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Epstein-Barr Virus Nuclear Antigen 1 (EBNA1) Confers Resistance to Apoptosis in EBV positive B-lymphoma Cells through Up-regulation of Survivin
Abstract
Resistance to apoptosis is an important component of the overall mechanism which drives the tumorigenic process. EBV is a ubiquitous human gamma-herpesvirus which preferentially establishes latent infection in viral infected B-lymphocytes. EBNA1 is typically expressed in most forms of EBV-positive malignancies and is important for replication of the latent episome in concert with replication of the host cells. Here, we investigate the effects of EBNA1 on survivin up-regulation in EBV-infected human B-lymphoma cells. We present evidence which demonstrates that EBNA1 forms a complex with Sp1 or Sp1-like proteins bound to their cis-element at the survivin promoter. This enhances the activity of the complex and up-regulates survivin. Knockdown of survivin and EBNA1 showed enhanced apoptosis in infected cells and thus supports a role for EBNA1 in suppressing apoptosis in EBV-infected cells. Here, we suggest that EBV encoded EBNA1 can contribute to the oncogenic process by up-regulating the apoptosis suppressor protein, survivin in EBV-associated B-lymphoma cells.
... In addition, EBNA1 is able to interact with premyelocytic leukemia proteins (PML) and induce their degradation, thereby preventing p53 activation and thus induction of apoptosis upon DNA damage 29 . Furthermore, EBNA1 contributes to the oncogenic process by inducing the apoptosis suppressor protein Survivin in EBV-associated lymphomas 30 . ...
... Finally, the EBV-based plasmid partitioning assay also allowed the group of L. Frappier to test the ability of other host proteins, Brd2, Brd4 (which are members of the bromodomain and extra terminal domain-(BET-)family) and MeCP2 that are implicated in the genome segregation of papillomavirus and Kaposi's sarcoma-associated herpesvirus, to support EBNA1-mediated segregation of EBV-based plasmids [30]. They found that Brd4 enabled EBNA1-based segregation of the EBV genome, while Brd2 and MeCP2 had a general stimulatory effect on the plasmid maintenance. ...
... Briefly, this oligonucleotide (GQ) was linked to biotin and pulldown experiments using 204 streptavidin-conjugated sepharose beads were performed. As a negative control, we used an 205 oligonucleotide (GM) with a similar sequence except that the four guanines forming the G4 were 206 replaced by adenines or uridines in order to completely abolish the G4 structure, as predicted 207 using the GQRS-H predictor software30 . As a positive control, we used ARPC2 30 nt-long 208 oligonucleotide which corresponds to a G4 found in the ARPC2 mRNA and that has been shown 209 to bind NCL 28 . ...
The Epstein-Barr Virus (EBV) is the first oncogenic virus described in humans. It is a ubiquitous virus which infects over 90% of the human population. EBV causes a latent infection of B lymphocytes and remains asymptomatic in most of infected individuals. However, insome conditions like immunosuppression, EBV can cause uncontrolled cell proliferation responsible for some types of cancers like Burkitt lymphoma, Hodgkin lymphoma and nasopharyngeal carcinoma. During latency, EBNA1 is the only protein expressed in all EBV-infected cells as it is essential for EBV genome replication and maintenance. EBNA1 is highly antigenic and T cells raised against EBNA1 exist ; nevertheless EBV-infected cells are not eliminated by the host immune system. This is due to the central GAr (Gly-Ala repeat) domain of EBNA1 which is able to inhibit the translation of its own mRNA in cis, there by preventing the recognition of EBV-infected cells by the immunesystem of the host. The mechanisms involved in this GAr-based translation inhibition were unknown when I started my PhD. During this thesis, we identified the first cellular factor, Nucleolin (NCL), able to affect GAr-based translation inhibition. We showed that NCL over expression enhances EBNA1 translation inhibition and inversely, that its down regulation decreases EBNA1 translation inhibition effect. Furthermore, we demonstrated that the interaction between NCL and GArRNA occurs throught NCL binding to GAr-encoding mRNA via a particular secondary structure called G-quadruplex (G4).We showed that disrupting NCL-GAr RNA interaction with small G4 ligands enhances EBNA1 translation and thus antigen presentation, which constitutes a new therapeutic avenue to treat EBV-associated cancers. Finally, we went deeper into the mechanism involved in this interaction by showing that NCL has a direct effect on the GAr-based translation inhibition and that the nuclear location of NCL-GAr RNA interaction is forced by the nuclear location of NCL in the cells. Taken together, these results have shed light upon the mechanism involved in the GAr domain translation inhibition and immune evasion, and have revealed a possible therapeutic target to eradicate EBV-associated cancers.
... The initial EBV infection is often asymptomatic and is transmitted through oral secretions during childhood. EBV then establishes a long-term latent infection in a small subset of human memory B cells (MemB), which can last throughout the lifetime of a healthy individual [5]. MemB cells express three latent proteins as a part of EBV's latency transcription program, with Epstein-Barr nuclear antigen 1 (EBNA1) being the only consistently expressed protein in all EBV-associated malignancies [6,7]. ...
... EBNA1 plays a crucial role in EBV episome replication and stable persistence and has been shown to possess the potential to modify the cellular environment. Moreover, EBNA1 promotes genomic instability and potentially acts as an oncogene [5], which might make cytotoxic T lymphocytes unable to effectively recognize and respond to infected cells, thereby allowing EBV-infected cells to evade immune surveillance [8]. Understanding these mechanisms may provide valuable insights that help in identifying effective drug targets, developing promising therapeutic strategies, and minimizing the toxicity associated with individual drugs. ...
Objective
Epstein-Barr virus (EBV)-associated gastric cancer (EBVaGC) is a distinct molecular subtype of gastric cancer (GC). At present, the clinical characteristics and prognostic implications of EBV infection and the potential clinical benefits of immune checkpoint blockade in GC remain to be clarified. Hence, this study was designed to analyze the clinical and pathological characteristics of GC patients with varying EBV infection states and compare their overall survival (OS).
Methods
A retrospective study was performed on 1031 consecutive GC patients who underwent gastrectomy at the Affiliated Hospital of Xuzhou Medical University from February 2018 to November 2022. EBV-encoded RNA (EBER) in situ hybridization (ISH) was used for EBV assessment, and immunohistochemical staining was used for evaluation of human epidermal growth factor receptor 2 (HER2), programmed death ligand 1 (PD-L1), and Ki67 expression. EBVaGC was defined as tumors with EBV positivity. In addition, EBV-negative GC (EBVnGC) patients were matched with EBVaGC patients based on seven clinicopathological parameters (age, gender, anatomic subsite, tumor size, Lauren classification, degree of differentiation, and tumor-node-metastasis [TNM] stage). The correlations of clinical features with HER2, PD-L1, and Ki67 expression were evaluated statistically. The survival of patients was assessed through medical records, telephone, or WeChat communication, and prognostic analysis was performed using the logrank test as well as univariable and multivariable regression analysis.
Results
Out of 1031 GC patients tested, 35 (3.4%) were diagnosed with EBVaGC. Notably, the EBVaGC group exhibited a distinct predominance of males and younger patients, significantly higher Ki67 and PD-L1 expression levels, and a lower prevalence of pericancerous nerve invasion than the EBVnGC group (P < 0.01). In the 35 EBVaGC cases, Ki67 expression was negatively correlated with age (P < 0.05), suggesting that a younger onset age was associated with higher Ki67 expression. In addition, PD-L1 expression was correlated with the degree of differentiation, T-stage, and clinical stage of the patient. Furthermore, PD-L1 expression was elevated in tumors with lower differentiation or at later stages (P < 0.05). Using univariate analysis, Ki67, PD-L1, and clinical stage were identified as significant factors influencing the overall survival (OS) of EBVaGC patients (P < 0.05). Moreover, multivariate survival analysis revealed that clinical stage and Ki67 expression were independent risk factors for the OS of the patients (P < 0.05), and the three-year OS rate of EBVaGC patients was 64.2%.
Conclusion
EBV-ISH is a practical and valuable method to identify EBVaGC. Owing to its unique etiological, pathological, and clinical characteristics, patients with EBVaGC might benefit from immune checkpoint blockade therapy.
... EBNA1 is another latent protein implicated in tumor formation. In B cell lymphoma, it has been shown to upregulate an anti-apoptotic protein named survivin [72,73]. In nasopharyngeal carcinoma, ENBA1 can inhibit NF-κB through inhibition of IKKα and β, which promotes development of squamous cell carcinoma by stimulating tissue hyperplasia [74]. ...
... Vasculogenic mimicry is a mechanism by which tumor cells secure a blood supply in the absence of angiogenesis or endothelial cells (reviewed in [72]). Recently, it was discovered that EBV promotes vasculogenic mimicry in epithelial cancers, including NPC and GC [84]. ...
Epstein–Barr virus (EBV) is one of eight known herpesviruses with the potential to infect humans. Globally, it is estimated that between 90–95% of the population has been infected with EBV. EBV is an oncogenic virus that has been strongly linked to various epithelial malignancies such as nasopharyngeal and gastric cancer. Recent evidence suggests a link between EBV and breast cancer. Additionally, there are other, rarer cancers with weaker evidence linking them to EBV. In this review, we discuss the currently known epithelial malignancies associated with EBV. Additionally, we discuss and establish which treatments and therapies are most recommended for each cancer associated with EBV.
... EBNA1 ne semble pas être indispensable pour la transformation des cellules B in vitro mais pourrait contribuer au l'oncogenèse. En effet, EBNA 1 interfère avec l'axe p53/Mdm2 via une interaction avec USP7 (Ubiquitin-specific protease 7) avec comme conséquence un effet anti-apoptotique par diminution de p53 et augmentation de la survivine (Frappier, 2012;Kennedy et al., 2003;Lu et al., 2011). Un autre effet anti-apoptotique a également été rapporté par inhibition de l'expression de la protéine pro-apoptotique Bim dans des cellules de LB . ...
... Des mutations fréquentes sur le gène TP53 inhibent l'apoptose p53dépendante. On peut également observer une augmentation de Mdm2, un régulateur négatif de p53(Eischen et al., 1999;Kelly and Rickinson, 2007;Lindström and Wiman, 2002); une mutation sur MYC affectant sa capacité à activer la protéine pro-apoptotique Bim(Adhikary and Eilers, 2005;Hemann et al., 2005) ou encore un effet anti-apoptotique exercé par les protéines du virus EBV telle que EBNA1(Lu et al., 2011). MYC confère aux cellules un phénotype non immunogénique avec diminution de l'expression des molécules du CMHI, une diminution des molécules d'adhésion, une altération de la voie NF-KB et une diminution de la réponse aux IFN de type 1 favorisant ainsi l'échappement de la cellule au système immunitaire(Polack et al., 1996;Schlee et al., 2007;Staege et al., 2002). ...
Le Lymphome de Burkitt est un lymphome non hodgkinien de type B caractérisé dans 80% des cas par une translocation chromosomique impliquant l’oncogène MYC sur le chromosome 8 et le gène des chaines lourdes d’immunoglobuline IGH sur le chromosome 14. Le développement de ce lymphome est étroitement lié au virus EBV, systématiquement présent dans les formes endémiques et au VIH qui lui définit une entité clinique à part. Cependant, le rôle joué par ces virus dans la formation de la translocation est peu connu. Des cassures double brins simultanées, une réparation non-homologue de ces cassures et une proximité spatiale sont les conditions nécessaires pour que deux loci forment une translocation. Nous avons ainsi investigué l’effet des virus EBV sur ces trois conditions dans les lymphocytes B. Nos résultats montrent que la réactivation du virus EBV dans les lymphocytes B infectées de façon latente augmente la proximité spatiale entre MYC et IGH, normalement situés dans des compartiments différents. Cette relocalisation semble être secondaire à l’induction de dommages d’ADN spécifiques sur MYC et serait médiée par la protéine de réparation de l’ADN MRE11. Nous montrons aussi que la protéine Tat du VIH-1 peut pénétrer dans les cellules B et y induire une expression aberrante du gène AICDA qui peut secondairement introduire des cassures doubles brins simultanées sur MYC et IGH. Ce travail fournit une preuve de l’implication directe du cycle lytique d’EBV dans la formation de la t(8;14) et renforce les données existantes sur le rôle de Tat dans cette translocation.
... These data correlate with the observation that the transcriptional activation function of EBNA1 is essential to drive transcription of the EBV transforming genes after infection of primary lymphocytes [118]. Many studies have now revealed that EBNA1 also regulates a wide variety of functionally distinct cellular genes [119][120][121][122][123][124][125] and binds numerous sites within cell chromatin [121,122,124]. ...
... Similarly, shRNA depletion of MEF2b resulted in significant loss of cell viability in both LCL and various BL EBV-positive cell lines, but not in EBV-negative B cell lines [126]. In BJAB cells (an EBV-negative BL cell line) constitutively expressing EBNA1, the viral protein has been found to directly up-regulate survivin, an inhibitor of apoptosis expressed in many human cancer cells [125]. Thus, EBNA1 function in transcriptional regulation of host cellular genes could account for the previously reported crucial role of EBNA1 in drastically enhancing B-cell immortalization by EBV [118,127] as well as providing survival signals to EBV-positive BL cells and inhibiting p53-induced apoptosis in non-infected cells [17]. ...
The presence of the Epstein-Barr virus (EBV)-encoded nuclear antigen-1 (EBNA1) protein in all EBV-carrying tumours constitutes a marker that distinguishes the virus-associated cancer cells from normal cells and thereby offers opportunities for targeted therapeutic intervention. EBNA1 is essential for viral genome maintenance and also for controlling viral gene expression and without EBNA1, the virus cannot persist. EBNA1 itself has been linked to cell transformation but the underlying mechanism of its oncogenic activity has been unclear. However, recent data are starting to shed light on its growth-promoting pathways, suggesting that targeting EBNA1 can have a direct growth suppressing effect. In order to carry out its tasks, EBNA1 interacts with cellular factors and these interactions are potential therapeutic targets, where the aim would be to cripple the virus and thereby rid the tumour cells of any oncogenic activity related to the virus. Another strategy to target EBNA1 is to interfere with its expression. Controlling the rate of EBNA1 synthesis is critical for the virus to maintain a sufficient level to support viral functions, while at the same time, restricting expression is equally important to prevent the immune system from detecting and destroying EBNA1-positive cells. To achieve this balance EBNA1 has evolved a unique repeat sequence of glycines and alanines that controls its own rate of mRNA translation. As the underlying molecular mechanisms for how this repeat suppresses its own rate of synthesis in cis are starting to be better understood, new therapeutic strategies are emerging that aim to modulate the translation of the EBNA1 mRNA. If translation is induced, it could increase the amount of EBNA1-derived antigenic peptides that are presented to the major histocompatibility (MHC) class I pathway and thus, make EBV-carrying cancers better targets for the immune system. If translation is further suppressed, this would provide another means to cripple the virus.
