EBV-associated Malignancies Research Articles

Interleukin 15 Provides Relief to CTLs from Regulatory T Cell–Mediated Inhibition: Implications for Adoptive T Cell–Based Therapies for Lymphoma

Systemic administration of recombinant interleukin (IL)-2 is used to support the expansion and persistence of adoptively transferred antigen-specific CTLs in patients with cancer. However, IL-2 also expands regulatory T cells (Treg) that in turn impair the antitumor activity of CTLs. As recombinant IL-15 is approaching clinical applications, we assessed the effects of this cytokine on the proliferation and antitumor activity of CTLs in the presence of Tregs. We used the model of adoptive transfer of Epstein-Barr virus (EBV)-CTLs, as these cells induce responses in patients with EBV-associated Hodgkin lymphoma, and Tregs are frequently abundant in these patients. Tregs were isolated from the peripheral blood of healthy donors and patients with Hodgkin lymphoma or from Hodgkin lymphoma tumors and assessed for their ability to inhibit the proliferation and antitumor activity of EBV-CTLs in the presence of IL-15 or IL-2. Specific molecular pathways activated by IL-15 were also explored. We found that in the presence of Tregs, IL-15, but not IL-2, promoted the proliferation, effector function, and resistance to apoptosis of effectors T cells and EBV-CTLs. IL-15 did not reverse or block Tregs but instead preferentially supported the proliferation of CTLs and effector T cells as compared with Tregs. IL-15 selectively favors the survival, proliferation, and effector function of antigen-specific CTLs in the presence of Tregs, and thus IL-15, unlike IL-2, would have a significant impact in sustaining expansion and persistence of adoptively transferred CTLs in patients with cancer, including those infused with EBV-CTLs for treatment of EBV-associated malignancies.

Epstein-Barr virus-driven lymphomagenesis in the context of human immunodeficiency virus type 1 infection

Epstein–Barr virus (EBV) is a ubiquitous human γ-herpes virus which establishes a life-long asymptomatic infection in immunocompetent hosts. In human immunodeficiency virus type 1 (HIV-1) infected patients, the impaired immunosurveillance against EBV may favor the development of EBV-related diseases, ranging from lymphoproliferative disorders to B cell non-Hodgkin’s lymphomas (NHL). Antiretroviral therapy (ART) has significantly modified the natural course of HIV-1 infection, resulting in decreased HIV-1 plasmaviremia, increased CD4 lymphocytes, and decreased opportunistic infections, indicating a restoration of immune functions. However, the impact of ART appears to be less favorable on EBV-related malignancies than on other AIDS-defining tumors, such as Kaposi’s sarcoma, and NHL remains the most common cancer during the ART era. EBV-driven tumors are associated with selective expression of latent oncogenic proteins, but uncontrolled lytic cycle with virus replication and/or reactivation may favor cell transformation, at least in the early phases. Several host’s factors may promote EBV reactivation and replication; besides immunodepression, inflammation/chronic immune stimulation may play an important role. Microbial pathogen-associated molecular patterns and endogenous damage-associated molecular patterns, through Toll-like receptors, activate the immune system and may promote EBV reactivation and/or polyclonal expansion of EBV-infected cells. A body of evidence suggests that chronic immune stimulation is a hallmark of HIV-1 pathogenesis and may persist even in ART-treated patients. This review focuses on lymphomagenesis driven by EBV both in the context of the natural history of HIV-1 infection and in ART-treated patients. Understanding the mechanisms involved in the expansion of EBV-infected cells is a premise for the identification of prognostic markers of EBV-associated malignancies.

