Latent Epstein-Barr Virus Infection Research Articles

Improving T-Cell Therapy for Epstein-Barr Virus Lymphoproliferative Disorders

More than 90% of the population is infected with Epstein-Barr virus (EBV), which is an enveloped herpesvirus. EBV infection in immunocompetent individuals causes a mild self-limiting illness, but the virus persists lifelong as a latent infection in B cells and spreads in the community by productive replication in B cells and oral epithelial cells. EBV latency varies in the number and type of EBV proteins expressed by B cells. In type 3 latency, all nine latency-associated EBV proteins are expressed. Such B cells express cell adhesion and costimulatory molecules rendering them immunogenic and susceptible to immune-mediated killing by EBV-specific cytotoxic T lymphocytes (CTLs). After hematopoietic stem-cell transplantation (HSCT) without functional CTLs, type 3 latency EBVinfected B cells proliferate unchecked. EBV-infected B cells cause a lymphoproliferative disorder that can progress to lymphoma with increased levels of EBV DNA detected by polymerase chain reaction (PCR). EBV-associated post-transplant lymphoproliferative disease (PTLD) occurs in less than 1% to 25% of HSCT recipients depending on the type of graft, the degree to which the graft is T-cell depleted, and the post-HSCT immunosuppression. Rituximab is an effective monotherapy even in established PTLD after HSCT, with response rates of 55% to 100%. However, this B-cell depleting antibody increases risk of infection, and PTLD can recur if EBV-specific T-cell immunity is not restored. Because of its immunogenicity and occurrence in the context of immunodeficiency, PTLD is highly amenable to immunotherapy with EBVspecific CTLs. In the article that accompanies this editorial, Icheva et al used donor-derived Epstein-Barr nuclear antigen 1 (EBNA1) –specific T cells to treat post-HSCT PTLD. CTLs were generated with antigenpresenting cells that were pulsed either with whole protein or synthetic overlapping peptides to elicit EBNA1-specific T cells, which were then selected by interferon alfa capture (Fig 1). The selected T cells were either transfused directly without in vitro expansion or cryopreserved for future use. These rapidly generated EBNA1 CTLs were effective in seven of 10 cases of EBV-associated PTLD. Immunotherapeutic strategies to restore EBV-specific T-cell function have been used for more than 15 years to improve the outcome of PTLD. Initially, unselected donor lymphocytes from the EBV seropositive stem-cell donors (assumed to have a high frequency of EBV-specific precursors) were used to restore the immune response to EBV. This simple strategy is readily available at any transplant center and yields responses of approximately 70%. However, unselected donor lymphocyte infusions carry significant risk of severe graft versus host disease. Because of this, several groups generated and infused donor-derived EBV-specific T cells in patients after SCT. More than 100 patients have received such EBV-specific T cells, either as prophylaxis or as treatment for EBV PTLD with complete remission rates also in the order of 70%. Thus far, to generate EBV-specific CTLs, investigators have used EBV-transformed B cells (lymphoblastoid cell lines [LCLs]) that express type III latency like PTLD. It takes 10 to 12 weeks to generate LCLs and sufficient EBV-specific CTLs for infusion and release testing. This prolonged manufacturing time limits the ready use of this therapy for the urgent treatment of EBVassociated PTLD. The importance of the study by Icheva et al is that it demonstrates that rapidly generated EBV-specific CTLs can control PTLD similarly to the traditionally manufactured EBVspecific CTLs. Other investigators have successfully reported the rapid manufacture of EBV-CTLs by using HLA-A2 restricted tetramer selection, immunomagnetic selection of interferon-gamma–secreting T cells after stimulation of donor mononuclear cells with a spectrum of class I and II EBV peptides or by using latent membrane protein 2 (LMP2), EBNA1, and BamH1 Z leftward reading frame 1 (BZLF1, an immediate early transcription protein responsible for switching between latent and lytic EBV infection) plasmid transfected dendritic cells to expand EBV-specific T cells. The novelty of the approach by Icheva et al is that they chose (more for regulatory rather than scientific reasons) to target only the immunosubdominant EBV antigen EBNA1 rather than the immunodominant EBV proteins EBNA3, EBNA2, or LMP2 expressed in PTLD. Nevertheless, EBNA1 was shown to be an effective target and, moreover, promises to broaden the applicability of this immunotherapy to other EBV type 1 and 2 latency tumors (including Burkitt’s lymphoma and Hodgkin’s lymphoma, respectively) because all EBVassociated cancers express EBNA1. However, there are still problems to be addressed in additional studies. First, the targeting of a single antigen can increase the likelihood of tumor immune escape. For this reason, the authors intend to target additional EBV antigens such as LMP2 in future studies. In addition, because EBV reactivation and lymphoproliferation usually occurs from endogenous virus, seropositive recipients of transplants from EBV-naive, seronegative donors are at greatest risk for EBV LPD. Although EBV-specific CTLs have been JOURNAL OF CLINICAL ONCOLOGY E D I T O R I A L VOLUME 31 NUMBER 1 JANUARY 1 2013

EBV-Specific Micro-RNA Via Exosome: A Key Inter-Cellular machinery between EBV+ Tumor and Tumor-Surrounding Immune Cells?

