EBV-infected Cells Research Articles

Infection of Epstein⁻Barr Virus in Type III Latency Modulates Biogenesis of Exosomes and the Expression Profile of Exosomal miRNAs in the Burkitt Lymphoma Mutu Cell Lines.

Infection of Epstein–Barr virus (EBV), a ubiquitous human gamma herpesvirus, is associated with various malignancies in B lymphocytes and epithelial cells. EBV encodes 49 microRNAs in two separated regions, termed the BART and BHRF1 loci. Although accumulating evidence demonstrates that EBV infection regulates the profile of microRNAs in the cells, little is known about the microRNAs in exosomes released from infected cells. Here, we characterized the expression profile of intracellular and exosomal microRNAs in EBV-negative, and two related EBV-infected Burkitt lymphoma cell lines having type I and type III latency by next-generation sequencing. We found that the biogenesis of exosomes is upregulated in type III latently infected cells compared with EBV-negative and type I latently infected cells. We also observed that viral and several specific host microRNAs were predominantly incorporated in the exosomes released from the cells in type III latency. We confirmed that multiple viral microRNAs were transferred to the epithelial cells cocultured with EBV-infected B cells. Our findings indicate that EBV infection, in particular in type III latency, modulates the biogenesis of exosomes and the profile of exosomal microRNAs, potentially contributing to phenotypic changes in cells receiving these exosomes.

Air-Liquid Interface System To Understand Epstein-Barr Virus-Associated Nasopharyngeal Carcinoma.

Epstein-Barr virus (EBV) infects epithelial cells and is associated with epithelial malignancies. Although EBV reactivation is induced by epithelial differentiation, the available methods for differentiation are not widely used. In a recent study, Caves et al. (mSphere 3:e00152-18, 2018, https://doi.org/10.1128/mSphere.00152-18) explored the use of a new transwell-based air-liquid interface (ALI) system to differentiate EBV-infected nasopharyngeal carcinoma cells. They found that cells cultured in the ALI system expressed markers of differentiation and supported complete EBV reactivation. This system offers an easy method for differentiation that could be widely adopted. This system could be extended to other epithelial cell types.

Air-Liquid Interface Method To Study Epstein-Barr Virus Pathogenesis in Nasopharyngeal Epithelial Cells.

Lifting adherent cells to the air-liquid interface (ALI) is a method conventionally used to culture airway epithelial cells into polarized apical and basolateral surfaces. Reactivation of Epstein-Barr virus (EBV) from monolayer epithelial cultures is sometimes abortive, which may be attributed to the lack of authentic reactivation triggers that occur in stratified epithelium in vivo In the present work, the ALI culture method was applied to study EBV reactivation in nasopharyngeal epithelial cells. The ALI culture of an EBV-infected cell line yielded high titers and can be dissected by a variety of molecular virology assays that measure induction of the EBV lytic cascade and EBV genome replication and assembly. EBV infection of polarized cultures of primary epithelial cells can be challenging and can have variable efficiencies. However, the use of the ALI method with established EBV-infected cell lines offers a readily available and reproducible approach for the study of EBV permissive replication in polarized epithelia.

Epstein-Barr Virus–Infected Plasma Cells Infiltrate Erosive Oral Lichen Planus

Epstein-Barr virus (EBV), in addition to its transforming properties, contributes to the pathogenesis of several inflammatory diseases. Here, we investigated its involvement in oral lichen planus (OLP), a common autoimmune-like disease of unknown etiopathogenesis that can display a malignant potential. EBV-infected cells (EBV+ cells) were sought in a large series of clinically representative OLPs (n = 99) through in situ hybridization to detect small noncoding EBV-encoded RNAs. Overall, our results demonstrated that EBV was commonly found in OLP (74%), with significantly higher frequency (83%) in the erosive form than in the reticular/keratinized type mild form (58%). Strikingly, many erosive OLPs were massively infiltrated by large numbers of EBV+ cells, which could represent a large part of the inflammatory infiltrate. Moreover, the number of EBV+ cells in each OLP section significantly correlated with local inflammatory parameters (OLP activity, infiltrate depth, infiltrate density), suggesting a direct relationship between EBV infection and inflammatory status. Finally, we characterized the nature of the infiltrated EBV+ cells by performing detailed immunohistochemistry profiles (n = 21). Surprisingly, nearly all EBV+ cells detected in OLP lesions were CD138+ plasma cells (PCs) and more rarely CD20+ B cells. The presence of EBV+ PCs in erosive OLP was associated with profound changes in cytokine expression profile; notably, the expression of key inflammatory factors, such as IL1-β and IL8, were specifically increased in OLP heavily infiltrated with EBV+ PCs. Moreover, electron microscopy–based experiments showed that EBV+ PCs actively produced EBV viral particles, suggesting possible amplification of EBV infection within the lesion. Our study thus brings conclusive evidence showing that OLP is commonly infiltrated with EBV+ PCs, adding a further puzzling element to OLP pathogenesis, given that PCs are now considered to be major regulatory immune cells involved in several autoimmune diseases (ClinicalTrials.gov NCT02276573).