CD44+ Cancer Stem-Like Cells in EBV-Associated Nasopharyngeal Carcinoma

Nasopharyngeal carcinoma (NPC) is a unique EBV-associated epithelial malignancy, showing highly invasive and metastatic phenotype. Despite increasing evidence demonstrating the critical role of cancer stem-like cells (CSCs) in the maintenance and progression of tumors in a variety of malignancies, the existence and properties of CSC in EBV-associated NPC are largely unknown. Our study aims to elucidate the presence and role of CSCs in the pathogenesis of this malignant disease. Sphere-forming cells were isolated from an EBV-positive NPC cell line C666-1 and its tumor-initiating properties were confirmed by in vitro and in vivo assays. In these spheroids, up-regulation of multiple stem cell markers were found. By flow cytometry, we demonstrated that both CD44 and SOX2 were overexpressed in a majority of sphere-forming C666-1 cells. The CD44+SOX2+ cells was detected in a minor population in EBV-positive xenografts and primary tumors and considered as potential CSC in NPC. Notably, the isolated CD44+ NPC cells were resistant to chemotherapeutic agents and with higher spheroid formation efficiency, showing CSC properties. On the other hand, microarray analysis has revealed a number of differentially expressed genes involved in transcription regulation (e.g. FOXN4, GLI1), immune response (CCR7, IL8) and transmembrane transport (e.g. ABCC3, ABCC11) in the spheroids. Among these genes, increased expression of CCR7 in CD44+ CSCs was confirmed in NPC xenografts and primary tumors. Importantly, blocking of CCR7 abolished the sphere-forming ability of C666-1 in vitro. Expression of CCR7 was associated with recurrent disease and distant metastasis. The current study defined the specific properties of a CSC subpopulation in EBV-associated NPC. Our findings provided new insights into developing effective therapies targeting on CSCs, thereby potentiating treatment efficacy for NPC patients.

Phase I Clinical Trial of Valacyclovir and Standard of Care Cyclophosphamide in Children With Endemic Burkitt Lymphoma in Malawi

Treatment options for Epstein-Barr virus (EBV)-associated Burkitt lymphoma in Africa are limited because of chemotherapy-associated toxicity. Since other EBV-associated diseases respond to antiviral agents, we investigated adding an antiviral agent, valacyclovir, to the current chemotherapy regimen in Malawi. In this phase I safety study, we showed that cyclophosphamide combined with valacyclovir was safe. Phase II efficacy trials should now be undertaken. Nucleoside analogues, including acyclovir, ganciclovir, and their precursors, have shown some efficacy against several Epstein-Barr virus (EBV)-associated diseases, including active EBV infection and posttransplantation lymphoproliferative disorder (PTLD). They have also been proposed as a possible treatment for EBV-associated malignancies, including endemic Burkitt lymphoma. The safety of nucleoside analogues in combination with chemotherapy in the developing world has not been studied and is necessary before any large scale efficacy trials are conducted. Children 3-15 years old meeting inclusion criteria were assigned to a 3+3 dose escalation trial of combination valacyclovir (15 and 30 mg/kg, 3 times daily for 40 days) and cyclophosphamide (CPM) (40 mg/kg day 1, 60 mg/kg on days 8, 18, and 28) or CPM monotherapy. Subjects were monitored for clinical and laboratory toxicity and had EBV levels measured regularly. Dose-limiting toxicity (DLT) was our primary outcome. We found that the combination of valacyclovir and CPM was safe and did not lead to any DLT compared with CPM monotherapy. The most common side effects were vomiting, abdominal pain, and tumor site pain, which were similar in both arms. Patients with measurable serum EBV showed decreased loads over their treatment course. We recommend a phase II valacyclovir dose of 30 mg/kg 3 times daily for 40 days. We also observed that 6 of our 12 patients with presumed Burkitt lymphoma had measurable EBV viral loads that decreased over the course of their treatment, suggesting that phase II studies should investigate this correlation further. This study paves the way for a phase II efficacy trial of combined valacyclovir and CPM in the treatment of endemic Burkitt lymphoma.

TRAF Binding Is Required for a Distinct Subset of In Vivo B Cell Functions of the Oncoprotein LMP1

EBV-encoded latent membrane protein 1 (LMP1) is important for EBV contributions to B cell transformation and many EBV-associated malignancies, as well as EBV-mediated exacerbation of autoimmunity. LMP1 functionally mimics TNF receptor (TNFR) superfamily member CD40, but LMP1 signals and downstream effects are amplified and sustained compared with CD40. CD40 and LMP1 both use TNFR-associated factor (TRAF) adaptor proteins, but in distinct ways. LMP1 functions require TRAFs 3, 5, and 6, which interact with LMP1. However, TRAFs can also contribute to signaling in the absence of direct interactions with cell surface receptors, so we investigated whether their roles in LMP1 in vivo functions require direct association. We show in this study that the LMP1 TRAF binding site was required for LMP1-mediated autoantibody production, the germinal center response to immunization, and optimal production of several isotypes of Ig, but not LMP1-dependent enlargement of secondary lymphoid organs in transgenic mice. Thus, LMP1 in vivo effects can be mediated via both TRAF binding-dependent and -independent pathways. Together with our previous findings, these results indicate that TRAF-dependent receptor functions may not always require TRAF-receptor binding. These data suggest that TRAF-mediated signaling pathways, such as those of LMP1, may be more diverse than previously appreciated. This finding has significant implications for receptor and TRAF-targeted therapies.