... Effect of Latency I EBV genes on BL cell survival. The literature mapping EBV protection to a Latency I gene product (namely EBNA1 protein, EBERs or miR-BARTs), is highly contradictory 18,25,[28][29][30][31][32][33] (reviewed in the Discussion). To broaden these assorted but limited analyses, each of the Latency I-associated genes were introduced into multiple EBV-loss clones derived from several tumours. ...
... EBNA1 is essential for virus genome maintenance and also reported to function in cell survival in certain circumstances. 31 EBER RNAs were shown to confer tumorigenicity to EBV-loss Akata-BL in vivo and to enhance the growth properties of cells in vitro, in part through upregulation of BCL-2 and IL-10. 28,54 Yet we found no evidence that BCL-2 or IL-10 were more highly expressed in EBER-expressing or EBV-positive cells compared with EBV-loss cells. ...
While the association of Epstein–Barr virus (EBV) with Burkitt lymphoma (BL) has long been recognised, the precise role of the virus in BL pathogenesis is not fully resolved. EBV can be lost spontaneously from some BL cell lines, and these EBV-loss lymphoma cells reportedly have a survival disadvantage. Here we have generated an extensive panel of EBV-loss clones from multiple BL backgrounds and examined their phenotype comparing them to their isogenic EBV-positive counterparts. We report that, while loss of EBV from BL cells is rare, it is consistently associated with an enhanced predisposition to undergo apoptosis and reduced tumorigenicity in vivo. Importantly, reinfection of EBV-loss clones with EBV, but surprisingly not transduction with individual BL-associated latent viral genes, restored protection from apoptosis. Expression profiling and functional analysis of apoptosis-related proteins and transcripts in BL cells revealed that EBV inhibits the upregulation of the proapoptotic BH3-only proteins, BIM and PUMA. We conclude that latent EBV genes cooperatively enhance the survival of BL cells by suppression of the intrinsic apoptosis pathway signalling via inhibition of the potent apoptosis initiators, BIM and PUMA.
... Вирус Эпштейн-Барр обладает белком EBNA1, состоящим из 60-300 аминокислотных остатков, богатых аланином и глицином (glucine-alanine repeat -GAr). Этот белок является «стоп-сигналом» для протеасомы, поэтому блокирование протеолиза нарушает презентацию антигенов вируса на MHC I класса, а значит, блокируется иммунный ответ, что позволяет вирусу персистировать в организме человека [16][17]. ...
Nuclear transcription factor NF-kB1 is a regulator of the expression of immune response genes, apoptosis, inflammatory reactions and cell proliferation. Microorganisms are able to change the activity of the nuclear transcription factor, which allows them to modulate the immune response of the macroorganism and/or have a pathogenic effect on organs and tissues. The aim of the study is to systematize data on microbial molecular factors regulating the transcription nuclear factor NFkB-1. The review describes microbial molecules, the mechanisms of their action on the nuclear transcription factor NF-kB1. The A52R protein of the smallpox vaccine virus acts as a homologue of MyD88, the Yersinia YopP/J protein acetylates the IKKß subunit, thereby inhibiting kB kinase (IKK) and preventing the activation of the nuclear factor NFkB. The EBNA1 protein of the Epstein-Barr virus is a "stop signal" for the proteasome, so blocking proteolysis disrupts the presentation of virus antigens on MHC I and the overall immune response. The OspG protein of microorganisms of the genus Shigella prevents ubiquitination, and also inhibits the degradation of phosphorylated IKKß, which disrupts the activation of NFkB-1. Enteropathogenic Escherichia coli (EPEC) produces three effectors to inhibit the function of NFkB. Two of these effectors, NleB and NleE block signaling at the level of TGF-beta-activated kinase 1 or IKKß kinase, the third NleH protein binds to the RPS3 (ribosomal protein S3) cofactor of the NFkB factor and suppresses the transcription of many genes. The components of bacterial cells – lipopolysaccharides of the cell walls of gram-negative bacteria, representatives of the Enterobacteriaceae family, participate in modulating the activity of the transcription factor. This polymer affects the presentation genes of MHC class I and II antigens, the death receptor, IL-6 signaling, IFN-γ signaling and the P38 signaling pathway. Conclusion. Protein and non-protein substances of microorganisms, components of bacterial cell structures are molecules that have a multidirectional effect on the activity of proteasomes that regulate inflammatory and immune responses in the macroorganism.
... EBNA1 was found to be critical for the immortalization of B cells (153,154). Survivin, an inhibitor of apoptosis, was also found to be activated in B lymphocytes by EBNA1 (155). In a recent study, it was demonstrated that EBNA1 results in a dose-dependent breakage at chromosome 11q23 (156). ...
Epstein-Barr virus (EBV), one of the most common human viruses, has been associated with both lymphoid and epithelial cancers. Undifferentiated nasopharyngeal carcinoma (NPC), EBV associated gastric cancer (EBVaGC) and lymphoepithelioma-like carcinoma (LELC) are amongst the few common epithelial cancers that EBV has been associated with. The pathogenesis of EBV-associated NPC has been well described, however, the same cannot be said for primary pulmonary LELC (PPLELC) owing to the rarity of the cancer. In this review, we outline the pathogenesis of EBV-associated NPC and EBVaGCs and their recent advances. By drawing on similarities between NPC and PPLELC, we then also postulated the pathogenesis of PPLELC. A deeper understanding about the pathogenesis of EBV enables us to postulate the pathogenesis of other EBV associated cancers such as PPLELC.
... According to our study, the expression level of BIRC5 gene was about tenfold higher in EBNA1 transfected HeLa cells (compared to control cells). In agreement with our study, Lu et al. stated that in Burkitt's lymphoma, EBNA1 increases the expression of the BIRC5 gene by forming a complex and binding to the promoter of this gene (37). BIRC5 (also named survivin) is a member of the inhibitors of apoptosis proteins (IAPs) family, which by interfering in the functions of caspases 3, 7 and also inhibiting Bax and Fas-induced apoptotic pathways, causes abnormal cells to survive (38). ...
Epstein-Barr virus (EBV) represents one of the most important viral carcinogens. EBV nuclear antigen-1 (EBNA1) can induce the expression of different cellular and viral genes. In this study, we evaluated the EBNA1 effects on the expression patterns of human papillomavirus type 18 (HPV-18) E6 and E7 oncogenes and three cellular genes, including BIRC5, c-MYC, and STMN1, in a cervical adenocarcinoma cell line. HeLa cells were divided into three groups: one transfected with a plasmid containing the EBNA1 gene, one transfected with a control plasmid, and one without transfection. In all three groups, the expression levels of E6, E7, BIRC5, c-MYC, and STMN1 genes were checked using real-time PCR. Pathological staining was used to examine changes in cell morphology. Real-time PCR results showed that the expression level of HPV-18 E6 (P=0.02) and E7 (P=0.02) oncogenes significantly increased in HeLa cells transfected with the EBNA1 plasmid compared to cells transfected with control plasmid. Also, the presence of EBNA1 induced the expression of BIRC5 and c-MYC, which increased tenfold (P=0.03) and threefold (P=0.02), respectively. Regarding the STMN1 cellular gene, although the expression level in HeLa cells transfected with EBNA1 plasmid showed a twofold increase, this change was insignificant (P=0.11). Also, EBNA1 expression caused the creation of large HeLa cells with abundant cytoplasm and numerous nuclei. The EBV-EBNA1 could increase the expression levels of HPV-18 E6 and E7 viral oncogenes as well as c-MYC and BIRC5 cellular genes in the HeLa cell line. These findings indicate that the simultaneous infection of cervical cells with HPV-18 and EBV might accelerate the progression of cervical cancer.
... EBNA1 is expressed in the lytic phase and all EBV latency types except latency type 0. EBNA1 is the only viral protein involved in DNA replication in latency and is expressed in all EBV-related tumors (20,21). Some reports showed that the expression of EBNA1 increased the tumorigenicity of EBV-negative tumors (20). ...
Background & objective:
Epstein-Barr virus nuclear antigen-1 (EBNA1) is one of the most important proteins of Epstein-Barr virus (EBV) that might be mutated in various related cancers. The purpose of this study was to compare EBNA1 mutations in the C-terminal region between patients with cervical and ovarian cancer and healthy individuals.
Methods:
As test and control groups, 18 EBV-positive paraffin-embedded samples of cervical and ovarian cancer and 10 age- and gender-matched healthy volunteers who did not have cancer but were EBV-positive were both used. Utilizing a commercial DNA extraction kit, total DNA was extracted following deparaffinization. The entire C-terminal region of the EBNA1 sequence was amplified using an in-house nested PCR. Phylogenetic analysis and Sanger sequencing were used to analyze the sequences using MEGA 7 software and through NJ method.
Results:
Sequence analysis revealed that the P-Ala subtype of EBNA1 was present in all samples. In two and one samples, respectively, of cervical cancer patients, the mutations A1887G and G1891A were found. The G1595T mutation was also detected in four sequences taken from ovarian cancer patients. No statistically significant difference could be found between the frequency of mutations in patients and controls (P>0.05). No known amino acid substitutions were found in the USP7-binding region and the DBD/DD domain.
Conclusion:
The findings showed that P-Ala is the predominant EBV subtype across all samples. Additionally, as the sequence of EBNA1's C-terminal region is so stable, it's possible that it had little impact on the pathogenesis of ovarian and cervical malignancies. It is advised to conduct additional research to verify these findings.
... In vivo experiments have revealed that EBNA-1 decreases the p53 level in GC, enhances apoptosis resistance of the cells, favors their survival and outgrowth, and thus promotes the pathogenesis of the malignancy [165]. In EBV-positive BL cells, EBNA-1 cooperates with Sp1 or Sp1-like proteins to enhance the expression of anti-apoptotic protein, survivin [169] that is known to block the activity of executioner caspases-3 and -7 and thus protect the cells from death via both extrinsic and intrinsic apoptosis pathway [170]. Another mechanism of the EBNA-1-mediated anti-apoptotic effect is to bind tumor suppressor NM23-H1, a protein that stimulates transcription of the genes encoding p53 and pro-apoptotic caspases-9 and -3. ...
Epstein-Barr virus (EBV), the representative of the Herpesviridae family, is a pathogen extensively distributed in the human population. One of its most characteristic features is the capability to establish latent infection in the host. The infected cells serve as a sanctuary for the dormant virus, and therefore their desensitization to apoptotic stimuli is part of the viral strategy for long-term survival. For this reason, EBV encodes a set of anti-apoptotic products. They may increase the viability of infected cells and enhance their resistance to chemotherapy, thereby contributing to the development of EBV-associated diseases, including Burkitt’s lymphoma (BL), Hodgkin’s lymphoma (HL), gastric cancer (GC), nasopharyngeal carcinoma (NPC) and several other malignancies. In this paper, we have described the molecular mechanism of anti-apoptotic actions of a set of EBV proteins. Moreover, we have reviewed the pro-survival role of non-coding viral transcripts: EBV-encoded small RNAs (EBERs) and microRNAs (miRNAs), in EBV-carrying malignant cells. The influence of EBV on the expression, activity and/or intracellular distribution of B-cell lymphoma 2 (Bcl-2) protein family members, has been presented. Finally, we have also discussed therapeutic perspectives of targeting viral anti-apoptotic products or their molecular partners.
... De plus, cette protéine serait essentielle dans la survie des LB, car elle inhibe l'apoptose en induisant l'expression de p53 (Kennedy et al., 2003) et de la survivine, une protéine anti-apoptotique (Lu et al., 2011). ...
Le virus Epstein-Barr (EBV) est un virus
humain ubiquitaire dont la persistance à l’état latent
est associée à de nombreux cancers, tels que le
lymphome de Burkitt (LB). BHRF1 est une orthologue
de BCL-2, qui s’exprime au cours de la latence et du
cycle productif. Cette protéine virale est retrouvée dans
15 % des LB liés à l’EBV, les rendant particulièrement
résistants au traitement. Initialement, BHRF1 était
connue pour son activité anti-apoptotique. Dans ce
travail, nous avons souhaité mieux caractériser le rôle
de BHRF1, notamment au regard de l’autophagie, un
processus de recyclage de constituants cellulaires.
Nous avons identifié que BHRF1 stimule l’autophagie
et altère la morphodynamique des mitochondries.
Plus précisément, BHRF1 augmente la fission
mitochondriale et exploite le réseau de microtubules
pour induire la formation de mito-agrésomes dans la
région périnucléaire. L’ensemble de ces altérations
cellulaires conduisent à la dégradation sélective
des mitochondries par autophagie, un processus
appelé mitophagie. Nous démontrons aussi que
BHRF1 constitue un facteur de restriction de
l’immunité antivirale puisqu’elle bloque la
production de cytokines antivirales, les interférons.
Dans la dernière partie de ce travail, nous avons
recherché si ces fonctions étaient retrouvées dans
des lymphomes EBV+ en utilisant des cellules de LB
exprimant BHRF1. Dans ce modèle, BHRF1 induit
des mito-agrésomes et est aussi à l’origine d’une
autophagie cytoprotectrice vis-à-vis de l’apoptose.
En conclusion, nous proposons que ces nouvelles
fonctions de BHRF1 pourraient participer à la
chimiorésistance et à la lymphomagenèse des LB
liés à l’EBV.
... Epstein-Barr viral NA1 activates the replication activity through its location (Sp1) in the promoter and induces survives in protein expression. By the inhibition of caspase pathways in the positive cells of the EBV, Therefore, the regulation of survival in expression will suppress apoptosis of the cell (Lu et al., 2011). The genome in stability, including DNA damage, chromosomal anomalies and the double-stranded DNA can be stimulated by EBNA1, (RAG-2) and (RAG-1) and ROS (increased by activation of transcription activity) (nX2) / gp91phox, catalytic susceptibility to NADPH oxidase (Gruhne et al., 2009). ...
Rheumatoid arthritis is a systemic autoimmune disease characterized by chronic, destructive, debilitating arthritis. The cause of rheumatoid arthritis (RA) is still unknown. Both genetic and environmental factors may help its development. For 25 years, the Epstein Barr virus has been suspected to contribute to RA pathogenesis. RA patients have higher levels of anti EBV antibodies than healthy controls. EBV, a widespread virus, highly recognized by antibodies but never eliminated, is an ideal candidate to trigger chronic immune complex disease. This virus stimulates polyclonal lymphocyte expansion and persists within B lymphocyte for the host's life, inhibited from reactivating by the immune response. Anti-EBV antibody responses should be considered as one of the chronic autoantibody responses that are the most relevant to the development of RA.Recent results link the removal of controls from Epstein Barr virus and autoimmune diseases, The Epstein virus causes continuous infection with programmed response time in B cells that enables immune defense to escape. A number of immune modified immune proteins and escape mechanisms have been described in EBV.
keyword: EBV, Epstein Barr virus, rheumatoid arthritis ,RA
... For example, it can cause chromosomal instability [66], and the transfection of mice with expression plasmids for EBNA1 resulted in a higher rate of lymphomas in the recipients [67]. EBNA1 can also enhance the expression of survivin (an apoptotic suppressor) and STAT1, while repressing TGF-b [68,69]. EBNA1 can bind a region located on chromosome 11 that contains several promoters, altering the expression of many genes, including HDAC3 and MAP3K1 [70]. ...
