Latent Epstein-Barr Virus Infection Research Articles

Epstein-Barr virus infection and nasopharyngeal carcinoma.

Epstein-Barr virus (EBV) is associated with multiple types of human cancer, including lymphoid and epithelial cancers. The closest association with EBV infection is seen in undifferentiated nasopharyngeal carcinoma (NPC), which is endemic in the southern Chinese population. A strong association between NPC risk and the HLA locus at chromosome 6p has been identified, indicating a link between the presentation of EBV antigens to host immune cells and NPC risk. EBV infection in NPC is clonal in origin, strongly suggesting that NPC develops from the clonal expansion of a single EBV-infected cell. In epithelial cells, the default program of EBV infection is lytic replication. However, latent infection is the predominant mode of EBV infection in NPC. The establishment of latent EBV infection in pre-invasive nasopharyngeal epithelium is believed to be an early stage of NPC pathogenesis. Recent genomic study of NPC has identified multiple somatic mutations in the upstream negative regulators of NF-κB signalling. Dysregulated NF-κB signalling may contribute to the establishment of latent EBV infection in NPC. Stable EBV infection and the expression of latent EBV genes are postulated to drive the transformation of pre-invasive nasopharyngeal epithelial cells to cancer cells through multiple pathways.This article is part of the themed issue 'Human oncogenic viruses'.

Malaria, Epstein-Barr virus infection and the pathogenesis of Burkitt's lymphoma.

A geographical and causal connection has long been recognized between malaria, Epstein-Barr virus (EBV) infection and Burkitt's lymphoma (BL), but the underlying mechanisms remain obscure. Potential clues are that the malaria parasite Plasmodium falciparum selectively absorbs vitamin A from the host and depends on it for its biological activities; secondly, alterations in vitamin A (retinoid) metabolism have been implicated in many forms of cancer, including BL. The first author has proposed that the merozoite-stage malaria parasite, emerging from the liver, uses its absorbed vitamin A as a cell membrane destabilizer to invade the red blood cells, causing anemia and other signs and symptoms of the disease as manifestations of an endogenous form of hypervitaminosis A (Mawson AR, Path Global Health 2013;107(3):122-9). Repeated episodes of malaria would therefore be expected to expose the tissues of affected individuals to potentially toxic doses of vitamin A. It is proposed that such episodes activate latent EBV infection, which in turn activates retinoid-responsive genes. Expression of these genes enhances viral replication and induces germinal center (GC) B cell expansion, activation-induced cytidine deaminase (AID) expression, and c-myc translocation, which in turn predisposes to BL. Thus, an endogenous form of retinoid toxicity related to malaria infection may be the common factor linking frequent malaria, EBV infection and BL, whereby prolonged exposure of lymphatic tissues to high concentrations of retinoids may combine to induce B-cell translocation and increase the risk of Burkitt's lymphoma.

Radiation-induced Epstein-Barr virus reactivation in gastric cancer cells with latent EBV infection.

Epstein-Barr virus, a ubiquitous human herpes virus with oncogenic activity, can be found in 6%-16% of gastric carcinomas worldwide. In Epstein-Barr virus-associated gastric carcinoma, only a few latent genes of the virus are expressed. Ionizing irradiation was shown to induce lytic Epstein-Barr virus infection in lymphoblastoid cell lines with latent Epstein-Barr virus infection. In this study, we examined the effect of ionizing radiation on the Epstein-Barr virus reactivation in a gastric epithelial cancer cell line (SNU-719, an Epstein-Barr virus-associated gastric carcinoma cell line). Irradiation with X-ray (dose = 5 and 10 Gy; dose rate = 0.5398 Gy/min) killed approximately 25% and 50% of cultured SNU-719 cells, respectively, in 48 h. Ionizing radiation increased the messenger RNA expression of immediate early Epstein-Barr virus lytic genes (BZLF1 and BRLF1), determined by real-time reverse transcription polymerase chain reaction, in a dose-dependent manner at 48 h and, to a slightly lesser extent, at 72 h after irradiation. Similar findings were observed for other Epstein-Barr virus lytic genes (BMRF1, BLLF1, and BcLF1). After radiation, the expression of transforming growth factor beta 1 messenger RNA increased and reached a peak in 12-24 h, and the high-level expression of the Epstein-Barr virus immediate early genes can convert latent Epstein-Barr virus infection into the lytic form and result in the release of infectious Epstein-Barr virus. To conclude, Ionizing radiation activates lytic Epstein-Barr virus gene expression in the SNU-719 cell line mainly through nuclear factor kappaB activation. We made a brief review of literature to explore underlying mechanism involved in transforming growth factor beta-induced Epstein-Barr virus reactivation. A possible involvement of nuclear factor kappaB was hypothesized.