Variations of Epstein-Barr virus nuclear antigen 1 in Epstein-Barr virus-associated gastric carcinomas from Guangzhou, southern China.

Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is the only viral protein consistently expressed in all EBV-associated malignancies, and play a critical role in the onset, progression, and/or maintenance of these tumors. Based on the signature changes at amino acid residue 487, EBNA1 is classified into five distinct subtypes: P-ala, P-thr, V-leu, V-val and V-pro. In the present study, the sequence variations of EBNA1 in EBV-associated gastric carcinoma (EBVaGC) and throat washing (TW) samples of healthy EBV carriers in Guangzhou, southern China, where nasopharyngeal carcinoma (NPC) is endemic, were analyzed by PCR and DNA sequencing. V-val subtype was the most predominant (53.6%, 15/28) in EBVaGC, followed by P-ala (42.9%, 12/28) and V-leu (32.1%, 9/28) subtypes. In TWs of healthy EBV carriers, V-val subtype was also predominant (85.7%, 18/21). The sequence variations of EBNA1 in EBVaGC were similar to those in TW of healthy EBV carriers (p>0.05), suggesting that the EBV strains in EBVaGC might originate from the viral strains prevalent within the background population. The predominance of V-val subtype in EBVaGC in Guangzhou was similar to that in EBVaGC in northern China and Japan, but was different from that in EBVaGC in America, suggesting that the variations of EBNA1 in EBVaGC represent geographic-associated polymorphisms rather than tumor-specific mutations. In addition, the EBNA1 variations in EBVaGC in gastric remnant carcinoma were also determined. V-leu subtype was detected in all 4 (100%) cases, although 2 cases occurred as mixed infection with P-ala subtype. This is different from the predominant V-val subtype in EBVaGC in conventional gastric carcinoma, suggesting that V-leu might be a subtype that adapts particularly well to the microenvironment within the gastric stump and enters the remnant gastric mucosa epithelia easily. This, to our best knowledge, is the first investigation of EBNA1 polymorphisms in EBVaGC from endemic area of NPC.

Epstein- Barr Virus Related Malignant and Non-Malignant Transformation in Two Paediatric Liver Transplant Patients

Introduction: Epstein-Barr virus (EBV)-associated smooth muscle tumors (EBV-SMT) are a distinctive group of mesenchymal tumors that develop exclusively in patients with clinical immunosuppression, predominantly in HIV and recipients of solid organ transplantation, particularly paediatric patients. The incidence of developing neoplastic disorders after solid organ transplantation is highly dependent on the intensity of the prophylactic anti-viral regimen, the induction therapy, as well as high levels of calcineurin inhibitors. Recently 2 of our liver transplant patients have been diagnosed with both benign and malignant EBV- related smooth muscle tumours presenting as significant gastrointestinal bleeds. Since the 1970‘s, there have been a small number of cases of leiomyomas in immunosuppressed patients post transplant. Case Presentation: Patient ZH underwent a liver transplant for missed biliary atresia at one year five months of age. Her course was complicated by portal vein thrombosis and a failed Meso-Rex shunt two months post transplant. Despite this, her liver remained well perfused on hepatic arterial blood supply with no problems with rejection. A meso-caval shunt was done at six years of age when she continued having significant gastrointestinal bleeds. A mesenteric lymph node removed incidentally during surgery was consistent with leiomyosarcoma on histology. Further imaging showed lung metastatic lesions and at a subsequent laparotomy a primary ileal lesion was resected. She completed chemotherapy and six weeks later had another gastrointestinal bleed which revealed a leiomyoma on histology from a rectal biopsy. She was commenced on IVI gancyclovir and tacrolimus was changed to sirolimus. Despite the above management, a repeat colonic biopsy following another gastrointestinal bleed confirmed the presence of a leiomyoma. Patient AJG underwent a liver transplant at four years of age for biliary atresia. He had an uncomplicated course post transplant except for raised EBV levels initially treated with three months of intravenous gancyclovir and then oral valgancyclovir. He had a massive gastrointestinal bleed two years later which revealed features consistent with post transplant lymphoproliferative disorder of terminal ileum and leiomyoma in the sigmoid colon. Discussion: Smooth muscle EBV induced tumours after organ transplantation are rare, occurring more often in the paediatric population. These tumours are generally insidious and their prognosis depends on the size, site and surgical options. There is no standard treatment. Although the benign variant carries a good prognosis, leiomyosarcoma carries a poor prognosis. We are highlighting these two cases to emphasise the need for ongoing vigilance to detect EBV-associated malignancies with regular EBV PCR monitoring and adjustment of immunosuppression if EBV PCR titres rise. Management involves an interdisciplinary approach with input from transplant physicians, surgeons and oncologists. Management involves surgical resection of all lesions if possible, reduction of immunosuppression and possibly changing from a calcineurin inhibitor to a MTOR inhibitor. Once malignant transformation has occurred, the prognosis is poor.