Increasing evidence suggests that microorganisms might represent at least highly interesting co‐factors in colorectal cancer (CRC) oncogenesis and progression. Still, associated mechanisms, specifically in colonocytes and their microenvironmental interactions, are still poorly understood. Although, currently, at least seven viruses are being recognized as human carcinogens, only three of these – Epstein‐Barr virus (EBV), human papillomavirus (HPV) and John Cunningham virus (JCV) – have been described, with varying levels of evidence, in CRC. In addition, cytomegalovirus (CMV) has been associated with CRC in some publications, albeit not being a fully acknowledged oncovirus. Moreover, recent microbiome studies set increasing grounds for new hypotheses on bacteriophages as interesting additional modulators in CRC carcinogenesis and progression. The present Review summarizes how particular groups of viruses, including bacteriophages, affect cells and the cellular and microbial microenvironment, thereby putatively contributing to foster CRC. This could be achieved, for example, by promoting several processes – such as DNA damage, chromosomal instability, or molecular aspects of cell proliferation, CRC progression and metastasis – not necessarily by direct infection of epithelial cells only, but also via interaction with the microenvironment of infected cells. In this context, there are striking common features of EBV, CMV, HPV and JCV that are able to promote oncogenesis, in terms of establishing latent infections and affecting p53‐/pRb‐driven, epithelial–mesenchymal transition (EMT)‐/EGFR‐associated, and especially Wnt/β‐catenin‐driven pathways. We speculate that, at least in part, such viral impacts on particular pathways might be reflected in lasting (e.g., mutational or further genomic) fingerprints of viruses in cells. Also, the complex interplay between several species within the intestinal microbiome, involving a direct impact on colorectal and microenvironmental cells but also between, for example, phages and bacterial and viral pathogens, certainly might, in part, explain ongoing difficulties to establish unequivocal monocausal links between specific viral infections and CRC.
... EBERs were also found to bind retinoic acid-inducible gene I (RIG-I) and thus activate its downstream signaling 33 . EBNA is essential for viral DNA replication and episome maintenance during cellular replication at latent stages 34 , EBNA1 also contribute to the oncogenic process by upregulating the apoptosis suppressor protein, survivin in EBV-associated B-lymphoma cells 35 , so EBNA1 is associated with the survival of Burkitt's lymphoma cells 34&35 . ...
... Oncogenic DNA viruses establish long-term persistent intracellular infections and can induce malignancy by providing a selective growth advantage to host cells (Moore et al., 2010). Epstein-Barr virus (EBV) is one of the first described human cancer viruses and is associated with up to 10 types of cancers, including Burkitt lymphoma, nasopharyngeal carcinoma (NPC) (Arvey et al., 2012;Xu et al., 2019), Hodgkin lymphomas, natural killer/T (NK/T) cell lymphomas (Lu et al., 2011;Peng et al., 2019) and a subset of gastric carcinomas (Iizasa et al., 2012;Nishikawa et al., 2018). ...
Motivation:
Epstein-Barr virus (EBV) is one of the most prevalent DNA oncogenic viruses. The integration of EBV into the host genome has been reported to play an important role in cancer development. The preference of EBV integration showed strong dependence on the local genomic environment, which enables the prediction of EBV integration sites.
Results:
An attention-based deep learning model, DeepEBV, was developed to predict EBV integration sites by learning local genomic features automatically. First, DeepEBV was trained and tested using the data from the dsVIS database. The results showed that DeepEBV with EBV integration sequences plus Repeat peaks and 2 fold data augmentation performed the best on the training dataset. Furthermore, the performance of the model was validated in an independent dataset. In addition, the motifs of DNA-binding proteins could influence the selection preference of viral insertional mutagenesis. Furthermore, the results showed that DeepEBV can predict EBV integration hotspot genes accurately. In summary, DeepEBV is a robust, accurate and explainable deep learning model, providing novel insights into EBV integration preferences and mechanisms.
Availability:
DeepEBV is available as open-source software and can be downloaded from https://github.com/JiuxingLiang/DeepEBV.gitSupplementary information Supplementary data are available at Bioinformatics online.
... EBNA1 binds to the viral origin of replication (OriP) and recruits cellular replication machinery to promote viral genome replication and episomal maintenance [10,14]. EBNA1 promotes immortalization and host cell survival by manipulating several cellular processes including p53 stabilization and pro-apoptotic pathways [15,16]. In addition to regulating the transcription of several viral latency genes, EBNA1 also binds directly to promoter regions and regulates the expression of several cellular genes, including the GCspecific tumor suppressor genes gastrokine 1 and 2 [17]. ...
Background and aims:
Epstein-Barr virus (EBV)-associated gastric carcinoma (EBVaGC) is the most common EBV-associated cancer and accounts for ~ 10% of all gastric cancers (GC). Epstein-Barr virus nuclear antigen 1 (EBNA1), which is critical for the replication and maintenance of the EBV latent genome, is consistently expressed in all EBVaGC tumors. We previously developed small molecule inhibitors of EBNA1. In this study, we investigated the efficacy and selectivity of an EBNA1 inhibitor in cell-based and animal xenograft models of EBV-positive and EBV-negative gastric carcinoma.
Methods:
We tested the potency of an EBNA1 inhibitor, VK-1727, in vitro and in xenograft studies, using EBV-positive (SNU719 and YCCEL1) and EBV-negative (AGS and MKN74) GC cell lines. After treatment, we analyzed cell viability, proliferation, and RNA expression of EBV genes by RT-qPCR.
Results:
Treatment with VK-1727 selectively inhibits cell cycle progression and proliferation in vitro. In animal studies, treatment with an EBNA1 inhibitor resulted in a significant dose-dependent decrease in tumor growth in EBVaGC xenograft models, but not in EBV-negative GC xenograft studies. Gene expression analysis revealed that short term treatment in cell culture tended towards viral gene activation, while long-term treatment in animal xenografts showed a significant decrease in viral gene expression.
Conclusions:
EBNA1 inhibitors are potent and selective inhibitors of cell growth in tissue culture and animal models of EBV-positive GC. Long-term treatment with EBNA1 inhibitors may lead to loss of EBV in mouse xenografts. These results suggest that pharmacological targeting of EBNA1 may be an effective strategy to treat patients with EBVaGC.
... Several factors associated with modulation of cell death pathways (e.g., autophagy-related genes 5 and survivin 6 ) have been shown to be dysregulated in virus-infected cells. One of the major molecular controllers of cell death, is microRNAs (miRNAs), which can be affected by viral infections. ...
Viral infections lead to the death of over a million people each year around the world, both directly and indirectly. Viruses interfere with many cell functions, particularly critical pathways for cell death, by affecting various intracellular mediators. MicroRNAs (miRNAs) are a major example of these mediators, because they are involved in many (if not most) cellular mechanisms. Virus-regulated miRNAs have been implicated in three cell death pathways, namely, apoptosis, autophagy, and anoikis. Several molecules (e.g., BECN1 and BCL2 family members) are involved in both apoptosis and autophagy, while activation of anoikis leads to cell death similar to apoptotsis. These mechanistic similarities suggest that common regulators, including some miRNAs (e.g., miR-21 and miR-192) are involved in different cell death pathways. Because the balance between cell proliferation and cell death is pivotal to the homeostasis of the human body, miRNAs that regulate cell death pathways have drawn much attention from researchers. MiR-21 is regulated by several viruses, and can affect both apoptosis and anoikis via modulating various targets, such as PDCD4, PTEN, IL-12, Maspin, and Fas-L. MiR-34 can be down-regulated by viral infection, and has different effects on apoptosis, depending on the type of virus and/or host cell. The present review summarizes the existing knowledge on virus-regulated miRNAs involved in the modulation of cell death pathways. Understanding the mechanisms for virus-mediated regulation of cell death pathways could provide valuable information to improve the diagnosis and treatment of many viral diseases.
... MicroRNAs (miRNA) are small, single stranded and noncoding RNAs that can be up to 25 nucleotides in length with 5′ phosphate and 3′ hydroxyl end. The miRNAs have vital roles in various biological processes like development, differentiation, growth, cell division, apoptosis, cancer, maintaining physiological homeostasis, and various biological process [1][2][3][4][5]. They play an essential regulatory role in animals and plants by targeting specific mRNA for degradation or translation repression [3]. ...
Previous literature supports the variations in microRNAs expression levels among lymphoma patients due to EBV infection. These alterations can be observed in both EBV-encoded-microRNAs and EBV-induced cellular microRNAs. Moreover, changes in the microRNA profile could be significant in disease progression. This study aimed to assess published literature to obtain a microRNA profile for both EBV-encoded microRNAs and EBV-induced cellular microRNAs among lymphoma patients. We searched common available electronic databases by using relevant keywords. The result demonstrated that EBV infection could alter the microRNA expression levels among lymphoma patients. In Burkitt lymphoma, hsa-miR197 and miR510 were most frequently assessed human micro RNAs. Also, miR-BART6-3P and miR-BART17-5P were the most frequent viral micro RNAs in Burkitt lymphoma. Other human important micro RNAs were hsa-miR155 (in Diffuse large B cell lymphoma (DLBCL)), hsa-miR145 (in Nasal natural killer T cell lymphoma (NNKTCL)), miR-96, miR-128a, miR-128b, miR-129, and miR-205 (in Classic Hodgkin lymphoma (CHL)), miR-21, miR-142-3P, miR-126, miR-451 and miR-494-3P (in Nasal natural killer cell lymphoma (NNKCL)). Also, viral assessed micro RNAs were miR-BART1-5P (in DLBCL and NNKTCL), miR-BART-5 (in CHL), and EBV-miR-BART20-5P (in NNKCL). In conclusion, it could be suggested that EBV-encoded-microRNAs and EBV-induced cellular-microRNAs can be utilized as helpful factors for different types of lymphoma diagnoses or prognostic factors. Moreover, the mentioned microRNAs can also be promising therapeutic targets and can be used to modulate the oncogenes.
... In addition, EBNA1 interacts with USP7, the regulator of p53, and depletes p53 through proteasome degradation. EBNA1 also blocks caspase activation through Survivin [172,173]. LMP1 triggers resistance in NPC cells through PI3K/Akt/FOXO3a pathway by altering human miR-21 expression [174]. ...
Simple Summary
Epstein-Barr virus (EBV) infection is known to contribute in nasopharyngeal carcinoma (NPC) carcinogenesis. The oncogenic roles of the EBV proteins and non-coding RNAs in NPC are becoming evident with the aid of current advances in genome-wide and in-depth molecular analyses. This current work provides a comprehensive overview, which covers recent understandings of the pathogenic role of EBV infection in NPC. Perspectives on molecular mechanisms, which are involved in the pathogenesis of NPC, focusing on the connection between EBV and NPC cells and the corresponding signaling pathways are highlighted. Cancer hallmarks associated with EBV in NPC development are also discussed herein.
Abstract
Nasopharyngeal carcinoma (NPC) is one of the most common tumors occurring in China and Southeast Asia. Etiology of NPC seems to be complex and involves many determinants, one of which is Epstein-Barr virus (EBV) infection. Although evidence demonstrates that EBV infection plays a key role in NPC carcinogenesis, the exact relationship between EBV and dysregulation of signaling pathways in NPC needs to be clarified. This review focuses on the interplay between EBV and NPC cells and the corresponding signaling pathways, which are modulated by EBV oncoproteins and non-coding RNAs. These altered signaling pathways could be critical for the initiation and progression of NPC.
... EBNA1 is also thought to contribute to the survival of EBV-positive Burkitt's lymphoma (BL) cells in vitro [16], potentially through upregulation of survivin, an inhibitor of apoptosis [17]. The observed interaction of EBNA1 with the ubiquitin-specific protease, USP7, is predicted to contribute to host cell immortalisation through sequestration of USP7 and destabilisation of p53 [18,19]. ...
The Epstein-Barr virus (EBV)-encoded nuclear antigen 1 (EBNA1) protein is expressed in all virus-associated malignancies, where it performs an essential role in the maintenance, replication and transcription of the EBV genome. In recent years, it has become apparent that EBNA1 can also influence cellular gene transcription. Here, we demonstrate that EBNA1 is able to stimulate the expression of the Transforming growth factor-beta (TGFβ) superfamily member, bone morphogenic protein 2 (BMP2), with consequential activation of the BMP signalling pathway in carcinoma cell lines. We show that BMP pathway activation is associated with an increase in the migratory capacity of carcinoma cells, an effect that can be ablated by the BMP antagonist, Noggin. Gene expression profiling of authentic EBV-positive nasopharyngeal carcinoma (NPC) tumours revealed the consistent presence of BMP ligands, established BMP pathway effectors and putative target genes, constituting a prominent BMP “signature” in this virus-associated cancer. Our findings show that EBNA1 is the major viral-encoded protein responsible for activating the BMP signalling pathway in carcinoma cells and supports a role for this pathway in promoting cell migration and possibly, metastatic spread.
... Myc-, flag-, GFP-and RFP-tagged EBNA3C constructs in pA3M, pA3F, pEGFP-C1 and pDsRED-Monomer-N1 vectors, respectively were previously described [27,29,36]. Myc-tagged EBNA1 expressing construct in pA3M was described earlier [90]. Myc-and flag-tagged EBNA3C truncation constructs expressing EBNA3C residues 1-365, 366-620 and 621-992 were previously described [27,29]. ...
Epstein-Barr virus (EBV) nuclear oncoprotein EBNA3C is essential for B-cell transformation and development of several B-cell lymphomas particularly those are generated in an immuno-compromised background. EBNA3C recruits ubiquitin-proteasome machinery for deregulating multiple cellular oncoproteins and tumor suppressor proteins. Although EBNA3C is found to be ubiquitinated at its N-terminal region and interacts with 20S proteasome, the viral protein is surprisingly stable in growing B-lymphocytes. EBNA3C can also circumvent autophagy-lysosomal mediated protein degradation and subsequent antigen presentation for T-cell recognition. Recently, we have shown that EBNA3C enhances autophagy, which serve as a prerequisite for B-cell survival particularly under growth deprivation conditions. We now demonstrate that proteasomal inhibition by MG132 induces EBNA3C degradation both in EBV transformed B-lymphocytes and ectopic-expression systems. Interestingly, MG132 treatment promotes degradation of two EBNA3 family oncoproteins–EBNA3A and EBNA3C, but not the viral tumor suppressor protein EBNA3B. EBNA3C degradation induced by proteasomal inhibition is partially blocked when autophagy-lysosomal pathway is inhibited. In response to proteasomal inhibition, EBNA3C is predominantly K63-linked polyubiquitinated, colocalized with the autophagy-lysosomal fraction in the cytoplasm and participated within p62-LC3B complex, which facilitates autophagy-mediated degradation. We further show that the degradation signal is present at the first 50 residues of the N-terminal region of EBNA3C. Proteasomal inhibition reduces the colony formation ability of this important viral oncoprotein, induces apoptotic cell death and increases transcriptional activation of both latent and lytic gene expression which further promotes viral reactivation from EBV transformed B-lymphocytes. Altogether, this study offers rationale to use proteasome inhibitors as potential therapeutic strategy against multiple EBV associated B-cell lymphomas, where EBNA3C is expressed.