NK Cell Recognition of Peptides Encoded by EBV Latent Cycle Proteins

Introduction Epstein–Barr virus (EBV) infects more than 90% of adults worldwide and is associated with several malignancies, including post-transplant lymphoproliferative disorder (PTLD). Although EBV infection is typically asymptomatic in immune-competent individuals, transplant recipients, especially children, are highly susceptible to EBV-associated PTLD due to the immunosuppression treatment required to prevent graft rejection. Innate immune responses, including Natural Killer (NK) cells, are considered vital early in the infection process. Our group has recently demonstrated that a specific NK cell subset can recognize and respond to autologous B cells latently infected with EBV. This NK subset expresses NKG2A/CD94, an inhibitory receptor that recognizes HLA-E. Since EBV-infected cells induced NK cell activation despite the expression of HLA-E, we hypothesize that EBV encoded peptides play a pivotal role in the recognition and response of NKG2A+ NK cells towards latently infected EBV+ cells. Methods and Results Using in silico analysis (NetMHCpan 4.0) a peptide library derived from EBV latent cycle proteins (LMPs and EBNAs) was generated. Initial assessment resulted in a library of 61 peptides which could potentially bind to HLA-E. Then, using target cells, we performed a peptide stabilization assay in vitro to test the ability of individual peptides to be presented by HLA-E. We identified 37 peptides, expressed during the latency III stage of EBV infection that bound and induced HLA-E expression at the surface of the target cells. Functional assays, using CD107a as a readout for NK cell activity, demonstrated HLA-E bound EBV-derived peptides were able to inhibit NK cell activation (15 peptides), or prevent NK cell inhibition via the NKG2A receptor, resulting in NK cell activation (12 peptides). We determined that, in general, peptides encoded by LMPs tended to favor NK cell degranulation (NK cell killing) while peptides encoded by EBNAs were mostly inhibitory. Conclusions Our results demonstrate that EBV latent cycle proteins can encode for peptides that bind to HLA-E which have the ability to promote NK cell degranulation and therefore may be involved in the elimination of EBV latently infected B cells. As NK cells are less susceptible to immunosuppression, understanding NK cell interactions with EBV-infected cells will lead to improvements in therapeutic strategies to control EBV diseases post-transplant. Stanford Transplantation and Tissue engineering center of Excellence (TTE) Fellowship.

Abstract 2565: EBV peptide-derived vaccine significantly enhanced in vitro cytotoxicity against EBV-positive B-cell lymphoma (EBV-BL) treatment using TMV-based delivery system