Cytolytic Virus Activation Therapy for Epstein-Barr Virus–Driven Tumors

Nasopharyngeal carcinoma (NPC) is causally linked to Epstein-Barr virus (EBV) infection. Because all tumor cells carry EBV, the virus itself is a potential target for therapy. In these tumor cells, EBV hides in a latent state and expresses only a few non-immunogenic proteins for EBV maintenance and contributes to tumor growth. We developed a cytolytic virus activation (CLVA) therapy for NPC treatment, reactivating latent EBV, triggering immune recognition, and inducing susceptibility to antiviral therapy. CLVA therapy combines gemcitabine (GCb) and valproic acid (VPA) for virus activation and tumor clearance with (val)ganciclovir (GCV) as the antiviral drug to block virus replication and kill proliferating virus-infected cells. CLVA treatment was optimized and validated in NPC cell lines and subsequently tested in 3 Dutch patients with NPC that was refractory to conventional treatment. In NPC cell lines, both GCb and VPA can induce the lytic cycle of EBV. Their combination resulted in a strong synergistic effect. The addition of GCV resulted in higher cytotoxicity compared with chemotherapy alone, which was not observed in EBV-negative cells. CLVA therapy was analyzed in 3 patients with end-stage NPC. Patients developed increased levels of viral DNA in the circulation originating from apoptotic tumor cells, had disease stabilization, and experienced improved quality of life. Our results in the initial CLVA-treated patients indicate that the therapy had a biological effect and was well tolerated with only moderate transient toxicity. This new virus-specific therapy could open a generic approach for treatment of multiple EBV-associated malignancies.

Epstein-Barr nuclear antigen 1 induces expression of the cellular microRNA hsa-miR-127 and impairing B-cell differentiation in EBV-infected memory B cells. New insights into the pathogenesis of Burkitt lymphoma

Epstein-Barr Virus (EBV) is a γ-herpesvirus that infects >90% of the human population. Although EBV persists in its latent form in healthy carriers, the virus is also associated with several human cancers. EBV is strongly associated with Burkitt lymphoma (BL), even though there is still no satisfactory explanation of how EBV participates in BL pathogenesis. However, new insights into the interplay between viruses and microRNAs (miRNAs) have recently been proposed. In particular, it has been shown that B-cell differentiation in EBV-positive BL is impaired at the post-transcriptional level by altered expression of hsa-miR-127. Here, we show that the overexpression of hsa-miR-127 is due to the presence of the EBV-encoded nuclear antigen 1 (EBNA1) and give evidence of a novel mechanism of direct regulation of the human miRNA by this viral product. Finally, we show that the combinatorial expression of EBNA1 and hsa-miR-127 affects the expression of master B-cell regulators in human memory B cells, confirming the scenario previously observed in EBV-positive BL primary tumors and cell lines. A good understanding of these mechanisms will help to clarify the complex regulatory networks between host and pathogen, and favor the design of more specific treatments for EBV-associated malignancies.