... EBNA1 binding with ubiquitin-specific protease 7 (USP7) influences p53 and Mdm2 expression. This results in the regulation of antiapoptotic activity, possibly through promoting survivin expression levels (37,38). Despite these critical activities, using recombinant virus, EBNA1 was shown to be not essential for in vitro B-cell transformation. ...
Epstein-Barr virus (EBV) is a ubiquitous gammaherpesvirus, which asymptomatically infects majority of the world population. In immune-compromised conditions, EBV can trigger human cancers of epithelial and lymphoid origin. The oncogenic potential of EBV is demonstrated by in-vitro infection and transformation of quiescent B-cells into lymphoblastoid cell lines (LCLs). These cell lines, along with primary infection using genetically engineered viral particles coupled with recent technological advancements have elucidated the underlying mechanisms of EBV-induced B-cell lymphomagenesis.
... Another factor associated with HL is EBV, an oncogenic virus that expresses latency proteins assumed to up-regulate proliferation signaling cascades (Vrzalikova et al., 2018). Additionally, EBNA1, LMP1 and LMP2, which are expressed during viral latency in HL (Carbone and Gloghini, 2018;Vrzalikova et al., 2018), were also described to upregulate survivin in in vitro studies (Hino et al., 2008;Lu et al., 2011;Sun et al., 2015). However, and in line with another study in HL biopsies (Aktas et al., 2007), we found no changes in the expression of survivin in our pediatric HL series in relation to EBV. ...
Survivin is abundantly expressed during fetal development but absent in most differentiated adult tissues; an exception being components of the immune system, such as B and T lymphocytes. Beyond acting as a master regulator of the cell cycle, survivin acts as an inhibitor of apoptosis and is overexpressed in almost all carcinoma types; however, its expression in lymphomas is lesser-explored. Survivin's role in carcinogenesis was subjected to its sub-cellular localization and splice transcripts expression, namely wild-type survivin, survivin-∆Ex3 and survivin-2B. To assess survivin's expression and sub-cellular localization in Epstein Barr virus positive and negative biopsies from treatment naïve pediatric patients with Hodgkin lymphoma (HL), samples were stained for survivin protein by immunofluorescence. The proportion of survivin+ cells was calculated, survivin sub-cellular localization assessed and its fluorescence intensity quantified. Transcription profile of survivin mRNA variants was studied by RT-qPCR. Survivin was overexpressed in the nucleus of tumor cells, and also in a greater proportion of tumor cells, in comparison with the non-tumoral infiltrating cells. Although a higher expression of survivin was observed in advanced clinical stages, no correlation was found between the expression level of survivin and a proliferation marker, or event-free survival. Instead, survivin was related to apoptosis inhibition in tumor cells. Additionally, survivin's transcriptional variants displayed similar expression levels. Present results suggest that although survivin is overexpressed in Hodgkin's tumor cells, it may not play a central role in the progression of classic HL, or act as a suitable progression biomarker, as suggested for most carcinomas.
... EBNA1 can also tether the EBV episomes to the cellular chromosomes by interacting with [151]. EBNA1 has roles in cell survival through regulating cellular gene expression, including upregulation of survivin expression and inhibition of the protein tyrosine phosphatase receptor kappa (PTPRK) [94]. EBNA1 also contributes to tumorigenesis through dysregulation of the host cell genome stability by inducing RAG1/RAG2 and increasing reactive oxygen species (ROS) [56,146]. ...
Herpesvirus-induced disease is one of the most lethal factors which leads to high mortality in HIV/AIDS patients. EBV, also known as human herpesvirus 4, can transform naive B cells into immortalized cells in vitro through the regulation of cell cycle, cell proliferation, and apoptosis. EBV infection is associated with several lymphoma and epithelial cancers in humans, which occurs at a much higher rate in immune deficient individuals than in healthy people, demonstrating that the immune system plays a vital role in inhibiting EBV activities. EBV latency infection proteins can mimic suppression cytokines or upregulate PD-1 on B cells to repress the cytotoxic T cells response. Many malignancies, including Hodgkin Lymphoma and non-Hodgkin’s lymphomas occur at a much higher frequency in EBV positive individuals than in EBV negative people during the development of HIV infection. Importantly, understanding EBV pathogenesis at the molecular level will aid the development of novel therapies for EBV-induced diseases in HIV/AIDS patients.
... Following EBV infection, the viral protein EBNA-1 causes genomic instability and at the same time counteracts the DNA damage response (DDR) activation by direct binding to the p53 regulator USP7 and upregulates the survivin protein by inhibiting the downstream caspase activation [29,30]. ...
Viral oncogenesis is a multistep process largely depending on the complex interplay between viruses and host factors. The oncoviruses are capable of subverting the cell signaling machinery and metabolic pathways and exploit them for infection, replication, and persistence. Several viral oncoproteins are able to functionally inactivate the tumor suppressor p53, causing deregulated expression of many genes orchestrated by p53, such as those involved in apoptosis, DNA stability, and cell proliferation. The Epstein⁻Barr virus (EBV) BZLF1, the high-risk human papillomavirus (HPV) E6, and the hepatitis C virus (HCV) NS5 proteins have shown to directly bind to and degrade p53. The hepatitis B virus (HBV) HBx and the human T cell lymphotropic virus-1 (HTLV-1) Tax proteins inhibit p53 activity through the modulation of p300/CBP nuclear factors, while the Kaposi's sarcoma herpesvirus (HHV8) LANA, vIRF-1 and vIRF-3 proteins have been shown to destabilize the oncosuppressor, causing a decrease in its levels in the infected cells. The large T antigen of the Merkel cell polyomavirus (MCPyV) does not bind to p53 but significantly reduces p53-dependent transcription. This review describes the main molecular mechanisms involved in the interaction between viral oncoproteins and p53-related pathways as well as in the development of therapeutic strategies targeting such interactions.
... In this respect, there are two possible scenarios of pathogenetic EBV involvement. In the first, the viral genes facilitate cell survival after MYC rearrangement has taken place by abrogating apoptosis through the activity of oncogenic EBERs, EBNA1 or BART-mRNAs and their interactions with anti-apoptotic proteins such as survivin or P53 regulator USP7 [102][103][104][105]. In addition, LMP2 has been shown to activate the PI3K pathway contributing to persistent B-cell receptor (BCR) signalling and thus aiding MYC driven lymphomagenesis [106][107][108]. ...
The contribution of Epstein-Barr virus (EBV) to the development of specific types of benign lymphoproliferations and malignant lymphomas has been extensively studied since the discovery of the virus over the last 50 years. The importance and better understanding of the EBV-associated lymphoproliferative disorders (LPD) of B, T or natural killer (NK) cell type has resulted in the recognition of new entities like EBV+ mucocutaneous ulcer or the addition of chronic active EBV (CAEBV) infection in the revised 2016 World Health Organization (WHO) lymphoma classification. In this article, we review the definitions, morphology, pathogenesis, and evolving concepts of the various EBV-associated disorders including EBV+ diffuse large B-cell lymphoma, not otherwise specified (DLBCL, NOS), EBV+ mucocutaneous ulcer, DLBCL associated with chronic inflammation, fibrin-associated DLBCL, lymphomatoid granulomatosis, the EBV+ T and NK-cell LPD of childhood, aggressive NK leukaemia, extranodal NK/T-cell lymphoma, nasal type, and the new provisional entity of primary EBV+ nodal T- or NK-cell lymphoma. The current knowledge regarding the pathogenesis of B-cell lymphomas that can be EBV-associated including Burkitt lymphoma, plasmablastic lymphoma and classic Hodgkin lymphoma will be also explored.
... Cette protéine est un inhibiteur de l'apoptose cellulaire, elle est surexprimée dans les cellules infectées par l'EBV. La surexpression de la survivine contribue à la survie des lymphocytes B immortalisés par le virus (Lu et al., 2011). Grossman et son équipe ont démontré que le transactivateur d'EBNA2 serait dirigé par des éléments de réponse par le signal de la protéine de liaison de la recombinaison de Jκ (Grossman et al., 1994). ...
Le virus d’Epstein-Barr (EBV) est le premier virus transformant à avoir été identifié chez l’Homme. Il infecte plus de 90% de la population adulte mondiale, persistant sous forme épisomale dans le compartiment B mémoire tout au long de la vie de l’hôte. Lors de la primo-infection et lors de phases de réactivation du virus, les cellules B immortalisées sont en programme de latence III, aussi appelée phase de prolifération, où l’ensemble des protéines de latence sont exprimées. Lorsque les hôtes sont immunocompétents, un équilibre entre hôte et virus s’établit et la plupart des cellules B infectées sont éliminées par le système immunitaire de l’hôte, principalement par les lymphocytes T cytotoxiques. En cas de déficit immunitaire, il peut y avoir émergence de lymphomes, tels que les désordres lymphoprolifératifs des patients immunodéprimés (PTLDs), les lymphomes non-Hodgkiniens (LNH) et Hodgkiniens (LH). Les travaux antérieurs du laboratoire ont permis de révéler que l’immuno-inhibiteur PD-L1/B7-H1/CD274 est surexprimé à la surface des lymphocytes B en latence III de l’EBV. L’interleukine-10 (IL-10) est également sécrétée par ces cellules. Ces caractéristiques sont communes aux cellules B régulatrices (Bregs). Le but de ma thèse était d’interroger les caractéristiques immuno-modulatrices des cellules B en latence III de l’EBV, dans le cadre des propriétés des Bregs. Nous montrons que les cellules B en latence III de l’EBV possèdent les déterminants antigéniques communs aux Bregs immatures (CD24High CD38High PD-L1High), associée à une surexpression des cytokines immunosuppressives cardinales des Bregs (IL-10, TGF-β1 et IL-35). Nous montrons que les cellules B en latence III de l’EBV peuvent conduire à la mort des cellules T CD4 autologues, ainsi qu’à l’inhibition de la prolifération des lymphocytes T CD4 et CD8, au profit de l’expansion de lymphocytes T régulateurs (Tregs). Nous avons trouvé que cette expansion est médiée par l’axe PD-1/PD-L1. Ces travaux mettent en évidence un nouveau mécanisme de l’EBV concernant le détournement du système immunitaire de l’hôte, augmentant ses capacités oncogéniques.
... EBNA-1 has been shown to cause genomic instability by triggering reactive oxygen species production and the DNA damage response (DDR) [78,79]. Conversely however, EBNA1 may stabilise p53 to counteract DDR by binding to the p53 regulator, USP7 [80] and further block downstream caspase activation by upregulation of Survivin [81]. EBNA-1 reportedly binds and regulates the promoters of many other cellular genes but the functional consequences and implications of these interactions for cell survival are not yet fully elucidated [82][83][84][85]. ...
Epstein–Barr virus (EBV) was first discovered in cells from a patient with Burkitt lymphoma (BL), and is now known to be a contributory factor in 1–2% of all cancers, for which there are as yet, no EBV-targeted therapies available. Like other herpesviruses, EBV adopts a persistent latent infection in vivo and only rarely reactivates into replicative lytic cycle. Although latency is associated with restricted patterns of gene expression, genes are never expressed in isolation; always in groups. Here, we discuss (1) the ways in which the latent genes of EBV are known to modulate cell death, (2) how these mechanisms relate to growth transformation and lymphomagenesis, and (3) how EBV genes cooperate to coordinately regulate key cell death pathways in BL and lymphoblastoid cell lines (LCLs). Since manipulation of the cell death machinery is critical in EBV pathogenesis, understanding the mechanisms that underpin EBV regulation of apoptosis therefore provides opportunities for novel therapeutic interventions.
... EBNA1 was reported to confer resistance to apoptosis in EBV-positive Blymphoma cells through up-regulation of apoptosis suppressor protein, survivin. 78 Besides, EBERs could also confer resistance to apoptosis via binding to PKR and inhibition of its phosphorylation in Burkitt's lymphoma. 79 In addition, prevention of apoptosis is a major function of miR-BARTs in EBV (1) epithelial cancers. ...
Epstein-Barr virus (EBV) is an important cancer causing virus. Cancer associated with EBV account for approximately 1.5% of all cancers, and represent 1.8% of all cancer deaths worldwide. EBV reactivation plays an important role in the development of EBV-related diseases and is closely related with patients' survival and clinical stages of EBV-related cancers. The therapy regarding to EBV-related cancers is very urgent, especially in endemic areas. Generating oxidative stress is a critical mechanism by which host cells defend against infection by virus. In addition, ROS-mediated oxidative stress plays a significant but paradoxical role acting as a "double-edged sword" to regulate cellular response to radiation, which is the main therapy strategy for EBV-related cancers, especially nasopharyngeal carcinoma. Therefore, in this review we primarily discuss the possible interplay among the oxidative stress, EBV lytic reactivation and radioresistance. Understanding the role of oxidative stress in EBV lytic reactivation and radioresistance will assist in the development of effective strategies for prevention and treatment of EBV-related cancers. This article is protected by copyright. All rights reserved.
... We focused on the Epstein-Barr nuclear antigen 1 (EBNA1) protein since EBNA1 is the only viral nuclear protein expressed in EBVaGC, and is also the only viral protein required to maintain viral latency, because of its various roles in the segregation and replication of the EBV episomes [36,37]. There is also increasing evidence that EBNA1 modifies the cellular environment in ways that promote genomic instability, thereby leading to tumorigenesis [38][39][40]. We relied on a high-throughput reverse transcription-PCR (RT-PCR)-based platform [41] to examine AS in 1200 cancer-associated genes. ...
Multiple human diseases including cancer have been associated with a dysregulation in RNA splicing patterns. In the current study, modifications to the global RNA splicing landscape of cellular genes were investigated in the context of Epstein-Barr virus-associated gastric cancer. Global alterations to the RNA splicing landscape of cellular genes was examined in a large-scale screen from 295 primary gastric adenocarcinomas using high-throughput RNA sequencing data. RT-PCR analysis, mass spectrometry, and co-immunoprecipitation studies were also used to experimentally validate and investigate the differential alternative splicing (AS) events that were observed through RNA-seq studies. Our study identifies alterations in the AS patterns of approximately 900 genes such as tumor suppressor genes, transcription factors, splicing factors, and kinases. These findings allowed the identification of unique gene signatures for which AS is misregulated in both Epstein-Barr virus-associated gastric cancer and EBV-negative gastric cancer. Moreover, we show that the expression of Epstein–Barr nuclear antigen 1 (EBNA1) leads to modifications in the AS profile of cellular genes and that the EBNA1 protein interacts with cellular splicing factors. These findings provide insights into the molecular differences between various types of gastric cancer and suggest a role for the EBNA1 protein in the dysregulation of cellular AS.
... Most EBVpositive BL displays a latency I viral gene expression program, with EBNA1, LMP2A, and viral microRNAs detected (Rowe et al, 1987). These viral factors have been shown to provide survival advantages to B cells by various mechanisms (Kennedy et al, 2003;Lu et al, 2011;Riley et al, 2012). ...