Epstein-Barr virus ensures B cell survival by uniquely modulating apoptosis at early and late times after infection.

Latent Epstein-Barr virus (EBV) infection is causally linked to several human cancers. EBV expresses viral oncogenes that promote cell growth and inhibit the apoptotic response to uncontrolled proliferation. The EBV oncoprotein LMP1 constitutively activates NFκB and is critical for survival of EBV-immortalized B cells. However, during early infection EBV induces rapid B cell proliferation with low levels of LMP1 and little apoptosis. Therefore, we sought to define the mechanism of survival in the absence of LMP1/NFκB early after infection. We used BH3 profiling to query mitochondrial regulation of apoptosis and defined a transition from uninfected B cells (BCL-2) to early-infected (MCL-1/BCL-2) and immortalized cells (BFL-1). This dynamic change in B cell survival mechanisms is unique to virus-infected cells and relies on regulation of MCL-1 mitochondrial localization and BFL-1 transcription by the viral EBNA3A protein. This study defines a new role for EBNA3A in the suppression of apoptosis with implications for EBV lymphomagenesis.

Regulatory network analysis of Epstein-Barr virus identifies functional modules and hub genes involved in infectious mononucleosis.

Epstein-Barr virus (EBV) is the most common cause of infectious mononucleosis (IM) and establishes lifetime infection associated with a variety of cancers and autoimmune diseases. The aim of this study was to develop an integrative gene regulatory network (GRN) approach and overlying gene expression data to identify the representative subnetworks for IM and EBV latent infection (LI). After identifying differentially expressed genes (DEGs) in both IM and LI gene expression profiles, functional annotations were applied using gene ontology (GO) and BiNGO tools, and construction of GRNs, topological analysis and identification of modules were carried out using several plugins of Cytoscape. In parallel, a human-EBV GRN was generated using the Hu-Vir database for further analyses. Our analysis revealed that the majority of DEGs in both IM and LI were involved in cell-cycle and DNA repair processes. However, these genes showed a significant negative correlation in the IM and LI states. Furthermore, cyclin-dependent kinase 2 (CDK2) - a hub gene with the highest centralityscore - appeared to be the key player in cell cycle regulation in IM disease. The most significant functional modules in the IM and LI states were involved in the regulation of the cell cycle and apoptosis, respectively. Human-EBV network analysis revealed several direct targets of EBV proteins during IM disease. Our study provides an important first report on the response to IM/LI EBV infection in humans. An important aspect of our data was the upregulation of genes associated with cell cycle progression and proliferation.

Development of a small molecule drug for EBV-associated gastric cancers.