Dasatinib therapy results in decreased B cell proliferation, splenomegaly, and tumor growth in a murine model of lymphoma expressing Myc and Epstein-Barr virus LMP2A

Epstein-Barr virus (EBV) infection and latency has been associated with malignant diseases including nasopharyngeal carcinoma, Hodgkin lymphoma, Burkitt lymphoma, and immune deficiency associated lymphoproliferative diseases. EBV-encoded latent membrane protein 2A (LMP2A) recruits Lyn and Syk kinases via its SH2-domain binding motifs, and modifies their signaling pathways. LMP2A transgenic mice develop hyperproliferative bone marrow B cells and immature peripheral B cells through modulation of Lyn kinase signaling. LMP2A/λ-MYC double transgenic mice develop splenomegaly and cervical lymphomas starting at 8weeks of age. We reasoned that targeting Lyn in LMP2A-expressing B cells with dasatinib would provide a therapeutic option for EBV-associated malignancies. Here, we show that dasatinib inhibits B cell colony formation by LMP2A transgenic bone marrow cells, and reverses splenomegaly and tumor growth in both a pre-tumor and a syngeneic tumor transfer model of EBV-associated Burkitt lymphoma. Our data support the idea that dasatinib may prove to be an effective therapeutic molecule for the treatment of EBV-associated malignancies.

Effects of Epstein-Barr Virus Latent Membrane Protein 2A (LMP2A) on cytokine production in primary and transformed B cells (105.40)

Abstract Epstein-Barr Virus (EBV) latently-infected B cells are precursors of EBV-associated malignancies. However, the transition from a latently-infected cell to a malignant tumor cell is poorly understood. Studies show that EBV modulates pro-survival and anti-inflammatory cytokine levels in EBV-transformed cell lines. However, gene expression in these cell lines may not reflect gene expression found in latent B cells and lymphomas. One EBV protein, LMP2A, is consistently detected in both latently-infected resting B cells, as well as in EBV-associated malignancies. Therefore, we hypothesized that LMP2A may modulate cytokines that aid in the transition between latency and malignancy. Cytokine array data analyzing B cells from LMP2A-transgenic mice and B cell lymphomas expressing LMP2A indicate that LMP2A does not globally alter B cell-produced cytokine levels, but rather targets a limited number of cytokines. Additional studies using ELISA and Real-time RT-PCR confirm that LMP2A increases IL-10 levels and flow cytometry reveals that LMP2A increases the amount of IL-10 produced per B cell. Inhibitors of the LMP2A signaling pathway are being tested to determine the signaling requirements for LMP2A-mediated increases in IL-10. These data identify a novel function of LMP2A in the alteration of a cytokine that is important for both tumor survival and anti-tumor responses.

Global expression analysis of EBV-infected B cells early and late after infection reveals a dynamic interplay between growth and survival signals

Epstein-Barr virus (EBV) is a member of the γ-herpesvirus family estimated to infect 90% of the world’s adult population. Despite the high prevalence of infection, EBV-associated malignancies are largely kept in check by a strong cytotoxic T cell immune response. However, EBV causes lymphoproliferative disease in immune-deficient individuals following transplant and CNS and other lymphomas in HIV-infected individuals. EBV also plays a role in the pathogenesis of endemic African Burkitt’s lymphoma, Hodgkin’s disease, and nasopharyngeal carcinoma. In vitro, EBV infection of primary human B cells results in proliferation and outgrowth of indefinitely proliferating lymphoblastoid cell lines, or LCLs, which represent a viable model for the pathogenesis of EBV-associated malignancies. Ongoing studies in our group have shown that the earliest EBV-infected proliferating B cells differ greatly from LCLs phenotypically. Using CFSE staining and flow cytometry-based sorting, we have isolated these early proliferating B cells and analyzed genome-wide exon level mRNA expression relative to uninfected resting B cells and LCLs. Gene ontology analysis of these expression data identified enrichment of genes associated with proliferation and the DNA damage response in early proliferation. Furthermore, c-Myc mRNA and activity, as inferred from its genome-wide expression signature, were also highly induced early. Most interestingly, however, analysis of changes from early proliferating to final LCL outgrowth revealed striking attenuation of proliferative gene sets and c-Myc, along with delayed induction kinetics of NFκB activation. Specifically, genes with NFκB motifs in their promoters were highly expressed from early proliferating B cells to LCL and many canonical NFκB targets and pathway components were induced at late times after infection. These results suggest a novel, dynamic EBV-driven growth pattern and expression program that relies on mutually exclusive signals from c-Myc and NFκB. Furthermore, our data suggest that the earliest stages of EBV-driven B cell immortalization may provide unique insight into the pathogenesis of EBV-associated malignancies.