Abstract Background: EBV latent proteins (LMP-1/2) increase the chemoresistance in Burkitt Lymphoma (BL) cells by constitutively activating the CD40 receptor. It has also been demonstrated that B- cell lymphoma resistant to rituximab is a result of LMP-1 induced miR-155 expression and survival mediated by AKT phosphorylation (Hong et al.Gene Ther, 2012). EBV peptides delivered by a virus carrier (Tobacco mosaic virus;TMV) has the potential to boost immunity and stimulate potent T cell responses against EBV-associated antigens and induce a virus specific T cell response against EBV infected malignant cells (Miles/Cairo et al. BJH,2012, Kemnade/McCormick et al. Vaccine, 2014).We previously observed that obinutuzumab (Anti-CD20 mAb) significantly enhanced cell death and increased overall survival against BL (Awasthi/Cairo et al. BJH, 2015) in xenografted NSG mice. However, synergistic effects of obinutuzumab in-combination with LMP1/2-peptide vaccine against mature-B-NHL are unknown. Objective: To determine the efficacy of peptide specific cytotoxic T-lymphocytes (CTL) or the EBV vaccine alone/ in-combination with obinutuzumab against mature-B-NHL Methods: Raji4RH (provided by M. Barth, MD, Roswell Park Cancer Institute) and Raji cells (ATCC, USA) were cultured in 10% RPMI. Tumor cells were incubated with TMV-RIED IgG (LMP2) with obinutuzumab (100ug/ml) for 4 hrs with NK cells. Cytotoxicity was determined by DELFIA cytotoxicity assay at 10:1 E:T ratio. Further, to generate LMP1/LMP2 peptide specific cytotoxic T cells (CTL), mature dendritic cells were pulsated with TMV-conjugated LMP1/LMP2 peptides for 24 hrs. PBMC isolated T cells were mixed with APC (LMPs-mature -DCs) at 1:20 ratio for activation and maturation. LMP-peptide specific CTLs were investigated in-vitro for cytotoxicity efficacy against BL cell lines. Results: LMP1 and LMP2 peptide were successfully conjugated with TMV. TMV conjugated and purified serum vaccine was tested against BL cell lines. Conjugated serum vaccine (LMP2-(RIED-TMV) +obinutuzumab+NK, compared to obinutuzumab+NK or vaccine+NK alone significantly enhanced in-vitro cytotoxicity 62.67±7.82% vs. 53.66±6.43% vs. 50.8±5.2% (p=0.04 and 0.02), respectively against BL (Raji). Furthermore, cytotoxicity of CTL (LMP1-MSD-TMV) or (LMP2-TYG-TMV) cells were significantly enhanced compare to IL2 treated T cells only, 58.5±8.6% or 48.56±15.18% vs. 21.42±13.3% in Raji (P=0.01, 0.02), and 57.8±7.9% or 45.52±19.4% vs. 14.48±4.2% in Raji4RH (P=0.003, 0.002), respectively. Conclusion: Our preliminary data indicates that LMP1 (MSD-TMV) and LMP2 (TYG-TMV) successfully conjugated with TMV and TMV-conjugated peptide specific CTL cells significantly enhanced cytotoxicity against BL cell lines compared to T cells only. Citation Format: Aradhana Awasthi Tiwari, Alison McCormick, Dina Edani, Aaron Newman, Christeen Azmy, Janet Ayello, Christian Klein, Mitchell S. Cairo. EBV peptide-derived vaccine significantly enhanced in vitro cytotoxicity against EBV-positive B-cell lymphoma (EBV-BL) treatment using TMV-based delivery system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2565.

Abstract 3079: Epstein Barr virus-encoded LMP1 reprograms glucose metabolism to enhance cell motility in nasopharyngeal epithelial cell

Abstract How oncogenic virus rewires glucose flux to regulate cell motility is not well-understood. Epstein-Barr virus (EBV) infection of preinvasive nasopharyngeal epithelium is believed to be an essential initiation event during nasopharyngeal carcinoma pathogenesis. Here we demonstrate that EBV-encoded LMP1 enhances glycolysis thereby accelerating cell mobility of nasopharyngeal epithelial (NPE) cells. Activation of IGF1/mTORC2/AKT/PDHE1α signaling which mediates Histone 3 acetylation is involved in cell motility in EBV-infected NPE cells. Blocking the glucose uptake by pharmacological inhibitors effectively suppress glycolysis as well as the cell motility, linking the glucose metabolism to cell movement. We further show that mTORC2/AKT mediated phosphorylation of pyruvate decarboxylase kinase 1 (PDHK1) and its substrate, pyruvate decarboxylase E1α (PDHE1α) component plays a significant role in cell motility. PDHE1α knockdown effectively blocks LMP1-induced cell motility while overexpression of its phosphomimetic mutant S293A but not S293D inhibits LMP1-induced cell motility, supporting the involvement of PDHE1α in enhanced cell motility is depended on Ser293 phosphorylation. Furthermore, PDHE1α translocation into nucleus enhances Histone 3 acetylation and its binding to the promoter region of snail to enhance cell motility. Finally, we find that LMP1 increases the mRNA expression and the secretion of the ligand IGF1, which promotes phosphorylation of IGF1R as the key regulator for LMP1-induced mTORC2/AKT activation. In summary, this study provides evidences of involvement of EBV infection in metabolic reprogramming of NPE cells to promote cell motility involving activation of mTORC2/AKT/PDHE1α. Acknowledgment: This project was supported by funding from the Research Grants Council, Hong Kong: General Research Fund (17120814, 17161116, 17111516, 171110315), Area of Excellence (AoE/M-06/08), Theme-Based Research Scheme (T12-401/13-R), Collaborative Research Fund (Project reference: C7027-16G); Health and Medical Research Fund of Hong Kong (12110782, 13120872, 04151726); and CRCG, University of Hong Kong, Hong Kong. Citation Format: Jun Zhang, Weitao Lin, Chi Man Tsang, Teng Fei Liu, Wing Chong Tang, Yim Ling Yip, Wen Deng, Lin Jia, Sai Wah Tsao. Epstein Barr virus-encoded LMP1 reprograms glucose metabolism to enhance cell motility in nasopharyngeal epithelial cell [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3079.