63 Background: Worldwide, EBV is associated with ~10% of gastric cancer. Currently, no pharmaceutical-based therapies exist that selectively target EBV associated gastric cancers. EBV, in its latent oncogenic form, is dependent on the continuous expression of Epstein-Barr Nuclear Antigen 1 (EBNA1), a multifunctional dimeric protein critical for viral replication, genome maintenance and viral gene expression. Methods: The aim of this program is to advance the development of a New Chemical Entity (NCE) for latent infection of Epstein-Barr Virus (EBV) to treat EBV-associated gastric cancer. We have used structure-based drug design and medicinal chemistry methods to identify and develop a small molecule clinical candidate that selectively inhibits the DNA-binding activity of EBNA1. Results: The clinical candidate inhibits EBNA1 function with nanomolar potency in biochemical assays and low micromolecular activity in several cell-based assays. We demonstrate that our clinical candidate provides protection in 4 different xenograft models of EBV-driven tumor growth, including patient-derived xenografts. Furthermore, RNA analysis experiments confirm in vivo target engagement by the elimination of EBV in treated tumor tissue. The clinical candidate is selective, showing no activity in an EBV-negative xenograft experiments. The clinical candidate has met industry-accepted criteria for drug suitability, safety and toxicology including physicochemical properties, metabolic stability, selectivity in broad-based screens and bioavailability, and lack in vivoliabilities, including genotoxicity and 14-day toxicity studies. Conclusions: IND-enabling studies including safety pharmacology and toxicology and GMP manufacturing have begun with a projected First-In-Human clinical trial in Q4 2017. These data establish proof-of-concept for targeting EBNA1—a protein previously thought to be undruggable.

Evaluation of the presence of Epstein-Barr virus (EBV) in Iranian patients with thyroid papillary carcinoma

Background and purposePapillary thyroid carcinoma (PTC) is the most common thyroid cancer. EBV is one of the most important viruses related to different types of malignancies. This study investigated the relationship between EBV and papillary thyroid carcinoma. Material and methodsIn this study the presence of Epstein-Barr Nuclear Antigen 1 (EBNA1) gene in papillary thyroid carcinoma tissues were examined by nested-PCR method. Paraffin-embedded tissues (N=41) blocks of thyroid cancer were used. DNA was extracted from all samples and then samples were evaluated for the presence of EBV gene. ResultsIn 41 samples, EBNA1 was detected in 65.8% of patients with papillary thyroid carcinoma which was significantly higher in younger ages. ConclusionThe significant presence of EBV genome in papillary thyroid carcinoma suggests that this virus may play a role in this cancer especially in younger ages. As a result, monitoring of patients with EBV latent infection for PTC can be very important.

Carcinoma-risk variant of EBNA1 deregulates Epstein-Barr Virus episomal latency.

Epstein-Barr Virus (EBV) latent infection is a causative co-factor for endemic Nasopharyngeal Carcinoma (NPC). NPC-associated variants have been identified in EBV-encoded nuclear antigen EBNA1. Here, we solve the X-ray crystal structure of an NPC-derived EBNA1 DNA binding domain (DBD) and show that variant amino acids are found on the surface away from the DNA binding interface. We show that NPC-derived EBNA1 is compromised for DNA replication and episome maintenance functions. Recombinant virus containing the NPC EBNA1 DBD are impaired in their ability to immortalize primary B-lymphocytes and suppress lytic transcription during early stages of B-cell infection. We identify Survivin as a host protein deficiently bound by the NPC variant of EBNA1 and show that Survivin depletion compromises EBV episome maintenance in multiple cell types. We propose that endemic variants of EBNA1 play a significant role in EBV-driven carcinogenesis by altering key regulatory interactions that destabilize latent infection.

EBV Infection and Glucose Metabolism in Nasopharyngeal Carcinoma.