Epstein-Barr virus (EBV) is a ubiquitous gammaherpesvirus that establishes a lifelong persistent infection in the oral cavity and is intermittently shed in the saliva. EBV exhibits a biphasic life cycle, supported by its dual tropism for B lymphocytes and epithelial cells, which allows the virus to be transmitted within oral lymphoid tissues. While infection is often benign, EBV is associated with a number of lymphomas and carcinomas that arise in the oral cavity and at other anatomical sites. Incomplete association of EBV in cancer has questioned if EBV is merely a passenger or a driver of the tumorigenic process. However, the ability of EBV to immortalize B cells and its prevalence in a subset of cancers has implicated EBV as a carcinogenic cofactor in cellular contexts where the viral lifecycle is altered. In many cases, EBV likely acts as an agent of tumor progression rather than tumor initiation, conferring malignant phenotypes observed in EBV-positive cancers. Given that the oral cavity serves as the main site of EBV residence and transmission, here we review the prevalence of EBV in oral malignancies and the mechanisms by which EBV acts as an agent of tumor progression. This article is protected by copyright. All rights reserved.
Epstein-Barr virus (EBV), the first human oncovirus discovered in 1964, has become a focal point in virology, immunology, and oncology because of its unique biological characteristics and significant role in human diseases. As we commemorate the 60th anniversary of EBV’s discovery, it is an opportune moment to reflect on the major advancements in our understanding of this complex virus. In this review, we highlight key milestones in EBV research, including its virion structure and life cycle, interactions with the host immune system, association with EBV-associated diseases, and targeted intervention strategies.
Background and Aim: Infection with Epstein-Barr virus (EBV) ranks as one of the most substantial risk factors associated with Glioblastoma multiforme (GBM). At the core of this intricate relationship lies the EBV nuclear antigen-1 (EBNA1) protein, a central figure with a remarkable ability to regulate the expression of both cellular and viral genes. This research delves into the impact of EBNA1 on the expression patterns of four cellular genes - MDMX, MDM2, MYC, and BIRC5 in the U87MG cell line.
Materials and Methods: We divided U87MG cells into two distinct groups. The first group involved cells that were transfected with a plasmid containing the EBNA1 gene, while the second group consisted of cells that were transfected with a control plasmid. To evaluate the transcriptional activity of MDMX, MDM2, MYC, and BIRC5 genes in both sets of cells, we employed a real-time PCR technique. Any observed differences were considered statistically significant if the associated P-values were less than 0.05.
Results: Our findings demonstrated a substantial three-fold increase in the expression of the MDMX gene when U87MG cells were transfected with EBNA1 plasmid (P=0.02). Although the cells transfected with EBNA1 plasmid displayed great elevations in the expression levels of MDM2, MYC, and BIRC5 genes, these alterations were not statistically significant.
Conclusion: The outcomes of this investigation have unveiled that EBNA1 has the ability to trigger the expression of four crucial cellular genes, which wield substantial influence in the genesis of GBM within glioblastoma astrocytoma cells. This underscores the potential impact of EBNA1 on the evolution of GBM, particularly in individuals harboring EBV.
Epstein-Barr virus (EBV) is a human tumor virus associated with a variety of malignancies, including nasopharyngeal carcinoma, gastric cancers, and B-cell lymphomas. N⁶-methyladenosine (m⁶A) modifications modulate a wide range of cellular processes and participate in the regulation of virus-host cell interactions. Here, we discovered that EBV infection downregulates toll-like receptor 9 (TLR9) m⁶A modification levels and thus inhibits TLR9 expression. TLR9 has multiple m⁶A modification sites. Knockdown of METTL3, an m⁶A “writer”, decreases TLR9 protein expression by inhibiting its mRNA stability. Mechanistically, Epstein-Barr nuclear antigen 1 increases METTL3 protein degradation via K48-linked ubiquitin-proteasome pathway. Additionally, YTHDF1 was identified as an m⁶A “reader” of TLR9, enhancing TLR9 expression by promoting mRNA translation in an m⁶A -dependent manner, which suggests that EBV inhibits TLR9 translation by “hijacking” host m⁶A modification mechanism. Using the METTL3 inhibitor STM2457 inhibits TLR9-induced B cell proliferation and immunoglobulin secretion, and opposes TLR9-induced immune responses to assist tumor cell immune escape. In clinical lymphoma samples, the expression of METTL3, YTHDF1, and TLR9 was highly correlated with immune cells infiltration. This study reveals a novel mechanism that EBV represses the important innate immunity molecule TLR9 through modulating the host m⁶A modification system.
Genomic instability facilitates the evolution of cells, tissues, organs, and species. The progression of human malignancies can be regarded as the accumulation of genomic instability, which confers a high evolutionary potential for tumor cells to adapt to continuous changes in the tumor microenvironment. Nasopharyngeal carcinoma (NPC) is a head-and-neck squamous-cell carcinoma closely associated with Epstein–Barr virus (EBV) infection. NPC progression is driven by a combination of accumulated genomic instability and persistent EBV infection. Here, we present a review of the key characteristics of genomic instability in NPC and the profound implications of EBV infection. We further discuss the significance of profiling genomic instability for the assessment of disease progression and treatment efficacy, as well as the opportunities and challenges of targeted therapies for NPC based on its unique genomic instability.
Epstein–Barr virus (EBV; human herpesvirus 4; HHV‐4) and Kaposi sarcoma‐associated herpesvirus (KSHV; human herpesvirus 8; HHV‐8) are human gammaherpesviruses that have oncogenic properties. EBV is a lymphocryptovirus, whereas HHV‐8/KSHV is a rhadinovirus. As lymphotropic viruses, EBV and KSHV are associated with several lymphoproliferative diseases or plasmacytic/plasmablastic neoplasms. Interestingly, these viruses can also infect epithelial cells causing carcinomas and, in the case of KSHV, endothelial cells, causing sarcoma. EBV is associated with Burkitt lymphoma, classic Hodgkin lymphoma, nasopharyngeal carcinoma, plasmablastic lymphoma, lymphomatoid granulomatosis, leiomyosarcoma, and subsets of diffuse large B‐cell lymphoma, post‐transplant lymphoproliferative disorder, and gastric carcinoma. KSHV is implicated in Kaposi sarcoma, primary effusion lymphoma, multicentric Castleman disease, and KSHV‐positive diffuse large B‐cell lymphoma. Pathogenesis by these two herpesviruses is intrinsically linked to viral proteins expressed during the lytic and latent lifecycles. This comprehensive review intends to provide an overview of the EBV and KSHV viral cycles, viral proteins that contribute to oncogenesis, and the current understanding of the pathogenesis and clinicopathology of their related neoplastic entities.
Whole genome sequence analysis of Epstein-Barr virus genomes from tumours and healthy individuals identified three amino acid changes in EBNA1 that are strongly associated with gastric carcinoma and nasopharyngeal carcinoma. Here we show that, while these mutations do not impact EBNA1 plasmid maintenance function, one of them (Thr85Ala) decreases transcriptional activation and results in a gain of function interaction with PLOD1 and PLOD3. PLOD family proteins are strongly linked to multiple cancers, and PLOD1 is recognized as a prognostic marker of gastric carcinoma. We identified the PLOD1 binding site in EBNA1as the N-terminal transactivation domain and show that lysine 83 is critical for this interaction. The results provide a novel link between EBV infection and the cancer-associated PLOD proteins.
Epstein-Bar virus (EBV) can directly cause lymphoproliferative disease (LPD), including AIDS-defining lymphomas such as Burkitt’s lymphoma and other non-Hodgkin lymphomas (NHL), as well as human immunodeficiency virus (HIV)-related Hodgkin lymphoma (HL). The prevalence of EBV in HL and NHL is elevated in HIV-positive individuals compared with the general population. Rates of incidence of AIDS-defining cancers have been declining in HIV-infected individuals since initiation of combination anti-retroviral therapy (cART) use in 1996. However, HIV-infected persons remain at an increased risk of cancers related to infections with oncogenic viruses. Proposed pathogenic mechanisms of HIV-related cancers include decreased immune surveillance, decreased ability to suppress infection-related oncogenic processes and a state of chronic inflammation marked by alteration of the cytokine profile and expanded numbers of cytotoxic T lymphocytes with down-regulated co-stimulatory molecules and increased expression of markers of senescence in the setting of treated HIV infection. Here we discuss the cooperation of EBV-infected B cell- and environment-associated factors that may contribute to EBV-related lymphomagenesis in HIV-infected individuals. Environment-derived lymphomagenic factors include impaired host adaptive and innate immune surveillance, cytokine dysregulation and a pro-inflammatory state observed in the setting of chronic, cART-treated HIV infection. B cell factors include distinctive EBV latency patterns and host protein expression in HIV-associated LPD, as well as B cell-stimulating factors derived from HIV infection. We review the future directions for expanding therapeutic approaches in targeting the viral and immune components of EBV LPD pathogenesis.
Summary
The Epstein-Barr virus (EBV) is strongly and causally associated with two malignancies of
epithelial origin, namely almost 100% of undifferentiated nasopharyngeal carcinoma (NPC) and 10% of all gastric carcinoma (GC) worldwide. EBV shows a latency gene expression profile in these tumors, meaning that only a few EBV genes are expressed that are essential for tumor cell growth and survival and viral persistence. One of these genes is the BamH-I rightward frame 1, known as BARF1 which is the main topic of this thesis. BARF1 is a viral oncogene with pleiotropic functions, contributing to cell growth and survival as well as to immune modulation. Interestingly, BARF1 is selectively expressed in EBV associated carcinomas and not in lymphomas, indicating that its role might differ depending on cell type. More insight in this role might support the development of treatment options for EBV associated cancers. Beside its usefulness for the virus and for the tumor, BARF1 has specific features that potentially have diagnostic implications, the most important being that BARF1 is a secreted protein, opening options for less invasive sampling and screening.
The aim of this thesis was to further investigate the function, regulation and diagnostic use of the BARF1 protein in EBV associated carcinomas.
Chapter 2 includes a state-of-the-art review about BARF1. Deciphering the role of BARF1 in
EBV biology will contribute to novel diagnostic and treatment options for EBV driven
carcinomas. Starting with a description of its protein structure and expression in EBV
associated malignancies, the aspect of BARF1 as a diagnostic tool is also briefly touched. The oncogenic potential and immune modulating properties of BARF1 are discussed in detail, including indications of aspects that need further research.
EBV-positive nasopharyngeal carcinoma cells express the viral BARF1 gene, which encodes a secreted hexameric protein. Our aim was to analyze if serum of patients with NPC contains antibodies against BARF1 protein, to use these for early diagnosis of NPC.
Chapter 3 describes the development of a serological assay to detect antibodies to BARF1
protein. Previous work by our group (2001-2005; VUmc thesis J. van Beek) showed that short peptides from predicted immunodominant domains of the BARF1 protein and the
recombinant whole protein purified from non-human expression systems provide suitable
tools for the analysis of immune responses. While antibodies could be generated against
these peptides and recombinant proteins in mice and guinea pigs, capable of recognizing
denatured BARF1 from a human expression system, human antibodies were not detected
with these tools. Since this could be due to the more recently defined (2006) complex
hexameric structure of BARF1, we expressed and purified native NPC derived BARF1 from a human expression system in which posttranslational modifications such as glycosylation and folding resembles the secreted BARF1 protein in vivo (sBARF1).
Using this purified native hexameric sBARF1 protein we could show that NPC patients have significantly higher antibody responses to sBARF1 compared to healthy volunteers, however the anti-BARF1 responses were relatively weak compared to the responses to other EBV proteins, such as VCA-p18 and EBNA1. The presence of antibodies against native BARF1 in patients with NPC indirectly proved that BARF1 may circulate in vivo as a hexameric protein released by tumor cells in these patients. However, due the limited immunogenicity of BARF1, this assay is not suitable as a diagnostic assay for early detection of NPC.
Sequence variation in the EBV genome can influence disease development and progression and cause changes in the interactions between the virus and the host immune system, which has been demonstrated for various key EBV genes such as EBNA1, LMP1 and the lytic switch protein Z.
In Chapter 4 the sequence variation of the BARF1 gene was investigated, in relation to EBV genotype, viral load and serology markers. The BARF1 gene from Indonesian donors shows minor single nucleotide variation with the prototype EBV B95.8 sequence, but most mutations did not result in amino acid substitutions at the protein level. The three most frequently detected amino acid changes (V29A, W72G, H130R) were not predicted to lead to gross tertiary structure alterations based on the BARF1 crystal structure. The evolutionary well conserved sequence implicates that BARF1 has functional relevance in EBV biology. Utilizing the immunoserology assay from chapter 3, we determined that the presence of amino acid substitutions in the BARF1 protein in NPC patients did not influence the detection of antibody responses to BARF1.
EBV persists lifelong under a dynamic balance with the human immune system and, like many other persistent herpes viruses, has acquired numerous mechanisms for subverting or evading immune surveillance.
In Chapter 5 the role of secreted BARF1 protein as an immune modulator was studied. The BARF1 protein has partial sequence homology with a conserved structural domain found in various growth factor receptors, one of which is the receptor for macrophage colony stimulating factor (M-CSF receptor). Utilizing our purified native sBARF1 from chapter 2, polyclonal antibodies were developed which were used to coimmunoprecipitate M-CSF together with sBARF1, demonstrating that sBARF1 is capable of capturing M-CSF. The cytokine dependent human myeloid MUTZ3 cell line was deployed to demonstrate that sBARF1 binding to M-CSF functionally interferes with cell proliferation. Antibodies specific to hexameric native sBARF1 were able to oppose this effect. From the amount BARF1 protein needed to inhibit the M-CSF action we could determine that one sBARF1 hexamer binds three M-CSF dimers. Site directed mutation of the predicted sBARF1/M-CSF interaction site revealed that sBARF1 binds M-CSF using three ‘tweezer’ like loops, located on the exterior of the rings. The effect of sBARF1 on M-CSF driven mononuclear phagocyte differentiation was functionally evaluated, revealing that sBARF1 exposure during differentiation negatively affected the ability to phagocytose apoptotic cells, produce oxygen radicals, and functional surface markers such as the Fc receptor CD16. We conclude that sBARF1 protein is a potent decoy receptor for M-CSF, hampering the function and differentiation of mononuclear phagocytes. BARF1 suppresses M2 macrophage differentiation, which are the first line of anti-viral defense. We hypothesize that inhibition of mononuclear phagocytes by sBARF1 decreases anti-viral defense during lytic replication, which might allow macrophages to act as virus transmission vehicle of internalized virus,opposed to inactivation of the virus. In EBV associated malignancies BARF1 immune modulation of mononuclear phagocytes might constitute a possible mechanism by which macrophage-mediated anti-tumor immunity is evaded.
The transcriptional regulation of the BARF1 gene has been an unexplored area so far. While BARF1 mRNA can be detected in all cell types during lytic replication, in latency the gene is only expressed in tumors with an epithelial cell background, indicating that activation of the BARF1 promoter is cell type specific.