Abstract 1108: M2-polarized macrophages increase invasiveness of EBV-associated nasopharyngeal carcinoma by inducing invadopodia formation

Abstract Cancer metastasis is a major cause of mortality, accounting for approximately 90% of cancer related deaths. Emerging evidences suggest that tumour microenvironment plays an indispensable role in cancer metastasis. Recruitment of innate and adaptive immune cells to the tumour results in chronic inflammation which favours tumour growth and progression. Particularly, tumour associated macrophages (TAMs) are found to promote the initiation, growth, and metastasis in several types of cancers. Nasopharyngeal carcinoma (NPC) is one of the highly invasive and metastatic cancers and is closely associated with EBV infection. We postulate that TAMs may increase the invasiveness of NPC by enhancing the formation of invadopodia, which are membranous actin-rich protrusions with digestive ability of extra-cellular matrix. In this study, monocyte THP-1 cells were polarized to M2-macrophages, and co-cultured with nasopharyngeal cells in a contained chamber-insert. The stimulated nasopharyngeal cells then formed massive numbers of invadopodia and digested larger area of gelatin compared with the unstimulated counterparts. A cytokine antibody array was used to identify the upregulated cytokines in the co-culture medium of the M2-macrohphages and the NPC cells. Tumour necrosis factor alpha (TNFα) was revealed to be the responsible cytokine to induce the formation of invadopodia and digestion of gelatin. Besides, western blot analysis identified that several actin-associated proteins such as Src, Erk, and cortactin were involved in the TNFα-mediated invadopodia formation. We also found that the expression of latent membrane protein 1 (LMP1), an EBV protein that has similar structure to TNF receptor was elevated in NPC cells after co-culturing with the M2-polarized macrophages. Through transient transfection and stable expression of LMP1 in the NPC cells, LMP1 could also promote the formation of invadopodia through cdc42 activation. Taken together, these data indicate macrophages can promote the invasiveness of EBV-infected NPC cells by enhancing their ability in forming invadopodia through an activated TNFα signaling axis and LMP1 upregulation. Citation Format: Wilson Wing Chung Tang, Chan Ping You, Gareth E. Jones, George Sai-Wah Tsao, Anna Chi Man Tsang. M2-polarized macrophages increase invasiveness of EBV-associated nasopharyngeal carcinoma by inducing invadopodia formation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1108.

Abstract 3084: Epstein Barr virus-encoded LMP1 activates the mTORC2 signaling pathway to reprogram glucose metabolism in nasopharyngeal epithelial cell

Abstract Epstein-Barr virus (EBV) infection of preinvasive nasopharyngeal epithelium is believed to be an essential initiation event during nasopharyngeal carcinoma pathogenesis, but the underlying mechanisms are largely undefined. Here we demonstrate that EBV-encoded LMP1 reprograms the glucose metabolism from oxidative phosphorylation to glycolysis of nasopharyngeal epithelial (NPE) is a major process for NPC pathogenesis. RNA-seq analysis indicates that EBV infection reprograms the glucose metabolism from glycolysis and oxidative phosphorylation and enhances the metabolic associated pathways. Our study show EBV encoded LMP1 is important for this event, EBV infection and LMP1 expression enhance the extracellular acidification rate and decrease the oxygen consumption rate in NPE cells. We further show that activation of mTORC2/AKT signaling is involved in this metabolic reprogramming in EBV-infected NPE cells. Genetic or pharmacologic inhibition of the mTORC2/AKT signaling blocks the LMP1-induced glycolysis. In summary, this study provides evidences of involvement of EBV infection in metabolic reprogramming of NPE cells. Acknowledgment: This project was supported by funding from the Research Grants Council, Hong Kong: General Research Fund (17120814, 17161116, 17111516, 171110315), Area of Excellence (AoE/M-06/08), Theme-Based Research Scheme (T12-401/13-R), Collaborative Research Fund (Project reference: C7027-16G); Health and Medical Research Fund of Hong Kong (12110782, 13120872, 04151726); and CRCG, University of Hong Kong, Hong Kong. Citation Format: Jun Zhang, Weitao Lin, Chi-Man Tsang, Teng Fei Liu, W C. Tang, Tim Ling Yip, Wen Deng, Lin Jia, Sai-Wah Tsao. Epstein Barr virus-encoded LMP1 activates the mTORC2 signaling pathway to reprogram glucose metabolism in nasopharyngeal epithelial cell [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3084.

