal oncogene of Epstein-Barr virus (EBV), latent membrane protein 1 (LMP1) upregulates SATB1 RNA and protein expression in human nasopharyngeal cell lines. Silencing of endogenously expressed SATB1 with specific short hairpin RNA decreases cell proliferation and resistance to apoptosis induced by growth factor withdrawal. Additionally, we provide evidence that LMP1-mediated expression of Survivin, a multifunctional protein involved in promoting cell growth and survival, is mediated at least in part by SATB1 in human nasopharyngeal cells. Finally, we show that SATB1 protein levels are elevated in tissue samples from patients with nasopharyngeal carcinoma (NPC), and are directly correlated with the expression of LMP1. Taken together, our results suggest that SATB1 functions as a pro-metastatic effector of LMP1 signalling in EBV-positive NPC.

Epstein-Barr virus latent membrane protein 1 regulates the function of interferon regulatory factor 7 by inducing its sumoylation.

Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) induces multiple signal transduction pathways during latent EBV infection via its C-terminal activating region 1 (CTAR1), CTAR2, and the less-studied CTAR3. One mechanism by which LMP1 regulates cellular activation is through the induction of protein posttranslational modifications, including phosphorylation and ubiquitination. We recently documented that LMP1 induces a third major protein modification by physically interacting with the SUMO-conjugating enzyme Ubc9 through CTAR3 and inducing the sumoylation of cellular proteins in latently infected cells. We have now identified a specific target of LMP1-induced sumoylation, interferon regulatory factor 7 (IRF7). We hypothesize that during EBV latency, LMP1 induces the sumoylation of IRF7, limiting its transcriptional activity and modulating the activation of innate immune responses. Our data show that endogenously sumoylated IRF7 is detected in latently infected EBV lymphoblastoid cell lines. LMP1 expression coincided with increased sumoylation of IRF7 in a CTAR3-dependent manner. Additional experiments show that LMP1 CTAR3-induced sumoylation regulates the expression and function of IRF7 by decreasing its turnover, increasing its nuclear retention, decreasing its DNA binding, and limiting its transcriptional activation. Finally, we identified that IRF7 is sumoylated at lysine 452. These data demonstrate that LMP1 CTAR3 does in fact function in intracellular signaling, leading to biologic effects. We propose that CTAR3 is an important signaling region of LMP1 that regulates protein function by sumoylation. We have shown specifically that LMP1 CTAR3, in cooperation with CTAR2, can limit the ability of IRF7 to induce innate immune responses by inducing the sumoylation of IRF7.

Epstein-Barr virus BPLF1 deubiquitinates PCNA and attenuates polymerase η recruitment to DNA damage sites.

PCNA is monoubiquitinated in response to DNA damage and fork stalling and then initiates recruitment of specialized polymerases in the DNA damage tolerance pathway, translesion synthesis (TLS). Since PCNA is reported to associate with Epstein-Barr virus (EBV) DNA during its replication, we investigated whether the EBV deubiquitinating (DUB) enzyme encoded by BPLF1 targets ubiquitinated PCNA and disrupts TLS. An N-terminal BPLF1 fragment (a BPLF1 construct containing the first 246 amino acids [BPLF1 1-246]) associated with PCNA and attenuated its ubiquitination in response to fork-stalling agents UV and hydroxyurea in cultured cells. Moreover, monoubiquitinated PCNA was deubiquitinated after incubation with purified BPLF1 1-246 in vitro. BPLF1 1-246 dysregulated TLS by reducing recruitment of the specialized repair polymerase polymerase η (Polη) to the detergent-resistant chromatin compartment and virtually abolished localization of Polη to nuclear repair foci, both hallmarks of TLS. Expression of BPLF1 1-246 decreased viability of UV-treated cells and led to cell death, presumably through deubiquitination of PCNA and the inability to repair damaged DNA. Importantly, deubiquitination of PCNA could be detected endogenously in EBV-infected cells in comparison with samples expressing short hairpin RNA (shRNA) against BPLF1. Further, the specificity of the interaction between BPLF1 and PCNA was dependent upon a PCNA-interacting peptide (PIP) domain within the N-terminal region of BPLF1. Both DUB activity and PIP sequence are conserved in the members of the family Herpesviridae. Thus, deubiquitination of PCNA, normally deubiquitinated by cellular USP1, by the viral DUB can disrupt repair of DNA damage by compromising recruitment of TLS polymerase to stalled replication forks. PCNA is the first cellular target identified for BPLF1 and its deubiquitinating activity.