In Chapter 6 and Chapter 7 we set out to unravel the transcriptional regulation of BARF1. EBV regulates many of its genes by promotor methylation. A bisulfite sequencing analysis showed that the BARF1 control region is highly methylated in multiple cell types as well as in C15 and C17 mouse xenograft NPC tumor material.
In Chapter 6 a BARF1 promoter luciferase construct was created and utilized to evaluate the transactivating activity of the immediate-early proteins R and Z. Methylation of the construct enabled us to examine how methylation affects its ability to be activated. We found that the EBV encoded transcriptional activator R , but not Z, induced 50- to 250-fold upregulation of luciferase activity. Chromatin Immune Precipitation (ChIP), Electrophoretic Mobility Shift Assay (EMSA) and specific mutagenesis of the R responsive elements (RREs) demonstrated direct binding of the R protein to RREs between -554 and -327 nucleotides relative to the BARF1 transcriptional ATG start site. Using quantitative reverse transcriptase (RT)-PCR we showed that R is capable of transactivating the BARF1 promoter in the context of the viral genome, within 6 hours after transfection with a R expression vector.
In Chapter 7 transcription factor binding site analysis of the BARF1 promotor area revealed that it contains potential binding sites for the p53 family of transcription factors. One member of the p53 family of transcription factors is the ΔNp63α isoform of p63, which is highly expressed in undifferentiated NPC and is regarded a marker for epithelial differentiation. Reporter assays performed with the BARF1 promoter luciferase construct revealed that specifically the ΔNp63α isoform, and none of the other p53 family members is capable of transactivation of the BARF1 promoter. We concluded that although ΔNp63α can transactivate the BARF1 promotor reporter construct, additional factors are required to transactivate BARF1 in the context of the viral genome. We concluded that in EBV-positive NPC and GC, BARF1 expression might be induced by the epithelial differentiation marker ΔNp63α.
In Chapter 8 we developed and evaluated different methods for the detection of BARF1 protein. While BARF1 mRNA is frequently detected in EBV associated carcinomas such as nasopharyngeal and gastric carcinoma, evidence of the actual protein being translated in vivo is limited. New monoclonal and polyclonal antibodies capable of recognizing and capturing hexameric native sBARF1 protein were developed and used to set up an antigen-capture enzyme-linked immunosorbent assay (ELISA) system for detection of sBARF1 in sera of patients with NPC. The same antibodies were assessed for immunofluorescence (IF) and immunohistochemistry (IHC) in cells and tissues. BARF1 was also evaluated for its potential as a serum biomarker using a Mass-Spec MicroSequencing (MS/MS) proteomic approach, revealing a main identifier peptide suitable for a targeted proteomic analysis. We concluded that the capture ELISA in its current form can detect sBARF1 levels above 10 ng/ml, which appeared not sensitive enough to conclusively state that sBARF1 is present in NPC patient sera at levels above those found in healthy EBV carriers. The MS/MS proteomic approach in the current format available to us at VUmc proved even less sensitive than the capture-ELISA. We further demonstrate that antibodies suitable for detecting BARF1 protein in transfected cells cannot detect BARF1 protein expression using immunohistochemistry of NPC and GC tissue, that express BARF1 mRNA. We hypothesize that BARF1 is rapidly and fully secreted from tumor cells, directly binding to M-CSF or other yet unknown factors to form complexes. These complexes are either rapidly degraded by local myeloid cells, or shield BARF1 epitopes, with the consequence that BARF1 protein can not be traced or eludes antibody capture. We suggest that future research should further focus on a targeted proteomics approach.
Background:
Restoring apoptosis dysregulation via survivin inhibition has been investigated in several cancers. In Epstein-Barr Virus (EBV)-driven nasopharyngeal cancer (NPC), virally induced oncogenes can upregulate survivin. Therefore, we seek to investigate the therapeutic efficacy of YM-155 (a survivin inhibitor) in NPC, both in vitro and in vivo models.
Methods:
Cytotoxicity, apoptosis, and active-caspase 3 expression assays were performed.
Results:
Both NPC tissue and cells expressed high levels of survivin which were inhibited by YM-155 in a dose-dependent manner. In addition, YM-155 induced apoptosis of NPC cells with an IC50 of 100 nM and inhibited tumor growth in vivo (P < 0.05). YM-155 in combination with cisplatin or radiation significantly increased overall cytotoxicity as compared to YM-155 monotherapy. In the xenograft model, YM-155 plus radiation additively achieved significantly higher percentage of active-caspase 3-positive tumor cells than radiation alone (P < 0.05).
Conclusions:
YM-155 is a potential therapeutic agent for NPC through inhibiting survivin and restoring apoptosis dysregulation.
Epstein-Barr virus (EBV) nuclear oncoprotein EBNA3C is essential for B-cell transformation and development of several B-cell lymphomas particularly those are generated in an immuno-compromised background. EBNA3C recruits ubiquitin-proteasome machinery for deregulating multiple cellular oncoproteins and tumor suppressor proteins. Although EBNA3C is found to be ubiquitinated at its N-terminal region and interacts with 20S proteasome, the viral protein is surprisingly stable in growing B-lymphocytes. EBNA3C can also circumvent autophagy-lysosomal mediated protein degradation and subsequent antigen presentation for T-cell recognition. Recently, we have shown that EBNA3C enhances autophagy, which serve as a prerequisite for B-cell survival particularly under growth deprivation conditions. We now demonstrate that proteasomal inhibition by MG132 induces EBNA3C degradation both in EBV transformed B-lymphocytes and ectopic-expression systems. Interestingly, MG132 treatment promotes degradation of two EBNA3 family oncoproteins – EBNA3A and EBNA3C, but not the viral tumor suppressor protein EBNA3B. EBNA3C degradation induced by proteasomal inhibition is partially blocked when autophagy-lysosomal pathway is inhibited. In response to proteasomal inhibition, EBNA3C is predominantly K63-linked polyubiquitinated, colocalized with the autophagy-lsyosomal fraction in the cytoplasm and participated within p62-LC3B complex, which facilitates autophagy-mediated degradation. We further show that the degradation signal is present at the first 50 residues of the N-terminal region of EBNA3C. Proteasomal inhibition reduces the colony formation ability of this important viral oncoprotein, increases transcriptional activation of both latent and lytic gene expression and induces viral reactivation from EBV transformed B-lymphocytes. Altogether, this study offers rationale to use proteasome inhibitors as potential therapeutic strategy against multiple EBV associated B-cell lymphomas, where EBNA3C is expressed.
Author Summary
Epstein-Barr virus (EBV) establishes latent infection in B-lymphocytes and is associated with a number of human malignancies, both of epithelial and lymphoid origin. EBV encoded EBNA3 family of nuclear latent antigens comprising of EBNA3A, EBNA3B, and EBNA3C are unique to immunoblastic lymphomas. While EBNA3A and EBNA3C are involved in blocking many important tumor suppressive mechanisms, EBNA3B exhibits tumor suppressive functions. Although EBNA3 proteins, in particular EBNA3C, interact with and employ different protein degradation machineries to induce B-cell lymphomagenesis, these viral proteins are extremely stable in growing B-lymphocytes. To this end, we now demonstrate that proteasomal inhibition leads to specifically degradation of oncogenic EBNA3A and EBNA3C proteins, whereas EBNA3B remains unaffected. Upon proteasomal inhibition, EBNA3C degradation occurs via autophagy-lysosomal pathway, through labeling with K63-linked polyubiquitination and participating in p62-LC3B complex involved in ubiquitin-mediated autophagy substrate selection and degradation through autolysosomal process. We also demonstrate that the N-terminal domain is responsible for autophgy-lysosomal mediated degradation, while the C-terminal domain plays a crucial role in cytoplasmic localization. Fascinatingly, while proteasomal inhibition reduces EBNA3C’s oncogenic property, it induces both latent and lytic gene expressions and promotes viral reactivation from EBV transformed B-lymphocytes. This is the first report which demonstrates a viral oncoprotein degrades through autophagy-lysosomal pathway upon proteasomal inhibition. In sum, the results promise development of novel strategies specifically targeting proteolytic pathway for the treatment of EBV associated B-cell lymphomas, particularly those are generated in immunocompromised individuals.
Several DNA viruses including Human Papillomavirus (HPV), Epstein-Barr virus (EBV), and Human cytomegalovirus (HCMV) are mechanistically associated with the development of human cancers (HPV, EBV) and/or modulation of the immune system (HCMV). Moreover, a number of distinct mechanisms have been described regarding the modulation of tumor cell response to ionizing radiation and evasion from the host immune system by viral factors. There is further accumulating interest in the treatment with immune-modulatory therapies such as immune checkpoint inhibitors for malignancies with a viral etiology. Also, patients with HPV-positive tumors have a significantly improved prognosis that is attributable to increased intrinsic radiation sensitivity and may also arise from modulation of a cytotoxic T cell response in the tumor microenvironment (TME). In this review, we will highlight recent advances in the understanding of the biological basis of radiation response mediated by viral pathogenic factors and evasion from and modulation of the immune system by viruses.
Le virus d’Epstein-Barr (EBV) est un herpèsvirus humain qui infecte plus de 90% de la population mondiale. Il est décrit comme associé à plusieurs pathologies cancéreuses humaines comme les carcinomes nasopharyngés et gastriques et divers lymphomes, comme le lymphome de Burkitt, les lymphomes NK/T et certains lymphomes de Hodgkin. Le lymphome T angio-immunoblastique (LTAI), un cancer des cellules T folliculaires helper TFH, contient souvent des cellules B porteuses de l’EBV. Mais jusqu’à présent le rôle de l’EBV dans la pathogenèse de cette maladie reste inconnu. Dans ce contexte, notre travail avait pour objectif de déterminer si l’EBV associé au LTAI présentait une particularité laissant envisager son rôle dans cette pathologie. Pour ce faire, nous avons étudié la séquence complète de l’EBV au sein d’échantillons de LTAI et comparé les résultats à ceux obtenus pour d’autres lymphomes (B, NK/T) ainsi qu’aux séquences publiées. Le séquençage a tout d’abord été réalisé sur 7 lignées cellulaires positives pour l’EBV, afin de valider la technique, et a ensuite été appliqué aux échantillons d’adénopathies de 40 patients atteints de syndrome lymphoprolifératif, parmi lesquels 20 souffraient de LTAI. L’enrichissement en génome viral a été réalisé par capture à l’aide de sondes spécifiques du génome de l’EBV. Ensuite les librairies ont été synthétisées et séquencées sur les plateformes Illumina MiSeq et NextSeq. Dans un deuxième temps, nous avons réalisé l’assemblage de novo des reads et déterminé la séquence complète du virus majoritaire dans chaque échantillon. Les données obtenues ont été analysées bioinformatiquement. D’une manière intéressante, le virus a été trouvé clonal ou quasi-clonal dans les LTAI alors que les lymphocytes B étaient dans la plupart des cas polyclonaux. En outre, le profil de mutations trouvé présentait des similitudes avec ce qui était trouvé pour les autres lymphomes associés à l’EBV, notamment au niveau des épitopes cibles des cellules de l’immunité suggérant un processus de sélection de la souche virale identique à celui d’une tumeur clonale associée à l’EBV. Ceci pourrait jouer un rôle important dans l’échappement au système immunitaire du virus dans ce contexte multicellulaire complexe. La présence de cellules B polyclonales avec un EBV clonal dans un compartiment T tumoral clonal pourrait relever d’une double sélection tumorale, endogène T et exogène EBV clonal, et pourrait suggérer l’existence de cross-talk entre les cellules B-T.
RYMV phytovirus genome codes for five proteins. Together they transform cellular activity allowing virus multiplication and spreading. Among of them, P1 is a multifunctional and small cystein rich protein. It is involve in virus replication and intercellular movement and in PTGS regulation, an antiviral pathway. If functions knowledge’s about P1 are acquired, little is known about how its molecular mechanism orchestrates. My phd works gives some comprehensive element by a biochemical approach. P1 is a polymorphic protein with a redox dependent conformational change. Reduction of P1 was accompanied by the binding of two zinc atoms while its oxidation provoked zinc release and disulfide bond formation. Furthermore, the oxidation state of P1 was directly related to its oligomerization state in vitro. In a pathosystem, a viral activity detection is fallowed by oxidative stress that induced host defenses pathways. We suppose that, in planta, P1 could act as a rheostat sensing redox imbalances in the host cell. This redox dependant structural polymorphism could contribute to explain the P1’s multifunctionality and how these functions could be regulated. This property is also a way for the virus to generate multiple functions from the same genetic information.
Key words : RYMV-P1, PTGS, redox, structural polymorphism, multifunctionality, zinc-binding domain
Epstein–Barr virus (EBV), originally discovered through its association with Burkitt lymphoma, is now aetiologically linked to a remarkably wide range of lymphoproliferative lesions and malignant lymphomas of B-, T- and NK-cell origin. Some occur as rare accidents of virus persistence in the B lymphoid system, while others arise as a result of viral entry into unnatural target cells. The early finding that EBV is a potent B-cell growth transforming agent hinted at a simple oncogenic mechanism by which this virus could promote lymphomagenesis. In reality, the pathogenesis of EBV-associated lymphomas involves a complex interplay between different patterns of viral gene expression and cellular genetic changes. Here we review recent developments in our understanding of EBV-associated lymphomagenesis in both the immunocompetent and immunocompromised host.
This article is part of the themed issue ‘Human oncogenic viruses’.
The identification of the cellular factors that control the transcription regulatory activity of the Epstein-Barr virus C promoter (Cp) is fundamental to the understanding of the molecular mechanisms that control virus latent gene expression. Using transient transfection of reporter plasmids in group I phenotype B-lymphoid cells, we have previously shown that the 2248 to 255 region (2248/255 region) of Cp contains elements that are essential for oriPI-EBNA1-dependent as well as oriPI-EBNA1-independent activation of the promoter. We now establish the importance of this region by a detailed mutational analysis of reporter plasmids carrying Cp regulatory sequences together with or without oriPI. The reporter plasmids were transfected into group I phenotype Rael cells and group III phenotype cbc-Rael cells, and the Cp activity measured was correlated with the binding of candidate transcription factors in electrophoretic mobility shift assays and further assessed in cotransfection experiments. We show that the NF-Y transcription factor interacts with the previously identified CCAAT box in the 271/263 Cp region (M. T. Puglielli, M. Woisetschlaeger, and S. H. Speck, J. Virol. 70:5758-5768, 1996). We also show that members of the C/EBP transcription factor family interact with a C/EBP consensus sequence in the 2119/2112 region of Cp and that this interaction is important for promoter activity. A central finding is the identification of a GC-rich sequence in the 299/291 Cp region that is essential for oriPI-EBNA1-independent as well as oriPI-EBNA1-dependent activity of the promoter. This region contains overlapping binding sites for Sp1 and Egr-1, and our results suggest that Sp1 is a positive and Egr-1 is a negative regulator of Cp activity. Furthermore, we demonstrate that a reporter plasmid that in addition to oriPI contains only the 2111/176 region of Cp still retains the ability to be activated by EBNA1.