Abstract 4694: The contribution of Epstein-Barr virus to the impaired T-cell responses in nasopharyngeal carcinoma

Abstract Undifferentiated nasopharyngeal carcinoma (NPC) is a geographically skewed disease in which more than 80% cases are from southern China and Southeast Asia. NPC is consistently associated with Epstein-Barr virus (EBV) infection and characterized by a prominent lymphocyte infiltration. The consistent expression of EBV proteins in NPC cells has led to several clinical trials of adoptive T-cell therapy or vaccination to boost the immune response to EBV antigens. However, these treatments yield relatively modest clinical benefit, which may be due to varying degrees of immunosuppression in the tumor microenvironment. The lack of model systems to analyze the ability of therapeutically relevant T cells to recognize and kill NPC cells represents a fundamental limitation in our attempts to harness the immune system for clinical benefit. We used three NPC cell lines as targets in T-cell assays, together with a panel of established HLA-matched EBV-specific CD8 and CD4 T-cell clones. T-cell activity and killing were measured by IFN-γ ELISA and outgrowth assays, respectively. We demonstrate, for the first time, that NPC cells can be recognized and killed by EBV-specific T-cell clones in vitro, which suggests that tumor-derived factors inhibit such responses in vivo. These observations are consistent with the previous reported functional inactivation of EBV-specific cytotoxic T lymphocytes (CTL) in NPC patients. We hypothesized that EBV infection of NPC cells induces the secretion of immunosuppressive factors. The effect of conditioned media derived from an EBV-infected premalignant nasopharyngeal cell line, NP460htert, on T-cell activity was examined by treating several clones of EBV-specific CTLs in vitro. The conditioned media inhibited the secretion of IFN-γ from peptide-stimulated CTLs, indicating that secreted factors that could suppress CTL functions are induced following EBV infection in NP460htert cells. RNA sequencing analyses on NP460hTert and three NPC cell lines following EBV infection revealed a significant enrichment of genes whose proteins are located at extracellular region/matrix, including a number of immunomodulators. Our results suggest that EBV infection might induce the production of soluble molecules, which in turn suppress the antitumor T-cell responses. In conclusion, we have developed a system with which to study NPC-specific immune responses and shown that soluble factors induced by EBV infection can suppress T-cell activity. These model systems will be critical in the future to examine the effects of checkpoint inhibitors, secreted factors from cancer-associated fibroblasts or NPC cells upon T-cell activity, which will ultimately enhance the success of EBV-targeted immunotherapy strategies. Citation Format: Hui Min Lee, Tracey Haigh, George SW Tsao, Paul G. Murray, Graham Taylor, Ian C. Paterson, Lee Fah Yap. The contribution of Epstein-Barr virus to the impaired T-cell responses in nasopharyngeal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4694.

Abstract 3098: Promotion of in vivo growth of Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma by miR-BARTs

Abstract Epstein-Barr virus (EBV) infection is nearly 100% associated with undifferentiated nasopharyngeal carcinoma (NPC). However, EBV episomes are rapidly lost under in vitro culturing if there is no selective advantage to their retention. The persistence of the viral episomes within the in vivo tumors therefore implies that the virus must be contributing to the growth and/or survival of the tumors. Since most of the growth-promoting EBV latent proteins are immunogenic, their expressions are usually low and heterogeneous within a tumor. Hence, the abundant expression of the non-immunogenic microRNAs derived from EBV-encoded BART transcripts, i.e. miR-BARTs, strongly implicates their pathogenic roles in this cancer. We found that EBV infected NPC cells could develop into bigger tumors in nude mice than the non-infected counterparts. Importantly, the miR-BARTs were all upregulated after the in vivo growth conditions. Nevertheless, EBV-infected and non-infected cells had comparative growth in in vitro culturing, suggesting EBV infection could provide growth advantage to NPC in vivo, but not in vitro. We have recently established a new and representative NPC cell line (NPC43) to study the pathogenesis of EBV in the development of NPC. In consistent will other previous publications, the EBV genomes were lost gradually when growing in vitro, resulting in a cell line with only half of the population is EBV-positive (at PD 50). Interestingly, after this mixture of EBV-positive and EBV-negative cells were grown as tumor in nude mice, the tumor cells were detected to be nearly 100% positive for EBV-infection. This suggests the in vivo growth conditions could select for the EBV-positive NPC cells. miR-BARTs are known to have a predominant role in suppressing cellular apoptosis. We hypothesized that miR-BARTs may help to resist apoptosis under metabolic stress in vivo, when tumor growth outstrips the nutrient supply. In our in vitro experiments, we observed that EBV infection protected the cells from apoptosis if they were grown in culture medium deprived of glucose and amino acids. Moreover, the expression levels of miR-BARTs were also upregulated in this metabolically stressed condition. To further investigate the roles of miR-BARTs, we infected NPC cells (NPC43) and immortalized nasopharyngeal epithelial cells (NP460) with either wild-type M81 EBV or mutant M81 EBV (with BART deleted). We found that NP460-M81WT showed a general trend of higher resistance to starvation-induced cell death than NP460-M81ΔBART. Besides in the spheroid cultures of the pair of NPC43-M81WT and NPC43-M81ΔBART, the WT cells could grow into bigger spheroids, reflecting they could sustain their growth under a gradient of nutrient deficiency which is present in the 3D structure of a spheroid. Taken together, all these suggest miR-BARTs could promote in vivo growth by enhancing survival of the EBV-infected cells. Citation Format: Anna Chi Man Tsang, Shaina Chor Mei Huang, Ming Han Tsai, Henri-Jacques Delecluse, Honglin Chen, George Sai Wah Tsao. Promotion of in vivo growth of Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma by miR-BARTs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3098.