Abstract 50The Epstein-Barr virus (EBV) is one of the most major human pathogen that establish long-term latent, or chronic infections, which is associated with a heterogeneous group of lymphoma, including Burkitt’s lymphoma, Hodgkin’s lymphoma (HL), NK-T lymphomas and lymphoproliferative disease. These malignancies are subdivided into in terms of EBV latent infection pattern, with typical three types of latency: type I to type III. HL is characterized by a minority of neoplastic Hodgkin and Reed-Sternberg (HRS), which are embedded in non-neoplastic bystanders, mostly B and T cells, but also macrophages. Without these bystander cells, these HL cells are incapable of being engrafted in immunodeficient mice. In this context, the non-tumor immune cells are tumor-supportive “inflammatory niche”. Because of the complexity of interplay between tumor and tumor surrounding immune cells, the detailed mechanism and how tumor cells escape from the attack of host immune cells remains an open question. Small RNAs including miRNAs are well known intra-cellular regulatory elements of gene expression. Recently, it was reported that they are conjugated in exosomes and transferred to cells and are involved in tumor metastasis by educating tumor surrounding niche. Moreover, it was also reported that EBV-infected lymphocytes produce exosomes that contain viral encoded, EBV specific miRNAs (BART-miRNA) and that these could be transferred in host cells and decrease the levels of known cellular targets. Accordingly, we hypothesized that EBV+ tumor derived exosomal BART-miRNA might redirect tumor surrounding immune cells from tumor reactive into tumor- -supportive “inflammatory niche”, which ultimately leads to tumor progression. To this aim, first, we evaluated tumor derived viral encoded BART-miRNA in EBV+HL clinical specimens by using BART-miRNA specific probe in situ hybridization. As expected, these EBV specific BART-miRNA could be detected in HRS as well as in tumor surrounding inflammatory niche, especially macrophage. This result indicated that tumor derived EBV specific BART-miRNA could transfer to the non-tumor cells in the tumor inflammatory niche, supporting the in vivo relevance of secretary EBV specific miRNA. Next, we evaluated the properties of exosomes produced by EBV+ cells (EBV-Ex). To this aim, EBV-Ex was harvested either from the media of the type III or type I EBV-transformed lymphoid cell line. Then, by using transwell co-culture system, we tested the delivery and the effect of EBV-Ex on human peripheral blood mononuclear cells (PBMC) derived monocyte/macrophage (Mo/Mf). As a result, we detected uptake of fluorochrome dye-labeled EBV-Exo in Mo/Mf. We also confirmed exosomal BART miRNA transfer in Mo/Mf. Surprisingly, exosome from Type III latency (Type III-Ex) was relatively enriched in BART miRNA, and were potent on Mo/Mf in inducing surface CD69 expression (Fig.A). This is in contrast to that of exosome from Type I latency (Type I-Ex), in which BART miRNA were relatively vacant and were weak in inducing surface CD69 expression (Fig.A). Panels of cytokine analysis by Q-PCR revealed that type III-Ex treated Mo/Mf displayed an anti-inflammatory/immunosuppressive cytokine rich signature, especially IL-10, compared to type I-Ex treated Mo/Mf, suggesting the possibility that type III-Ex might polarize macrophage into immunosuppressive M2-like phenotype. Intriguingly, type III-Ex from BART miRNA deletion mutant derivative cell lines totally lack the type III -Ex signature. Moreover, ectopic expression of a part of BART in Type I cells changed the EBV-Ex signature from type III to type I (Fig.B), suggesting the importance of specific BART lesion in functional EBV-Ex production in terms of Mo/Mf polarization. Taken these together, secretary tumor derived miRNAs in EBV associated malignancy, specifically in EBV+HL, might play a certain role in tumor inflammation niche. EBV might utilize the exosomal machinery to secrete key viral-encoded miRNAs, through which a small number of neoplastic EBV+ cells could modulate the tumor microenvironment. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Clonal expansion of Epstein–Barr virus (EBV)-infected γδ T cells in patients with chronic active EBV disease and hydroa vacciniforme-like eruptions

Chronic active Epstein-Barr virus (EBV) disease (CAEBV) is a systemic EBV-positive lymphoproliferative disorder characterized by fever, lymphadenopathy, and splenomegaly. Patients with CAEBV may present with cutaneous symptoms, including hypersensitivity to mosquito bites and hydroa vacciniforme (HV)-like eruptions. HV is a rare photodermatosis characterized by vesicles and crust formation after exposure to sunlight, with onset in childhood, and is associated with latent EBV infection. While γδ T cells have recently been demonstrated to be the major EBV-infected cell population in HV, the immunophenotypic features of EBV-infected γδ T cells in CAEBV with HV-like eruptions or HV remain largely undetermined. We describe three patients with CAEBV whose γδ T cells were found to be the major cellular target of EBV. HV-like eruptions were observed in two of these patients. A clonally expanded subpopulation of γδ T cells that were highly activated and T cell receptor Vγ9- and Vδ2-positive cells was demonstrated in all patients. We also show that the clonally expanded γδ T cells infiltrated into the HV-like eruptions in one patient from whom skin biopsy specimens were available. These results suggest the pathogenic roles of clonally expanded γδ T cells infected by EBV in patients with CAEBV and HV-like eruptions.