Hepatocellular carcinoma (HCC) is one of the most common cancers in the world which is highly chemoresistant to currently available chemotherapeutic agents. Thus, novel therapeutic targets are needed to be sought for the successful treatment of HCC. Peptaibols, a family of peptides synthesized non-ribosomally by the Trichoderma species and other fungi, exhibit antibiotic activities against bacteria and fungi. Few studies recently showed that peptaibols exerted cytotoxicity toward human lung epithelial and breast carcinoma cells. However, the mechanism involved in peptaibol-induced cell death remains poorly understood.
Here, we showed that Trichokonin VI (TK VI), a peptaibol from Trichoderma pseudokoningii SMF2, induced growth inhibition of HCC cells in a dose-dependent manner. It did not obviously impair the viability of normal liver cells at lower concentration. Moreover, the suppression of cell viability resulted from the programmed cell death (PCD) with characteristics of apoptosis and autophagy. An influx of Ca2+ triggered the activation of mu-calpain and proceeded to the translocation of Bax to mitochondria and subsequent promotion of apoptosis. On the other hand, typically morphological characteristics consistent with autophagy were also observed by punctate distribution of MDC staining and the induction of LC3-II, including extensive autophagic vacuolization and enclosure of cell organelles by these autophagosomes. More significantly, specific depletion of Bak expression by small RNA interfering (siRNA) could partly attenuate TK VI-induced autophagy. However, siRNA against Bax led to increased autophagy.
Taken together, these findings showed for the first time that peptaibols were novel regulators involved in both apoptosis and autophagy, suggesting that the class of peptaibols might serve as potential suppressors of tumor cells.
Expression of survivin, a member of the inhibitor of apoptosis protein family, is elevated in human cancers and considered as a new therapeutic target. Mechanism upregulating survivin expression in tumour cells is poorly understood. In this study, we show that breast cancer patients harbouring a polymorphism G235A in the survivin promoter present a higher level of survivin expression. This polymorphism creates a binding site for the transcription factor GATA-1 inducing a second GATA-1-binding site in survivin promoter. At the mRNA level, GATA-1 was present in breast carcinomas and adjacent normal tissues, whereas the protein was only detected in carcinomas by western blot and immunohistochemistry. Transfection of wild-type and different constitutively active GATA-1 mutants (serine 26, 178 or 310) showed that only phospho-serine 26 GATA-1 was able to increase survivin expression. This increase was higher in G235A than in G235G cell lines. Phospho-serine 26 GATA-1 bound directly survivin promoter, with a stronger interaction in G235A than in G235G polymorphism indicating that both GATA-1-binding sites are functional. These data identify GATA-1 as a key feature in tumour aggressiveness by enhancing survivin expression and delineate its targeting as a possible new therapeutic strategy in breast carcinomas.
Epstein-Barr virus (EBV) is associated with several types of lymphomas and epithelial tumors including Burkitt's lymphoma (BL), HIV-associated lymphoma, posttransplant lymphoproliferative disorder, and nasopharyngeal carcinoma. EBV nuclear antigen 1 (EBNA1) is expressed in all EBV associated tumors and is required for latency and transformation. EBNA1 initiates latent viral replication in B cells, maintains the viral genome copy number, and regulates transcription of other EBV-encoded latent genes. These activities are mediated through the ability of EBNA1 to bind viral-DNA. To further elucidate the role of EBNA1 in the host cell, we have examined the effect of EBNA1 on cellular gene expression by microarray analysis using the B cell BJAB and the epithelial 293 cell lines transfected with EBNA1. Analysis of the data revealed distinct profiles of cellular gene changes in BJAB and 293 cell lines. Subsequently, chromatin immune-precipitation revealed a direct binding of EBNA1 to cellular promoters. We have correlated EBNA1 bound promoters with changes in gene expression. Sequence analysis of the 100 promoters most enriched revealed a DNA motif that differs from the EBNA1 binding site in the EBV genome.
The Epstein-Barr virus (EBV)-encoded EBNA1 protein is expressed in all EBV-associated tumours, including undifferentiated nasopharyngeal carcinoma (NPC), where it is indispensable for viral replication, genome maintenance and viral gene expression. EBNA1's transcription factor-like functions also extend to influencing the expression of cellular genes involved in pathways commonly dysregulated during oncogenesis, including elevation of AP-1 activity in NPC cell lines resulting in enhancement of angiogenesis in vitro. In this study we sought to extend these observations by examining the role of EBNA1 upon another pathway commonly deregulated during carcinogenesis; namely NF-kappaB.
In this report we demonstrate that EBNA1 inhibits the canonical NF-kappaB pathway in carcinoma lines by inhibiting the phosphorylation of IKKalpha/beta. In agreement with this observation we find a reduction in the phosphorylation of IkappaBalpha and reduced phosphorylation and nuclear translocation of p65, resulting in a reduction in the amount of p65 in nuclear NF-kappaB complexes. Similar effects were also found in carcinoma lines infected with recombinant EBV and in the EBV-positive NPC-derived cell line C666-1. Inhibition of NF-kappaB was dependent upon regions of EBNA1 essential for gene transactivation whilst the interaction with the deubiquitinating enzyme, USP7, was entirely dispensable. Furthermore, in agreement with EBNA1 inhibiting p65 NF-kappaB we demonstrate that p65 was exclusively cytoplasmic in 11 out of 11 NPC tumours studied.
Inhibition of p65 NF-kappaB in murine and human epidermis results in tissue hyperplasia and the development of squamous cell carcinoma. In line with this, p65 knockout fibroblasts have a transformed phenotype. Inhibition of p65 NF-kappaB by EBNA1 may therefore contribute to the development of NPC by inducing tissue hyperplasia. Furthermore, inhibition of NF-kappaB is employed by viruses as an immune evasion strategy which is also closely linked to oncogenesis during persistent viral infection. Our findings therefore further implicate EBNA1 in playing an important role in the pathogenesis of NPC.
The Epstein-Barr nuclear antigen 1 (EBNA1) protein enables the stable persistence of Epstein-Barr virus episomal genomes during latent infection, in part by tethering the episomes to the cellular chromosomes in mitosis. A host nucleolar protein, EBNA1-binding protein 2 (EBP2), has been shown to be important for interactions between EBNA1 and chromosomes in metaphase and to associate with metaphase chromosomes. Here, we examine the timing of the chromosome associations of EBNA1 and EBP2 through mitosis and the regions of EBNA1 that mediate the chromosome interactions at each stage of mitosis. We show that EBP2 is localized to the nucleolus until late prophase, after which it relocalizes to the chromosome periphery, where it remains throughout telophase. EBNA1 is associated with chromosomes early in prophase through to telophase and partially colocalizes with chromosomal EBP2 in metaphase through to telophase. Using EBNA1 deletion mutants, the chromosome association of EBNA1 at each stage of mitosis was found to be mediated mainly by a central glycine-arginine region, and to a lesser degree by N-terminal sequences. These sequence requirements for chromosome interaction mirrored those for EBP2 binding. Our results suggest that interactions between EBNA1 and chromosomes involve at least two stages, and that the contribution of EBP2 to these interactions occurs in the second half of mitosis.
The EBNA1 protein of Epstein-Barr virus (EBV) plays essential roles in enabling the replication and persistence of EBV genomes in latently infected cells and activating EBV latent gene expression, in all cases by binding to specific recognition sites in the latent origin of replication, oriP. Here we show that EBNA1 binding to its recognition sites in vitro is greatly stimulated by binding to the cellular deubiquitylating enzyme, USP7, and that USP7 can form a ternary complex with DNA-bound EBNA1. Consistent with the in vitro effects, the assembly of EBNA1 on oriP elements in human cells was decreased by USP7 silencing, whereas assembly of an EBNA1 mutant defective in USP7 binding was unaffected. USP7 affinity column profiling identified a complex between USP7 and human GMP synthetase (GMPS), which was shown to stimulate the ability of USP7 to cleave monoubiquitin from histone H2B in vitro. Accordingly, silencing of USP7 in human cells resulted in a consistent increase in the level of monoubquitylated H2B. The USP7-GMPS complex formed a quaternary complex with DNA-bound EBNA1 in vitro and, in EBV infected cells, was preferentially detected at the oriP functional element, FR, along with EBNA1. Down-regulation of USP7 reduced the level of GMPS at the FR, increased the level of monoubiquitylated H2B in this region of the origin and decreased the ability of EBNA1, but not an EBNA1 USP7-binding mutant, to activate transcription from the FR. The results indicate that USP7 can stimulate EBNA1-DNA interactions and that EBNA1 can alter histone modification at oriP through recruitment of USP7.
Survivin is a master regulator of cell proliferation and cell viability and is highly expressed in most human tumors. The molecular network linked to survivin expression in tumors has not been completely elucidated. In this study, we show that latency-associated nuclear antigen (LANA), a multifunctional protein of Kaposi's sarcoma-associated herpesvirus (KSHV) that is found in Kaposi's sarcoma tumors, upregulates survivin expression and increases the proliferation of KSHV-infected B cells. Analysis of pathway-specific gene arrays showed that survivin expression was highly upregulated in BJAB cells expressing LANA. The mRNA levels of survivin were also upregulated in HEK 293 and BJAB cells expressing LANA. Similarly, protein levels of survivin were significantly higher in LANA-expressing, as well as KSHV-infected, cells. Survivin promoter activity assays identified GC/Sp1 and p53 cis-acting elements within the core promoter region as being important for LANA activity. Gel mobility shift assays revealed that LANA forms a complex with Sp1 or Sp1-like proteins bound to the GC/Sp1 box of the survivin promoter. In addition, a LANA/p53 complex bound to the p53 cis-acting element within the survivin promoter, indicating that upregulation of survivin expression can also occur through suppression of p53 function. Furthermore, immunohistochemistry analyses revealed that survivin expression was upregulated in KSHV-associated Kaposi's sarcoma tissue, suggesting that LANA plays an important role in the upregulation of survivin expression in KSHV-infected endothelial cells. Knockdown of survivin expression by lentivirus-delivered small hairpin RNA resulted in loss of cell proliferation in KSHV-infected cells. Therefore, upregulation of survivin expression in KSHV-associated human cells contributes to their proliferation.
The Epstein-Barr virus (EBV) nuclear antigen (EBNA)-1 is the only viral protein expressed in all EBV-carrying malignancies, but its contribution to oncogenesis has remained enigmatic. We show that EBNA-1 induces chromosomal aberrations, DNA double-strand breaks, and engagement of the DNA damage response (DDR). These signs of genomic instability are associated with the production of reactive oxygen species (ROS) and are reversed by antioxidants. The catalytic subunit of the leukocyte NADPH oxidase, NOX2/gp91(phox), is transcriptionally activated in EBNA-1-expressing cells, whereas inactivation of the enzyme by chemical inhibitors or RNAi halts ROS production and DDR. These findings highlight a novel function of EBNA-1 and a possible mechanism by which expression of this viral protein could contribute to malignant transformation and tumor progression.
The Epstein-Barr virus (EBV)-encoded EBNA1 protein is expressed in all virus-associated tumours, including nasopharyngeal carcinoma (NPC), where it plays an essential role in EBV genome maintenance, replication and transcription. Previous studies suggest that EBNA1 may have additional effects relevant to oncogenesis, including enhancement of cell survival, raising the possibility that EBNA1 may influence cellular gene expression. We have recently demonstrated by gene expression microarray profiling in an NPC cell model that EBNA1 influences the expression of a range of cellular genes, including those involved in transcription, translation and cell signalling. Here, we report for the first time that EBNA1 enhances activity of the AP-1 transcription factor in NPC cells and demonstrate that this is achieved by EBNA1 binding to the promoters of c-Jun and ATF2, enhancing their expression. In addition, we demonstrate elevated expression of the AP-1 targets interleukin 8, vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1alpha in response to EBNA1 expression, which enhances microtubule formation in an in vitro angiogenesis assay. Furthermore, we confirm elevation of VEGF and the phosphorylated isoforms of c-Jun and ATF2 in NPC biopsies. These findings implicate EBNA1 in the angiogenic process and suggest that this viral protein might directly contribute to the development and aggressively metastatic nature of NPC.
The EBNA1 protein of Epstein-Barr virus (EBV) is essential for EBV latent infection in ensuring the replication and stable
segregation of the EBV genomes and in activating the transcription of other EBV latency genes. We have tested the ability
of four host proteins (Brd2, Brd4, DEK, and MeCP2) implicated in the segregation of papillomavirus and Kaposi's sarcoma-associated
herpesvirus to support EBNA1-mediated segregation of EBV-based plasmids in Saccharomyces cerevisiae. We found that Brd4 enabled EBNA1-mediated segregation while Brd2 and MeCP2 had a general stimulatory effect on plasmid maintenance.
EBNA1 interacted with Brd4 in both yeast and human cells through N-terminal sequences previously shown to mediate transcriptional
activation but not segregation. In keeping with this interaction site, silencing of Brd4 in human cells decreased transcriptional
activation by EBNA1 but not the mitotic chromosome attachment of EBNA1 that is required for segregation. In addition, Brd4
was found to be preferentially localized to the FR enhancer element regulated by EBNA1, over other EBV sequences, in latently
EBV-infected cells. The results indicate that EBNA1 can functionally interact with Brd4 in native and heterologous systems
and that this interaction facilitates transcriptional activation by EBNA1 from the FR element.
Latent Epstein-Barr virus (EBV) infection is strongly associated with several cancers, including nasopharyngeal carcinoma (NPC), a tumor that is endemic in several parts of the world. We have investigated the molecular basis for how EBV latent infection promotes the development of NPC. We show that the viral EBNA1 protein, previously known to be required to maintain the EBV episomes, also causes the disruption of the cellular PML (promyelocytic leukemia) nuclear bodies (or ND10s). This disruption occurs both in the context of a native latent infection and when exogenously expressed in EBV-negative NPC cells and involves loss of the PML proteins. We also show that EBNA1 is partially localized to PML nuclear bodies in NPC cells and interacts with a specific PML isoform. PML disruption by EBNA1 requires binding to the cellular ubiquitin specific protease, USP7 or HAUSP, but is independent of p53. We further observed that p53 activation, DNA repair and apoptosis, all of which depend on PML nuclear bodies, were impaired by EBNA1 expression and that cells expressing EBNA1 were more likely to survive after induction of DNA damage. The results point to an important role for EBNA1 in the development of NPC, in which EBNA1-mediated disruption of PML nuclear bodies promotes the survival of cells with DNA damage.