To establish persistent infection in cells, viruses evolve strategies to alter host cellular pathways to regulate cell proliferation and energy metabolism which support viral infection. Epstein-Barr virus (EBV) undergoes both lytic and latent infection to achieve persistent and lifelong infection in human. EBV readily infects human B cells, driving their transformation to proliferative lymphoblastoid cell lines (LCL), and eventually establishes lifelong latent infection in memory B cells. In contrary, EBV undergoes lytic replication upon infection into normal epithelial cells which is essential for the replication of EBV genome and production of infectious viral particles for transmission through saliva. EBV shuttles between B cells and epithelial cells to complete its infection cycle. EBV infection is closely associated with nasopharyngeal carcinoma (NPC) and is present in practically 100% of undifferentiated NPC. In contrast to undergo lyticinfection of normal pharyngeal epithelium, EBV establishes latent infection in NPC. The switch from lytic infection to latent infection may represent an early and essential step in the development of NPC. Recent studies in both B cells and NPC cells latently infected with EBV reveal alterations in cell metabolism to support persistentand latent EBV infection. Events underlying the switching of lytic to latent EBV infection in NPC cells are largely undefined. Molecular events and alterations of cell metabolism are likely to play crucial roles in switching EBV infection from lytic to latent in NPC cells. Latent EBV infection and expression of viral genes, including LMP1, LMP2, and possibly EBV-encoded micro RNAs, may play essential roles in alterations of cell metabolism to support NPC pathogenesis. Alteration of energy metabolism is an essential hallmark of cancer. The role of altered energy metabolism in host cells in modulating latent and lytic EBV infection in NPC cells is unclear. In this review, we will discuss the impact of genetic alterations in NPC to module cellular metabolism and its influence on latent infection and lytic reactivation of EBV infection in NPC cells. In particular, the role of EBV-encoded genes in driving glucose metabolism and their contribution to NPC pathogenesis will be discussed. This new perspective on the interplay between EBV infection and altered host metabolic pathways in NPC pathogenesis may offer novel and effective therapeutic strategies in the treatment of NPC and other EBV-associated malignancies.

Quantitative multi-target RNA profiling in Epstein-Barr virus infected tumor cells

Epstein-Barr virus (EBV) is etiologically linked to multiple acute, chronic and malignant diseases. Detection of EBV-RNA transcripts in tissues or biofluids besides EBV-DNA can help in diagnosing EBV related syndromes. Sensitive EBV transcription profiling yields new insights on its pathogenic role and may be useful for monitoring virus targeted therapy. Here we describe a multi-gene quantitative RT-PCR profiling method that simultaneously detects a broad spectrum (n=16) of crucial latent and lytic EBV transcripts. These transcripts include (but are not restricted to), EBNA1, EBNA2, LMP1, LMP2, BARTs, EBER1, BARF1 and ZEBRA, Rta, BGLF4 (PK), BXLF1 (TK) and BFRF3 (VCAp18) all of which have been implicated in EBV-driven oncogenesis and viral replication. With this method we determine the amount of RNA copies per infected (tumor) cell in bulk populations of various origin. While we confirm the expected RNA profiles within classic EBV latency programs, this sensitive quantitative approach revealed the presence of rare cells undergoing lytic replication. Inducing lytic replication in EBV tumor cells supports apoptosis and is considered as therapeutic approach to treat EBV-driven malignancies. This sensitive multi-primed quantitative RT-PCR approach can provide broader understanding of transcriptional activity in latent and lytic EBV infection and is suitable for monitoring virus-specific therapy responses in patients with EBV associated cancers.

LMP1 signaling pathway activates IRF4 in latent EBV infection and a positive circuit between PI3K and Src is required.

Interferon (IFN) regulatory factors (IRFs) have crucial roles in immune regulation and oncogenesis. We have recently shown that IRF4 is activated through c-Src-mediated tyrosine phosphorylation in virus-transformed cells. However, the intracellular signaling pathway triggering Src activation of IRF4 remains unknown. In this study, we provide evidence that Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) promotes IRF4 phosphorylation and markedly stimulates IRF4 transcriptional activity, and that Src mediates LMP1 activation of IRF4. As to more precise mechanism, we show that LMP1 physically interacts with c-Src, and the phosphatidylinositol 3 kinase (PI3K) subunit P85 mediates their interaction. Depletion of P85 by P85-specific short hairpin RNAs disrupts their interaction and diminishes IRF4 phosphorylation in EBV-transformed cells. Furthermore, we show that Src is upstream of PI3K for activation of both IRF4 and Akt. In turn, inhibition of PI3K kinase activity by the PI3K-speicfic inhibitor LY294002 impairs Src activity. Our results show that LMP1 signaling is responsible for IRF4 activation, and further characterize the IRF4 regulatory network that is a promising therapeutic target for specific hematological malignancies.