Analysis of the miRNA targetome in EBV-infected B cells

microRNAs (miRNAs) are ~22 nt, non-coding regulatory RNAs expressed by all metazoans and several viruses. During latent infection, Epstein-Barr virus (EBV) expresses 25 pre-miRNAs and influences the expression of cellular miRNAs, such as miR-155 and miR-21, all of which potentially have roles in viral oncogenesis. To date, only a limited number of EBV miRNA targets have been identified; thus, the role of viral miRNAs in viral pathogenesis and/or oncogenesis is not well defined. Using photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) [1] combined with high-throughput sequencing and computational analysis [2], we interrogated the miRNA targetome in EBV-infected B cells. We identified miRNA binding sites in over 5,700 cellular 3’ untranslated regions (UTRs), 25% of which contained sites for EBV miRNAs. miRNA binding sites were also identified at a lower frequency in coding regions. Our results reveal that EBV miRNAs predominantly target host cellular transcripts, thereby reshaping the host environment. Furthermore, viral miRNA targets are involved in multiple biological processes that are directly relevant to EBV infection, including modulation of immune responses, cell proliferation, and cell survival. Finally, we identified a number of viral transcripts that contained conserved binding sites for cellular miRNAs, including members of the myc-regulated miR-17/92 cluster. This comprehensive survey of the miRNA targetome in EBV-infected B cells is a positive step towards identifying novel therapeutic targets for EBV-associated malignancies.

EBV Latency II-derived Peptides Induce A Specific CD4+ Cytotoxic T-cell Activity and Not A CD4+ Regulatory T-cell Response

Epstein-Barr virus (EBV) is associated with several malignant diseases that can be distinguished by their patterns of viral latent gene expression. We developed here an original peptidic approach to favor the induction of a specific CD4+ T-cell response against EBV latency II malignancies (Hodgkin's lymphoma, nasopharyngeal carcinoma, T/NK lymphoma). Previously, we selected 6 peptides derived from EBV nuclear antigen-1, latency membrane proteins (LMP)-1, and LMP-2 highly promiscuous for major histocompatibility complex class II molecules and showed their ability to induce interferon-γ-secreting CD4+ T cells. We confirmed here that all peptides used in cocktail are recognized by human CD4+ memory T cells from healthy donors, inducing a broad T-helper (Th)1 cytokine secretion interferon-γ, interleukin-2. Furthermore, we have generated EBV-specific CD4+ T-cell lines and proved their cytotoxic potential, not only on original models expressing latency II antigens (EBV-transformed T cell or monocyte), but also on lymphoblastoid cell lines expressing latency III antigens (lymphoblastoid cell lines). In addition, granzyme B enzyme-linked immunospot assays suggested that a part of this specific cytotoxic activity could be linked to the granule lytic pathway. Very importantly, we have showed that neither phenotypical changes nor functional activities of CD4+CD25+CD127(low)-regulatory T cells were observed in response to EBV+ peptides, avoiding any risk of aggravation of the preexisting immunosuppressive environment reported in EBV-associated malignancies. In conclusion, our promiscuous peptide cocktail could be used safely in immunotherapeutic approaches against EBV latency II malignancies, mainly to prevent relapse in high-risk patients further to classic treatments.