The Epstein-Barr virus (EBV) nuclear antigen-1 (EBNA-1) is a pleiotropic protein which has been characterized extensively both biochemically and functionally. It is the only one of the identified latent protein-encoding genes to be consistently expressed in viral-associated endemic Burkitt's lymphoma cells. As such, it is the only candidate viral protein to possibly perform a maintenance function in the tumour pathology. Despite this, no oncogenic activity has been attributed to the protein in tissue culture assays. The experiments described here were initiated to explore the activity of the protein in B cells in vivo. EBNA-1 transgenic mice were generated with transgene expression directed to the B cell compartment using the mouse Ig heavy chain intron enhancer. Transgene expression was demonstrated in the lymphoid tissues of mice of two independent lines. Transgenic positive mice of both lines succumb to B cell lymphoma. The B cell tumours are monoclonal, frequently of follicular centre cell origin and remarkably similar to those induced by transgenic c-myc expression. These results demonstrate that EBNA-1 is oncogenic in vivo and suggest that the gene product may play a direct role in the pathogenesis of Burkitt's lymphoma and possibly other EBV-associated malignancies.
Inhibitors of programmed cell death (apoptosis) aberrantly prolonging cell viability may contribute to cancer by facilitating the insurgence of mutations and by promoting resistance to therapy. Despite the identification of several new apoptosis inhibitors related to bcl-2 or to the baculovirus IAP gene, it is not clear whether apoptosis inhibition plays a general role in neoplasia. Here, we describe a new human gene encoding a structurally unique IAP apoptosis inhibitor, designated survivin. Survivin contains a single baculovirus IAP repeat and lacks a carboxyl-terminal RING finger. Present during fetal development, survivin is undetectable in terminally differentiated adult tissues. However, survivin becomes prominently expressed in transformed cell lines and in all the most common human cancers of lung, colon, pancreas, prostate and breast, in vivo. Survivin is also found in approximately 50% of high-grade non-Hodgkin's lymphomas (centroblastic, immunoblastic), but not in low-grade lymphomas (lymphocytic). Recombinant expression of survivin counteracts apoptosis of B lymphocyte precursors deprived of interleukin 3 (IL-3). These findings suggest that apoptosis inhibition may be a general feature of neoplasia and identify survivin as a potential new target for apoptosis-based therapy in cancer and lymphoma.
We show clear evidence for direct infection of various human epithelial cells by Epstein-Barr virus (EBV) in vitro. The successful infection was achieved by using recombinant EBV (Akata strain) carrying a selective marker gene but without any other artificial operations, such as introduction of the known EBV receptor (CD21) gene or addition of polymeric immunoglobulin A against viral gp350 in culture. Of 21 human epithelial cell lines examined, 18 became infected by EBV, as ascertained by the detection of EBV-determined nuclear antigen (EBNA) 1 expression in the early period after virus exposure, and the following selection culture easily yielded a number of EBV-infected clones from 15 cell lines. None of the human fibroblasts and five nonhuman-derived cell lines examined was susceptible to the infection. By comparison, cocultivation with virus producers showed approximately 800-fold-higher efficiency of infection than cell-free infection did, suggesting the significance of direct cell-to-cell contact as a mode of virus spread in vivo. Most of the epithelial cell lines infectable with EBV were negative for CD21 expression at the protein and mRNA levels. The majority of EBV-infected clones established from each cell line invariably expressed EBNA1, EBV-encoded small RNAs, rightward transcripts from the BamHI-A region of the virus genome, and latent membrane protein (LMP) 2A, but not the other EBNAs or LMP1. This restricted form of latent viral gene expression, which is a central issue for understanding epithelial oncogenesis by EBV, resembled that seen in EBV-associated gastric carcinoma and LMP1-negative nasopharyngeal carcinoma. The results indicate that direct infection of epithelial cells by EBV may occur naturally in vivo, and this could be mediated by an unidentified, epithelium-specific binding receptor for EBV. The EBV convertants are viewed, at least in terms of viral gene expression, as in vitro analogs of EBV-associated epithelial tumor cells, thus facilitating analysis of an oncogenic role(s) for EBV in epithelial cells.
The Epstein-Barr virus (EBV) genome can persist in dividing human B cells as multicopy circular episomes. Viral episomes replicate in synchrony with host cell DNA and are maintained at a relatively constant copy number for a long time. Only two viral elements, the replication origin OriP and the EBNA-1 protein, are required for the persistence of viral genomes during latency. EBNA-1 activates OriP during the S phase and may also contribute to the partition and/or retention of viral genomes during mitosis. Indeed, EBNA-1 has been shown to interact with mitotic chromatin. Moreover, viral genomes are noncovalently associated with metaphase chromosomes. This suggests that EBNA-1 may facilitate the anchorage of viral genomes on cellular chromosomes, thus ensuring proper partition and retention. In the present paper, we have investigated the chromosome-binding activity of EBV EBNA-1, herpesvirus papio (HVP) EBNA-1, and various derivatives of EBV EBNA-1, fused to a variant of the green fluorescent protein. The results show that binding to metaphase chromosomes is a common property of EBV and HVP EBNA-1. Further studies indicated that at least three independent domains (CBS-1, -2, and -3) mediate EBNA-1 binding to metaphase chromosomes. In agreement with the anchorage model, two of these domains mapped to a region that has been previously demonstrated to be required for the long-term persistence of OriP-containing plasmids.
Latent Epstein-Barr virus (EBV) genomes are maintained in human cells as low copy number episomes that are thought to be partitioned by attachment to the cellular mitotic chromosomes through the viral EBNA1 protein. We have identified a human protein, EBP2, which interacts with the EBNA1 sequences that govern EBV partitioning. Here we show that, in mitosis, EBP2 localizes to the condensed cellular chromosomes producing a staining pattern that is indistinguishable from that of EBNA1. The localization of EBNA1 proteins with mutations in the EBP2 binding region was also examined. An EBNA1 mutant (delta325-376) disrupted for EBP2 binding and segregation function was nuclear but failed to attach to the cellular chromosomes in mitosis. Our results indicate that amino acids 325-376 mediate the binding of EBNA1 to mitotic chromosomes and strongly suggest that EBNA1 mediates EBV segregation by attaching to EBP2 on the cellular mitotic chromosomes.
Telomerase is a multi-subunit ribonucleoprotein holoenzyme that stabilizes telomere length through the addition of new repeat sequence to the ends of chromosomes. Telomerase reverse transcriptase is the subunit of this complex responsible for the enzymatic activity of telomerase. Expression of the reverse transcriptase is regulated at the level of transcription through the action of transcription factors that target its promoter. Most Kaposi's sarcoma tumor cells are latently infected with the Kaposi's sarcoma-associated herpesvirus, and the constitutive expression of a viral-encoded latency-associated nuclear antigen has been shown to be important for the maintenance of the viral episome. The proliferative nature of Kaposi's sarcoma suggests that this antigen may also play a critical role in viral-mediated oncogenesis. In this study telomerase reverse transcriptase promoter elements cloned into a luciferase reporter plasmid were analyzed to determine the ability of the latency-associated nuclear antigen to regulate transcription. The latency-associated nuclear antigen transactivated the full-length promoter in 293T, 293, and BJAB cell lines. Furthermore, truncation promoter studies implicated sequence from -130 to +5 in viral-mediated activation. This region contains five Sp1 transcription factor-binding sites. Electrophoretic mobility shift assays indicated that the latency-associated nuclear antigen targets and affects the Sp1-DNA complex in the context of BJAB nuclear extracts.
We have described transgenic mice expressing Epstein-Barr virus (EBV) nuclear antigen-1 (EBNA-1) in B-cells which show a predisposition to lymphoma. To investigate the underlying oncogenic mechanisms, we have cross bred transgenic strains of mice, examined the pre-tumour B-cell phenotype and investigated the expression levels of selected cellular genes as a response to EBNA-1 expression. We have found that bcl-xL and the recombination activating genes (RAG) 1 and 2 are induced in pre-neoplastic samples of EBNA-1 expressing mice. Induction of bcl-xL may explain the observed redundancy in lymphomagenesis between transgenic EBNA-1 and bcl-2. In addition, bone marrow cells derived from the EmuEBNA-1 mice show a greater capacity for cultured growth compared to controls, particularly in the presence of IL-2. Notably, bcl-xL expression is responsive to IL-2. These data shed new light on the potential contribution of EBNA-1 to EBV associated tumorigenicity as well as to the viral life cycle and open a potential avenue for therapeutic intervention.
Survivin is an anti-apoptotic protein that is overexpressed in most human cancers. We show that survivin forms complexes with a cellular protein, hepatitis B X-interacting protein (HBXIP), which was originally recognized for its association with the X protein of hepatitis B virus (HBX). Survivin-HBXIP complexes, but neither survivin nor HBXIP individually, bind pro-caspase-9, preventing its recruitment to Apaf1, and thereby selectively suppressing apoptosis initiated via the mitochondria/cytochrome c pathway. Viral HBX protein also interacts with the survivin- HBXIP complex and suppresses caspase activation in a survivin-dependent manner. Thus, HBXIP functions as a cofactor for survivin, and serves as a link between the cellular apoptosis machinery and a viral pathogen involved in hepatocellular carcinogenesis.
Epstein-Barr virus (EBV) has been causally associated with at least five human malignancies. The exact contributions made by EBV to these cancers remain unknown. We demonstrate that one viral protein found in all EBV-associated malignancies, Epstein-Barr nuclear antigen 1 (EBNA-1), is required for survival of one of these cancers, EBV-positive Burkitt's lymphoma. Inhibition of EBNA-1 decreases survival of these tumor cells by inducing apoptosis. Expression of EBNA-1 in uninfected cells also can inhibit apoptosis induced by expression of p53 in the absence of the EBV genome. Our findings demonstrate that EBNA-1 is critical for the continued survival of EBV-associated Burkitt's lymphoma, and, by extension, for the other B cell tumors with which EBV is associated. Efficient inhibitors of EBNA-1's functions would likely prove useful in the therapy of EBV-associated malignancies.
Kaposi's sarcoma (KS)-associated herpesvirus (KSHV), a human oncogenic gamma-2-herpesvirus, transforms human endothelial cells and establishes latent infection at a low efficiency in vitro. During latent infection, only a limited number of genes are expressed, and the circularized viral genome is maintained as a multicopy episome. Latency-associated nuclear antigen (LANA), exclusively expressed during latency, has been shown to have a multifunctional role in KS pathogenesis. LANA tethers the viral episome to the host chromosome, thus ensuring efficient persistence of the viral genome during successive rounds of cell division. Besides episome maintenance, LANA modulates the expression of genes of various cellular and viral pathways, including those of retinoblastoma protein and p53. Herpesvirus saimiri (HVS), another gamma-2-herpesvirus, primarily infects New World primates. Orf73, encoding the nuclear antigen of HVS, is the positional homolog of the LANA gene, and the ORF73 protein has some sequence homology to KSHV LANA. However, the function of ORF73 of HVS has not been thoroughly investigated. In this report, we show that HVS ORF73 may be important for episome persistence and colocalizes with the HVS genomic DNA on metaphase chromosomes. Furthermore, HVS terminal repeats (TRs) contain a cis-acting sequence similar to that in KSHV TRs, suggesting that the LANA binding sequence is conserved between these two viruses. This cis-acting element is sufficient to bind HVS ORF73 from strains C488 and A11, and plasmids containing the HVS C488 TR element are maintained and replicate in HVS C488 ORF73-expressing cells.
Increasing evidence indicates that survivin, an inhibitor of apoptosis protein (IAP), is expressed in human cancer cells but is absent from most normal adult tissues. Here, we examined the feasibility of using a survivin promoter (Sur-P) to direct therapeutic expression of a proapoptotic gene specifically in human tumor cells. First, we demonstrated that this promoter was highly active in human tumor cells but not in normal cells. Second, we found that Sur-P activity was upregulated by hypoxia in tumor cells. Third, to further enhance this promoter's activity under hypoxia, we added a hypoxia-responsive element (HRE) from the vascular endothelial growth factor gene promoter in its 5' region, and showed that this combination resulted in a further increase in the level of gene expression in hypoxic tumor cells. Finally, we demonstrated that expression of an autocatalytic reverse caspase-3 gene by this promoter specifically induced apoptotic cell death in human tumor cells but not in normal cells. These findings support the use of promoters Sur-P or chimeric HRE-Sur-P for generating novel vectors for cancer gene therapy.
Survivin is expressed in many cancers but not in normal adult tissues and is transcriptionally regulated. To test the feasibility of using the survivin promoter to induce cancer-specific transgene expression in lung cancer gene therapy, a vector expressing a luciferase gene driven by the survivin promoter was constructed and evaluated in vitro and in vivo. We found that the survivin promoter was generally more highly activated in cancer cell lines than in normal and immortalized normal cell lines. When delivered intravenously by DNA:liposome complexes, the survivin promoter was more than 200 times more cancer specific than the cytomegalovirus promoter in vivo. To identify lung cancer patients who may benefit from gene therapy with the survivin promoter, we measured survivin protein expression in surgical specimens of 75 non-small-cell lung cancers and 10 normal lung tissues by immunohistochemical staining and found that survivin is expressed in most of the non-small-cell lung cancers tested (81%, 61 of 75) but none of the normal lung tissues. The survivin promoter also induced transgene expression of a mutant Bik in cancer cells, which suppressed the growth of cancer cells in vitro and in vivo. These results indicate that the survivin promoter is a cancer-specific promoter for various cancers and that it may be useful in cancer gene therapy.
During latency, Epstein-Barr virus (EBV) is stably maintained as a circular plasmid that is replicated once per cell cycle
and partitioned at mitosis. Both these processes require a single viral protein, EBV nuclear antigen 1 (EBNA1), which binds
two clusters of cognate binding sites within the latent viral origin, oriP. EBNA1 is known to associate with cellular metaphase chromosomes through chromosome-binding domains within its amino terminus,
an association that we have determined to be required not only for the partitioning of oriP plasmids but also for their replication. One of the chromosome-binding domains of EBNA1 associates with a cellular nucleolar
protein, EBP2, and it has been proposed that this interaction underlies that ability of EBNA1 to bind metaphase chromosomes.
Here we demonstrate that EBNA1's chromosome-binding domains are AT hooks, a DNA-binding motif found in a family of proteins
that bind the scaffold-associated regions on metaphase chromosomes. Further, we demonstrate that the ability of EBNA1 to stably
replicate and partition oriP plasmids correlates with its AT hook activity and not its association with EBP2. Finally, we examine the contributions of
EBP2 toward the ability of EBNA1 to associate with metaphase chromosomes in human cells, as well as support the replication
and partitioning of oriP plasmids in human cells. Our results indicate that it is unlikely that EBP2 directly mediates these activities of EBNA1 in
human cells.
Epstein–Barr nuclear antigen 1, or EBNA‐1, is required for the replication of the EBV genome as an extra‒chromosomal element and is a key transcriptional regulator of this virus's latent gene expression. In this review we will describe the salient features of EBNA‐1 and oriP, the latent origin of EBV to which EBNA‐1 binds site‐specifically. EBNA‐1's association with host cellular factors, its association with metaphase chromosomes, and its ability to link DNAs to which it binds will be discussed in relation to its roles in replication and transcriptional activation. Although the mechanisms by which EBNA‐1 facilitates replication and transcription largely remain enigmatic, EBV's viral replicon has been exploited successfully for applications in gene therapy and in the design of eukaryotic vectors for use in cell culture. Copyright © 2000 John Wiley & Sons, Ltd.