Lenalidomide, Thalidomide, and Pomalidomide Reactivate the Epstein-Barr Virus Lytic Cycle through Phosphoinositide 3-Kinase Signaling and Ikaros Expression.

Lenalidomide, thalidomide, and pomalidomide (LTP) are immunomodulatory agents approved for use in multiple myeloma, but in some settings, especially with alkylating agents, an increase in Hodgkin lymphoma and other secondary primary malignancies (SPM) has been noted. Some of these malignancies have been linked to Epstein-Barr virus (EBV), raising the possibility that immunomodulatory drugs disrupt latent EBV infection. We studied the ability of LTP to reactivate latently infected EBV-positive cell lines in vitro and in vivo, and evaluated the EBV viral load in archived serum samples from patients who received a lenalidomide, thalidomide, and dexamethasone (LTD) combination. Treatment of EBV-infected B-cell lines with LTP at physiologically relevant concentrations induced the immediate early gene BZLF1, the early gene BMRF1, and the late proteins VCA and BCFR1. This occurred in the potency order pomalidomide > lenalidomide > thalidomide, and the nucleoside analogue ganciclovir enhanced the cytotoxic effects of lenalidomide and pomalidomide in Burkitt lymphoma cells in vitro and in vivo EBV reactivation was related to PI3K stimulation and Ikaros suppression, and blocked by the PI3Kδ inhibitor idelalisib. Combinations of lenalidomide with dexamethasone or rituximab increased EBV reactivation compared with lenalidomide alone and, importantly, lenalidomide with melphalan produced even greater reactivation. We conclude LTP may reactivate EBV-positive resting memory B cells thereby enhancing EBV lytic cycle and host immune suppression. Clin Cancer Res; 22(19); 4901-12. ©2016 AACR.

Anti-Cancer Effect of Quercetin in Xenograft Models with EBV-Associated Human Gastric Carcinoma.

Licorice extracts have been widely used in herbal and folk medications. Glycyrrhiza contains diverse range of biological compounds including triterpenes (glycyrrhizin, glycyrrhizic acid) and flavonoids (quercetin, liquiritin, liquiritigenin, glabridin, licoricidin, isoliquiritigenin). The flavonoids in licorice are known to have strong anti-cancer activities. Quercetin, the most abundant flavonoid, has been shown to have anti-ulcer, anti-cancer, antioxidant, and anti-inflammatory properties. Latent Epstein-Barr virus (EBV) infection can lead to serious malignancies, such as, Burkitt’s lymphoma, Hodgkin’s disease and gastric carcinoma(GC), and (Epstein-Barr virus associated gastric carcinoma) EBVaGC is one of the most common EBV-associated cancers. In this study, the authors first examined the anti-cancer effects of quercetin and isoliquiritigenin in vivo xenograft animal models implanted with EBV(+) human gastric carcinoma (SNU719) or EBV(−) human gastric carcinoma (MKN74), and then explored the molecular mechanisms responsible for their anti-cancer activities. The results obtained showed that anti-cancer effect of quercetin was greater than isoliquiritigenin in mice injected with EBV(+) human gastric carcinoma (SNU719) cells. On the other hand, quercetin and isoliquiritigenin had similar anti-cancer effects in mice injected with EBV(−) human gastric carcinoma (MKN74) cells. Interestingly, quercetin inhibited EBV viral protein expressions, including EBNA-1 and LMP-2 proteins in tumor tissues from mice injected with EBV(+) human gastric carcinoma. Quercetin more effectively induced p53-dependent apoptosis than isoliquiritigenin in EBV(+) human gastric carcinoma, and this induction was correlated with increased expressions of the cleaved forms of caspase-3, -9, and Parp. In EBV(−)human gastric carcinoma (MKN74), both quercetin and isoliquiritigenin induced the expressions of p53, Bax, and Puma and the cleaved forms of caspase-3 and -9 and Parp at similar levels.