Molecular signature of Epstein-Barr virus infection in MS brain lesions.

ObjectiveWe sought to confirm the presence and frequency of B cells and Epstein-Barr virus (EBV) (latent and lytic phase) antigens in archived MS and non-MS brain tissue by immunohistochemistry.MethodsWe quantified the type and location of B-cell subsets within active and chronic MS brain lesions in relation to viral antigen expression. The presence of EBV-infected cells was further confirmed by in situ hybridization to detect the EBV RNA transcript, EBV-encoded RNA-1 (EBER-1).ResultsWe report the presence of EBV latent membrane protein 1 (LMP-1) in 93% of MS and 78% of control brains, with a greater percentage of MS brains containing CD138+ plasma cells and LMP-1–rich populations. Notably, 78% of chronic MS lesions and 33.3% of non-MS brains contained parenchymal CD138+ plasma cells. EBV early lytic protein, EBV immediate-early lytic gene (BZLF1), was also observed in 46% of MS, primarily in association with chronic lesions and 44% of non-MS brain tissue. Furthermore, 85% of MS brains revealed frequent EBER-positive cells, whereas non-MS brains seldom contained EBER-positive cells. EBV infection was detectable, by immunohistochemistry and by in situ hybridization, in both MS and non-MS brains, although latent virus was more prevalent in MS brains, while lytic virus was restricted to chronic MS lesions.ConclusionsTogether, our observations suggest an uncharacterized link between the EBV virus life cycle and MS pathogenesis.

Ovum collection following ovarian stimulation in patients with chronic active Epstein-Barr virus infection

The only curative treatment for chronic active Epstein-Barr virus infection (CAEBV) is hematopoietic stem cell transplantation. For young female patients, ovulation induction and oocyte cryopreservation may be performed prior to transplantation to provide for future pregnancies. However, the effects of this ovum treatment on CAEBV and EBV infections have not been reported. Attempts were made to collect ova from three female CAEBV patients before transplantation conditioning, but this was only successful in two cases. Ovarian stimulation did not induce disease progression, and there was no change in the peripheral blood EBV DNA load. In one patient, 460 copies/ml of EBV DNA were detected in the follicular fluid by real-time PCR. Red blood cells were also present in the follicular fluid but not mononuclear cells. EBV protein mRNA was not detected in the RNA extracted from the same fluid, suggesting that the EBV DNA resulted from peripheral blood contamination. Moreover, there were no EBV-infected cells in the follicular fluid. Therefore cryopreservation of oocytes from CAEBV patients is possible and may be used to provide for future pregnancies.

EBV infection modulates the cell viability of gastric cancer cell through modulating miR34a-NOX2-ROS signaling

Abstract Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is a viral protein expressed in all EBV-infected cells that induces malignant transformation. EBNA1 is reported to contribute to tumor progression through an increase in reactive oxygen species via nicotinamide adenine dinucleotide phosphate oxidase. However, the underlying molecular mechanism of EBNA1-induced ROS accumulation in gastric cancer is poorly understood. Here, we demonstrated that miR34a regulation by EBNA1 determined cell fate in EBV-infected gastric cancer cells. ROS content and NOX2 expression were higher in EBNA1-expressing SNU719 cells than in EBNA1-nonexpressing SNU638 cells. Downregulation of NOX2 using siRNA technology in SNU719 cells decreased cell viability and ROS content. Regulation of EBNA1 expression in EBV-associated gastric cancers modulated NOX2 expression, ROS content and cell viability. We also showed that upregulation of NOX2 by EBNA1 was mediated by downregulating miRNA34a. Finally, overexpression of miR34a in EBNA1-expressing SNU719 cells induced typical apoptosis, suggesting that reactivation of miR34a in EBNA1-expressing gastric cancer cells could be a strategy for treatment of EBV-infected gastric cancer cells.

Blockage of store-operated Ca2+ entry antagonizes Epstein-Barr virus-promoted angiogenesis by inhibiting Ca2+ signaling-regulated VEGF production in nasopharyngeal carcinoma.