Methylation profiling of Epstein-Barr virus immediate-early gene promoters, BZLF1 and BRLF1in tumors of epithelial, NK- and B-cell origins

BackgroundEpstein-Barr virus (EBV) establishes its latency in EBV-associated malignancies, accompanied by occasionally reactivated lytic cycle. Promoter CpG methylation of EBV genome plays an essential role in maintaining viral latency. Two immediate-early (IE) genes, BZLF1 and BRLF1, induce the switch from latent to lytic infection. Studies of methylation-dependent binding of BZLF1 and BRLF1 to EBV promoters have been well reported, but little is known about the methylation status of BZLF1 and BRLF1 promoters (Zp and Rp) in tumor samples.MethodsWe evaluated the methylation profiles of Zp and Rp by methylation-specific PCR (MSP) and bisulfite genomic sequencing (BGS), as well as BZLF1 and BRLF1 expression by semiquantitative reverse transcription (RT)-PCR in tumors of epithelial, NK- and B-cell origins.ResultsWe found that both Zp and Rp were hypermethylated in all studied EBV-positive cell lines and tumors of lymphoid (B- or NK cell) or epithelial origin, while unmethylated Zp and Rp alleles were detected in cell lines expressing BZLF1 and BRLF1. Following azacytidine treatment or combined with trichostatin A (TSA), the expression of BZLF1 and BRLF1 was restored along with concomitant promoter demethylation, which subsequently induced the reactivation of early lytic gene BHRF1 and late lytic gene BLLF1.ConclusionsHypermethylation of Zp and Rp mediates the frequent silencing of BZLF1 and BRLF1 in EBV-associated tumors, which could be reactivated by demethylation agent and ultimately initiated the EBV lytic cascade.

Sequence Variations of Latent Membrane Protein 2A in Epstein-Barr Virus-Associated Gastric Carcinomas from Guangzhou, Southern China

Latent membrane protein 2A (LMP2A), expressed in most Epstein-Barr virus (EBV)-associated malignancies, has been demonstrated to be responsible for the maintenance of latent infection and epithelial cell transformation. Besides, it could also act as the target for a CTL-based therapy for EBV-associated malignancies. In the present study, sequence variations of LMP2A in EBV-associated gastric carcinoma (EBVaGC) and healthy EBV carriers from Guangzhou, southern China, where nasopharyngeal carcinoma (NPC) is endemic, were investigated. Widespread sequence variations in the LMP2A gene were found, with no sequence identical to the B95.8 prototype. No consistent mutation was detected in all isolates. The immunoreceptor tyrosine-based activation motif (ITAM) and PY motifs in the amino terminus of LMP2A were strictly conserved, suggesting their important roles in virus infection; while 8 of the 17 identified CTL epitopes in the transmembrane region of LMP2A were affected by at least one point mutation, which may implicate that the effect of LMP2A polymorphisms should be considered when LMP2A-targeted immunotherapy is conducted. The polymorphisms of LMP2A in EBVaGC in gastric remnant carcinoma (GRC) were for the first time investigated in the world. The LMP2A sequence variations in EBVaGC in GRC were somewhat different from those in EBVaGC in conventional gastric carcinoma. The sequence variations of LMP2A in EBVaGC were similar to those in throat washing of healthy EBV carriers, indicating that these variations are due to geographic-associated polymorphisms rather than EBVaGC-associated mutations. This, to our best knowledge, is the first detailed investigation of LMP2A polymorphisms in EBVaGC in Guangzhou, southern China, where NPC is endemic.

THE INFLUENCE OF POINT MUTATIONS IN THE EPSTEIN-BARR VIRUS LMP1 ONCOGENE ON THE CELL CYTOSKELETON AND ACTIVATION OF INDUCIBLE FORM OF NO SYNTHASE

One of the latent proteins encoded by the Epstein-Barr virus (EBV), the latent membrane protein 1 (LMP1), plays a key role in developing of EBV-associated human malignancies. Polymorphism of LMP1 protein is its characteristic feature. Some specific mutations in LMP1 genome have previously been detected in different geographic regions, however, the influence of these mutations on functional activity of LMP1 was not still determined. In this study we demonstrated for the first time the significance of individual point mutations among common ones observed in LMP1 and their combination on activation of the inducible form of nitric oxide synthase (iNOS). In addition, the influence of above mutations localized in the CTAR regions of the LMP1 molecule has also been investigated on structural components of the fibroblasts of the Rat1cell line.