Active CMV and EBV infections in renal transplant recipients with unexplained fever and elevated serum creatinine

Proper identification of active cytomegalovirus (CMV) and Epstein-Barr virus (EBV) infections are helpful for monitoring antiviral treatment in transplant recipients. Qualitative and quantitative CMV, EBV DNA PCR techniques in the context of serological tests are performed for early detection and differentiation of active and latent CMV and EBV infections in renal transplantation. Basically, 129 renal transplanted recipients monitored carefully and hospitalized for unexplained elevated creatinine levels or high fever and 21 of their donors were studied. CMV DNA was detected in 63.5% of the febrile episodes following transplantation and in 46.42% of readmitted patients using qualitative PCR method. In the first group, 15% of the patients and in the second group 42.85% of the patients had copy numbers more than cutoff point (900 copies/mL). Cutoff point had 100% sensitivity and 82.5% specificity for active and symptomatic CMV infection. Only 15.5% of the subjects were positive for EBV infection by qualitative PCR method. Among them 5% had >2000 copies/mL and were symptomatic. One subject with a history of three times hospitalization had higher EBV viral load and developed post-transplant lymphoproliferative disorder. CMV load was significantly correlated with elevated creatinine levels (OR = 3.1, p = 0.006), abnormal heart sounds (OR = 4.7; p = 0.02) and hypertension (OR = 3.6; p = 0.03). Only qRT-PCR could differentiate between latent and active infections and might be clinically useful for monitoring symptomatic CMV and EBV infections and initiation of the antiviral therapy. Elevated creatinine levels, hypertension, and abnormal heart sounds could be considered as main manifestations of HCMV infection in kidney recipients.

Epstein–Barr virus and regulatory T cells in Egyptian paediatric patients with acute B lymphoblastic leukaemia

ObjectiveAcute B lymphoblastic leukaemia (B-ALL) is the most common type of childhood malignancy worldwide but little is known of its origin. Recently, many studies showed both a high incidence of...

Epstein-Barr Virus Oncoprotein LMP1 Mediates Epigenetic Changes in Host Gene Expression through PARP1.

The latent infection of Epstein-Barr virus (EBV) is associated with 1% of human cancer incidence. Poly(ADP-ribosyl)ation (PARylation) is a posttranslational modification catalyzed by poly(ADP-ribose) polymerases (PARPs) that mediate EBV replication during latency. In this study, we detail the mechanisms that drive cellular PARylation during latent EBV infection and the effects of PARylation on host gene expression and cellular function. EBV-infected B cells had higher PAR levels than EBV-negative B cells. Moreover, cellular PAR levels were up to 2-fold greater in type III than type I latently infected EBV B cells. We identified a positive association between expression of the EBV genome-encoded latency membrane protein 1 (LMP1) and PAR levels that was dependent upon PARP1. PARP1 regulates gene expression by numerous mechanisms, including modifying chromatin structure and altering the function of chromatin-modifying enzymes. Since LMP1 is essential in establishing EBV latency and promoting tumorigenesis, we explored the model that disruption in cellular PARylation, driven by LMP1 expression, subsequently promotes epigenetic alterations to elicit changes in host gene expression. PARP1 inhibition resulted in the accumulation of the repressive histone mark H3K27me3 at a subset of LMP1-regulated genes. Inhibition of PARP1, or abrogation of PARP1 expression, also suppressed the expression of LMP1-activated genes and LMP1-mediated cellular transformation, demonstrating an essential role for PARP1 activity in LMP1-induced gene expression and cellular transformation associated with LMP1. In summary, we identified a novel mechanism by which LMP1 drives expression of host tumor-promoting genes by blocking generation of the inhibitory histone modification H3K27me3 through PARP1 activation. EBV is causally linked to several malignancies and is responsible for 1% of cancer incidence worldwide. The EBV-encoded protein LMP1 is essential for promoting viral tumorigenesis by aberrant activation of several well-known intracellular signaling pathways. We have identified and defined an additional novel molecular mechanism by which LMP1 regulates the expression of tumor-promoting host genes. We found that LMP1 activates the cellular protein PARP1, leading to a decrease in a repressive histone modification, accompanied by induction in expression of multiple cancer-related genes. PARP1 inhibition or depletion led to a decrease in LMP1-induced cellular transformation. Therefore, targeting PARP1 activity may be an effective treatment for EBV-associated malignancies.