BackgroundEpstein–Barr virus (EBV) actively contributes to the pathological process of nasopharyngeal carcinoma (NPC) by enabling NPC cells to acquire various capacities required for their malignant biological actions. Our earlier works demonstrated that EBV-encoded latent membrane protein 1 (LMP1) enhanced vascular endothelial growth factor (VEGF)-mediated angiogenesis by boosting store-operated Ca2+ entry (SOCE) upon extracellular epidermal growth factor (EGF) stimulation. However, the antagonistic effects of SOCE blockage on EBV-promoted angiogenesis must be appropriately evaluated in vivo, and the global effect of EBV infection on the EGF-elicited cytosolic Ca2+ signaling, which regulates VEGF-mediated angiogenesis remains to be further clarified.Materials and methodsTwo EBV-infected NPC cell lines, CNE2-EBV and HK1-EBV, along with their parental cell lines were employed in the present study. Dynamic cytosolic Ca2+ changes were measured in individual fluorescent Ca2+ indicator-loaded cells. Amounts of VEGF production were determined by enzyme-linked immunosorbent assay (ELISA). Human umbilical vein endothelial cells (HUVECs)-formed tube networks were quantitatively evaluated as an in vitro angiogenesis assay. A mouse model concurrently bearing EBV-positive/negative xenografts was utilized to evaluate the tumor growth and angiogenesis in vivo.ResultsEBV infection reliably promoted transplanted tumor growth while enhancing angiogenesis. Introduction of EBV into EBV-negative NPC cells increased the EGF-stimulated VEGF production while amplifying the EGF-evoked Ca2+ responses. Inhibition of the EBV-boosted Ca2+ signaling using 2-aminoethyl diphenylborinate (2-APB), a specific SOCE inhibitor, effectively antagonized the EBV-promoted VEGF production and endothelial tube formation in vitro. Pharmacological blockage of SOCE exhibited anti-angiogenic effect in the EBV-positive xenografts.ConclusionSOCE can serve as a candidate pharmacological target for treating NPC, as blockage of the Ca2+ signaling via SOCE is a feasible strategy to suppress the EBV-driven malignant profiles in NPC cells.

Epigenetic Regulation of Tumor Suppressors by Helicobacter pylori Enhances EBV-Induced Proliferation of Gastric Epithelial Cells.

ABSTRACTHelicobacter pylori and Epstein-Barr virus (EBV) are two well-known contributors to cancer and can establish lifelong persistent infection in the host. This leads to chronic inflammation, which also contributes to development of cancer. Association with H. pylori increases the risk of gastric carcinoma, and coexistence with EBV enhances proliferation of infected cells. Further, H. pylori-EBV coinfection causes chronic inflammation in pediatric patients. We have established an H. pylori-EBV coinfection model system using human gastric epithelial cells. We showed that H. pylori infection can increase the oncogenic phenotype of EBV-infected cells and that the cytotoxin-associated gene (CagA) protein encoded by H. pylori stimulated EBV-mediated cell proliferation in this coinfection model system. This led to increased expression of DNA methyl transferases (DNMTs), which reprogrammed cellular transcriptional profiles, including those of tumor suppressor genes (TSGs), through hypermethylation. These findings provide new insights into a molecular mechanism whereby cooperativity between two oncogenic agents leads to enhanced oncogenic activity of gastric cancer cells.

NF-κB Signaling Regulates Epstein-Barr Virus BamHI-Q-Driven EBNA1 Expression.

Epstein–Barr virus (EBV) nuclear antigen 1 (EBNA1) is one of the few viral proteins expressed by EBV in nasopharyngeal carcinoma (NPC), most likely because of its essential role in maintaining the viral genome in EBV-infected cells. In NPC, EBNA1 expression is driven by the BamHI-Q promoter (Qp), which is regulated by both cellular and viral factors. We previously determined that the expression of another group of EBV transcripts, BamHI-A rightward transcripts (BARTs), is associated with constitutively activated nuclear factor-κB (NF-κB) signaling in NPC cells. Here, we show that, like the EBV BART promoter, the EBV Qp also responds to NF-κB signaling. NF-κB p65, but not p50, can activate Qp in vitro, and NF-κB signaling regulates Qp-EBNA1 expression in NPC cells, as well as in other EBV-infected epithelial cells. The introduction of mutations in the putative NF-κB site reduced Qp activation by the NF-κB p65 subunit. Binding of p65 to Qp was shown by chromatin immunoprecipitation (ChIP) analysis, while electrophoretic mobility shift assays (EMSAs) demonstrated that p50 can also bind to Qp. Inhibition of NF-κB signaling by the IκB kinase inhibitor PS-1145 resulted in the downregulation of Qp-EBNA1 expression in C666-1 NPC cells. Since EBNA1 has been reported to block p65 activation by inhibiting IKKα/β through an unknown mechanism, we suggest that, in NPC, NF-κB signaling and EBNA1 may form a regulatory loop which supports EBV latent gene expression, while also limiting NF-κB activity. These findings emphasize the role of NF-κB signaling in the regulation of EBV latency in EBV-associated tumors.