Unique variations of Epstein–Barr virus-encoded BARF1 gene in nasopharyngeal carcinoma biopsies

The Epstein–Barr virus (EBV) BamHI-A rightward frame 1 (BARF1) gene is frequently expressed in EBV-associated epithelial malignancies and involves in oncogenicity and immunomodulation. To characterize the variations of BARF1 gene in different populations, the sequences of BARF1 gene in Northern Chinese nasopharyngeal carcinoma (NPC), EBV-associated gastric carcinoma (EBVaGC) and healthy donors were analyzed. The correlation of BARF1 variation with polymorphisms of BamHI F fragment (type F and f variants) and EBV-coded viral interleukin-10 (vIL-10) gene (B95-8 and SPM patterns) was also explored. Two major subtypes of BARF1 gene, designated as B95-8 and V29A, were identified. B95-8 subtype had identical amino acid sequence to B95-8 and was the dominant subtype among the EBV isolates from Northern China. V29A subtype, with one consistent amino acid change at residue 29 (V→A) and several nucleotide changes, showed higher frequency in NPC cases (25.3%, 20/79) than in EBVaGC cases (0/45) or healthy donors (4.3%, 2/46) (NPC vs. EBVaGC: P=0.0001; NPC vs. healthy donor: P=0.004). A preferential linkage between BamHI F and BARF1/vIL-10 polymorphisms was found. Type f isolates was specially correlated with the V29A/SPM genotype in NPC isolates and type f/V29A/SPM was preferentially found in NPC. BARF1/c-fms homology domain, transforming domain and cytotoxic T lymphocyte (CTL) epitopes of BARF1 were highly conserved in most isolates, suggesting the important role of BARF1 in virus infection and the potential usefulness in EBV-targeting immunotherapy of EBV-associated tumors. The relatively higher prevalence of type f/V29A/SPM strains in NPC may also suggest the association between these variations in multiple viral genes and NPC.

Emerging therapeutic strategies for Epstein-Barr virus+ post-transplant lymphoproliferative disorder.

De novo malignancies represent an increasing concern in the transplant population, particularly as long-term graft and patient survival improves. EBV-associated B-cell lymphoma in the setting of PTLD is the leading malignancy in children following solid organ transplantation. Therapeutic strategies can be categorized as pharmacologic, biologic, and cell-based but the variable efficacy of these approaches and the complexity of PTLD suggest that new treatment options are warranted. Here, we review current therapeutic strategies for treatment of PTLD. We also describe the life cycle of EBV, addressing the viral mechanisms that contribute to the genesis and persistence of EBV+ B-cell lymphomas. Specifically, we focus on the oncogenic signaling pathways activated by the EBV LMP1 and LMP2a to understand the underlying mechanisms and mediators of lymphomagenesis with the goal of identifying novel, rational therapeutic targets for the treatment of EBV-associated malignancies.

The viral and cellular microRNA targetome in lymphoblastoid cell lines.

Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus linked to a number of B cell cancers and lymphoproliferative disorders. During latent infection, EBV expresses 25 viral pre-microRNAs (miRNAs) and induces the expression of specific host miRNAs, such as miR-155 and miR-21, which potentially play a role in viral oncogenesis. To date, only a limited number of EBV miRNA targets have been identified; thus, the role of EBV miRNAs in viral pathogenesis and/or lymphomagenesis is not well defined. Here, we used photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) combined with deep sequencing and computational analysis to comprehensively examine the viral and cellular miRNA targetome in EBV strain B95-8-infected lymphoblastoid cell lines (LCLs). We identified 7,827 miRNA-interaction sites in 3,492 cellular 3′UTRs. 531 of these sites contained seed matches to viral miRNAs. 24 PAR-CLIP-identified miRNA:3′UTR interactions were confirmed by reporter assays. Our results reveal that EBV miRNAs predominantly target cellular transcripts during latent infection, thereby manipulating the host environment. Furthermore, targets of EBV miRNAs are involved in multiple cellular processes that are directly relevant to viral infection, including innate immunity, cell survival, and cell proliferation. Finally, we present evidence that myc-regulated host miRNAs from the miR-17/92 cluster can regulate latent viral gene expression. This comprehensive survey of the miRNA targetome in EBV-infected B cells represents a key step towards defining the functions of EBV-encoded miRNAs, and potentially, identifying novel therapeutic targets for EBV-associated malignancies.