Abstract 632: Establishment and characterization of xenografts and cell lines trom nasopharyngeal carcinoma

Abstract Nasopharyngeal carcinoma (NPC) is a common cancer among ethnic Cantonese living in Hong Kong and southern China. It is closely associated with Epstein-Barr virus (EBV) infection. Unfortunately, there are very few representative xenografts and cell lines established from NPC available for investigation. Most of the NPC xenografts established have been passaged in immune deficient animals for over 20 years and may not be representative of the original NPC in patients. For in vitro NPC cell lines, there is only one cell line which retains the EBV genomes. Other NPC cell lines have all lost their EBV episomes. Furthermore, many of these NPC cell lines are found to be contaminated with genetic components from HeLa cells (HPV16 genome) which raised issues on their origins and limited their uses as NPC cells. There is great urgency in establishment new NPC cell lines both in vivo and in vitro for various research investigations and for the study of pathogenic role of EBV infection in NPC. We have carried out continuous efforts since 2010 to attempt establishment of new NPC xenografts and cell lines from surgical and biopsies NPC tissues. NPC tissues from resected recurrent NPC and biopsies from primary NPC were explanted to subrenal capsular sites and maintained for four months to one year to observe for growth of new NPC xenografts. Attempts to establish new NPC celles were also carried out. At present, we have successfully established 3 NPC xenografts (Xeno 23, Xeno 32, Xeno 47) which could be passages at subcutaneously sites and one in vitro NPC cells (NPC43) which harbors EBV genomes. The growth properties and genetic alterations of these newly established NPC cell lines have been characterized. Novel genetic alterations and growth signaling pathways were observed in these newly established NPC cell lines and may represent driver mutations and signaling pathways essential for progression of NPC. Profiling of EBV gene expression of these newly established NPC cell lines revealed predominant latent EBV infection and expression of EBV-encoded mRNA from the BART transcripts which are characteristic of EBV infection in NPC. The establishment of new NPC cells will contribute to investigate the pathogenesis of NPC and its intimate relationship with EBV infection. Funding acknowledgement: University Grant Council (HK) grants (AoE NPC (AoE/M-06/08); TBRS (T12-401/13R); GRF); University of Hong Kong (CRCG and SRT cancer) Citation Format: WT Lin, L Xia, Dan Dan Wen, CM Tsang, KW Lo, ML Lung, George Sai-Wah Tsao. Establishment and characterization of xenografts and cell lines trom nasopharyngeal carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 632.

BZLF1 Attenuates Transmission of Inflammatory Paracrine Senescence in Epstein-Barr Virus-Infected Cells by Downregulating Tumor Necrosis Factor Alpha