The Microenvironment in Epstein-Barr Virus-Associated Malignancies.

The Epstein–Barr virus (EBV) can cause a wide variety of cancers upon infection of different cell types and induces a highly variable composition of the tumor microenvironment (TME). This TME consists of both innate and adaptive immune cells and is not merely an aspecific reaction to the tumor cells. In fact, latent EBV-infected tumor cells utilize several specific mechanisms to form and shape the TME to their own benefit. These mechanisms have been studied largely in the context of EBV+ Hodgkin lymphoma, undifferentiated nasopharyngeal carcinoma, and EBV+ gastric cancer. This review describes the composition, immune escape mechanisms, and tumor cell promoting properties of the TME in these three malignancies. Mechanisms of susceptibility which regularly involve genes related to immune system function are also discussed, as only a small proportion of EBV-infected individuals develops an EBV-associated malignancy.

Baicalein inhibits growth of Epstein-Barr virus-positive nasopharyngeal carcinoma by repressing the activity of EBNA1 Q-promoter

Epstein-Barr virus (EBV) can establish a life-long latent infection in the host and is associated with various human malignancies, including nasopharyngeal carcinoma (NPC), the most common cancer originated from nasopharynx. EBV nuclear antigen 1 (EBNA1) is the only viral protein absolutely demanded for segregation, replication, transcription and maintenance of EBV viral genome in host cells. Baicalein, a bioactive flavonoid compound purified from the root of Scutellariae baicaleinsis, displays anti-inflammatory, immunosuppressive, and anti-tumor properties. In this study, the therapeutic effects and functional mechanism of baicalein on EBV-positive human NPC were determined. Cell Counting Kit-8 assays and cell formation colony were performed to investigate that baicalein can suppress proliferation of EBV-infected human NPC cells. Flow cytometric and hoechst 33258 staining results indicated that baicalein induced cell cycle arrest and apoptosis. Western blotting results demonstrated that baicalein down-regulates EBNA1 expression but not reduces the stability and half-life of EBNA1 in EBV-infected NPC cells. Additionally, the mRNA level of EBNA1 was examined by real time-PCR, the activity of EBNA1 Q promoter (Qp) was determined by dual luciferase reporter assay. Considering that transcription factor specificity protein 1 (Sp1) can maintain EBNA1 Qp active. Further analyses also elucidated that baicalein inhibits the expression of Sp1 while knock-down Sp1 by specific shRNAs decreases the expression and transcription levels of EBNA1. Therefore, the results suggested that baicalein may decrease EBNA1 expression level in EBV-positive NPC cells via inhibiting the activity of EBNA1 Q-promoter while over-expression of EBNA1 attenuate the inhibitory effect of baicalein. Finally, it was found that baicalein may strongly reduce growth of tumor in the mouse xenograft model of EBV-positive NPC. These results indicated that baicalein inhibits growth of EBV-positive NPC by repressing the activity of EBNA1 Q-promoter. Baicalein may be used as a therapeutic agent to treat EBV-positive NPC.

Interplay of Viral Infection, Host Cell Factors and Tumor Microenvironment in the Pathogenesis of Nasopharyngeal Carcinoma.

Undifferentiated nasopharyngeal carcinoma (NPC) is strongly associated with Epstein-Barr virus (EBV) infection. In addition, heavy infiltration of leukocytes is a common characteristic of EBV-associated NPC. It has long been suggested that substantial and interactive impacts between cancer and stromal cells create a tumor microenvironment (TME) to promote tumorigenesis. The coexistence of tumor-infiltrating lymphocytes with EBV-infected NPC cells represents a distinct TME which supports immune evasion and cancer development from the early phase of EBV infection. Intracellularly, EBV-encoded viral products alter host cell signaling to facilitate tumor development and progression. Intercellularly, EBV-infected cancer cells communicate with stromal cells through secretion of cytokines and chemokines, or via release of tumor exosomes, to repress immune surveillance and enhance metastasis. Although high expression of miR-BARTs has been detected in NPC patients, contributions of these more recently discovered viral products to the establishment of TME are still vaguely defined. Further investigations are needed to delineate the mechanistic linkage of the interplay between viral and host factors, especially in relation to TME, which can be harnessed in future therapeutic strategies.