Recent studies have shown that inflammatory responses trigger and transmit senescence to neighboring cells and activate the senescence-associated secretory phenotype (SASP). Latent Epstein-Barr virus (EBV) infection induces increased secretion of several inflammatory factors, whereas lytic infections evade the antiviral inflammatory response. However, the changes in and roles of the inflammatory microenvironment during the switch between EBV life cycles remain unknown. In the present study, we demonstrate that latent EBV infection in EBV-positive cells triggers the SASP in neighboring epithelial cells. In contrast, lytic EBV infection abolishes this phenotype. BZLF1 attenuates the transmission of paracrine senescence during lytic EBV infection by downregulating tumor necrosis factor alpha (TNF-α) secretion. A mutant BZLF1 protein, BZLF1Δ207-210, that cannot inhibit TNF-α secretion while maintaining viral transcription, fails to block paracrine senescence, whereas a neutralizing antibody against TNF-α is sufficient to restore its inhibition. Furthermore, latent EBV infection induces oxidative stress in neighboring cells, while BZLF1-mediated downregulation of TNF-α reduces reactive oxygen species (ROS) levels in neighboring cells, and ROS scavengers alleviate paracrine senescence. These results suggest that lytic EBV infection attenuates the transmission of inflammatory paracrine senescence through BZLF1 downregulation of TNF-α secretion and alters the inflammatory microenvironment to allow virus propagation and persistence. The senescence-associated secretory phenotype (SASP), an important tumorigenic process, is triggered and transmitted by inflammatory factors. The different life cycles of Epstein-Barr virus (EBV) infection in EBV-positive cells employ distinct strategies to modulate the inflammatory response and senescence. The elevation of inflammatory factors during latent EBV infection promotes the SASP in uninfected cells. In contrast, during the viral lytic cycle, BZLF1 suppresses the production of TNF-α, resulting in the attenuation of paracrine inflammatory senescence. This finding indicates that EBV evades inflammatory senescence during lytic infection and switches from facilitating tumor-promoting SASP to generating a virus-propagating microenvironment, thereby facilitating viral spread in EBV-associated diseases.

Sensing of latent EBV infection through exosomal transfer of 5′pppRNA

Complex interactions between DNA herpesviruses and host factors determine the establishment of a life-long asymptomatic latent infection. The lymphotropic Epstein-Barr virus (EBV) seems to avoid recognition by innate sensors despite massive transcription of immunostimulatory small RNAs (EBV-EBERs). Here we demonstrate that in latently infected B cells, EBER1 transcripts interact with the lupus antigen (La) ribonucleoprotein, avoiding cytoplasmic RNA sensors. However, in coculture experiments we observed that latent-infected cells trigger antiviral immunity in dendritic cells (DCs) through selective release and transfer of RNA via exosomes. In ex vivo tonsillar cultures, we observed that EBER1-loaded exosomes are preferentially captured and internalized by human plasmacytoid DCs (pDCs) that express the TIM1 phosphatidylserine receptor, a known viral- and exosomal target. Using an EBER-deficient EBV strain, enzymatic removal of 5'ppp, in vitro transcripts, and coculture experiments, we established that 5'pppEBER1 transfer via exosomes drives antiviral immunity in nonpermissive DCs. Lupus erythematosus patients suffer from elevated EBV load and activated antiviral immunity, in particular in skin lesions that are infiltrated with pDCs. We detected high levels of EBER1 RNA in such skin lesions, as well as EBV-microRNAs, but no intact EBV-DNA, linking non-cell-autonomous EBER1 presence with skin inflammation in predisposed individuals. Collectively, our studies indicate that virus-modified exosomes have a physiological role in the host-pathogen stand-off and may promote inflammatory disease.

Epidemiology of Epstein-Barr virus-associated pediatric lymphomas from Argentina

More than 90% of the population is infected by Epstein-Barr virus (EBV), which has sophisticatedly evolved to survive silently in B cells for the life of infected individuals. However, if the virus-host balance is disturbed, latent EBV infection could be associated with several lymphomas. The age at primary infection varies substantially worldwide, and exposure to EBV is likely to be due to socioeconomic factors. In Argentina, EBV infection is mostly subclinical and 90% of patients are seropositive by the age of 3 years; therefore, its epidemiological characteristics resemble those of an underdeveloped or developing population. EBV-positive Hodgkin lymphoma (HL) in young adults from developed populations has been attributed to delayed primary EBV infection as suggested by the association with recent mononucleosis development. EBV-associated Burkitt lymphoma and Hodgkin lymphoma in children from Argentina display frequencies similar to those observed in developed countries, whereas EBV presence in pediatric diffuse large B-cell lymphoma is slightly increased compared to those populations. However, EBV presence is statistically associated particularly with patients < 10 years of age in all three entities. Therefore, a relationship between low age of EBV seroconversion and B-cell lymphoma development risk could be suggested in children from Argentina.