Epstein–Barr virus and methotrexate-related CNS lymphoma in a patient with rheumatoid arthritis

Patients with rheumatoid arthritis (RA), especially those treated with methotrexate (MTX), might have an increased risk of lymphoproliferative disorders that are associated with Epstein-Barr virus (EBV). We describe a case of EBV-associated central nervous system (CNS) lymphoma (diffuse large B-cell lymphoma) in a patient with RA on a short course of MTX treatment. The neoplastic cells express the B-cell surface markers (CD20, Pax-5 and CD30), and EBV-encoded RNA was demonstrated by in situ hybridization. The patient's lymphoma did not recur for the 8-year follow-up period after the tumor resection and cessation of MTX. MTX may promote EBV-positive CNS lymphoma in RA patient due to its immunosuppressive properties as well as reactivating latent EBV infection.

Small molecule and peptide-mediated inhibition of Epstein-Barr virus nuclear antigen 1 dimerization

Latent Epstein-Barr virus (EBV) infection is associated with human B cell lymphomas and certain carcinomas. EBV episome persistence, replication, and gene expression are dependent on EBV-encoded nuclear antigen 1 (EBNA1)’s DNA binding domain (DBD)/dimerization domain (DD)-mediated sequence-specific DNA binding activity. Homodimerization of EBNA1 is essential for EBNA1 DNA binding and transactivation. In this study, we characterized a novel small molecule EBNA1 inhibitor EiK1, screened from the previous high throughput screening (HTS). The EiK1 compound specifically inhibited the EBNA1-dependent, OriP-enhanced transcription, but not EBNA1-independent transcription. A Surface Plasmon Resonance Biacore assay revealed that EiK1 associates with EBNA1 amino acid 459–607 DBD/DD. Consistent with the SPR data, in vitro gel shift assays showed that EiK1 suppressed the activity of EBNA1 binding to the cognate familial repeats (FR) sequence, but not control RBP-Jκ binding to the Jκ site. Subsequently, a cross-linker-mediated in vitro multimerization assay and EBNA1 homodimerization-dependent yeast two-hybrid assay showed that EiK1 significantly inhibited EBNA1 dimerization. In an attempt to identify more highly specific peptide inhibitors, small peptides encompassing the EBNA1 DBD/DD were screened for inhibition of EBNA1 DBD-mediated DNA binding function. The small peptide P85, covering EBNA1 a.a. 560–574, significantly blocked EBNA1 DNA binding activity in vitro, prevented dimerization in vitro and in vivo, associated with EBNA1 in vitro, and repressed EBNA1-dependent transcription in vivo. Collectively, this study describes two novel inhibitors of EBNA1 dimerization. This study demonstrates that EBNA1 homodimerization can be effectively targeted by a small molecule or peptide.

Human Solid Tumor Xenografts in Immunodeficient Mice Are Vulnerable to Lymphomagenesis Associated with Epstein-Barr Virus

Xenografting primary human solid tumor tissue into immunodeficient mice is a widely used tool in studies of human cancer biology; however, care must be taken to prove that the tumors obtained recapitulate parent tissue. We xenografted primary human hepatocellular carcinoma (HCC) tumor fragments or bulk tumor cell suspensions into immunodeficient mice. We unexpectedly observed that 11 of 21 xenografts generated from 16 independent patient samples resembled lymphoid neoplasms rather than HCC. Immunohistochemistry and flow cytometry analyses revealed that the lymphoid neoplasms were comprised of cells expressing human CD45 and CD19/20, consistent with human B lymphocytes. In situ hybridization was strongly positive for Epstein-Barr virus (EBV) encoded RNA. Genomic analysis revealed unique monoclonal or oligoclonal immunoglobulin heavy chain gene rearrangements in each B-cell neoplasm. These data demonstrate that the lymphoid neoplasms were EBV-associated human B-cell lymphomas. Analogous to EBV-associated lymphoproliferative disorders in immunocompromised humans, the human lymphomas in these HCC xenografts likely developed from reactivation of latent EBV in intratumoral passenger B lymphocytes following their xenotransplantation into immunodeficient recipient mice. Given the high prevalence of latent EBV infection in humans and the universal presence of B lymphocytes in solid tumors, this potentially confounding process represents an important pitfall of human solid tumor xenografting. This phenomenon can be recognized and avoided by routine phenotyping of primary tumors and xenografts with human leukocyte markers, and provides a compelling biological rationale for exclusion of these cells from human solid tumor xenotransplantation assays.

Endoscopic Mucosal Resection of an Epstein–Barr Virus-Associated Lymphoepithelioma-Like Gastric Carcinoma

Epstein–Barr virus (EBV) usually causes a life-long persistent infection of the B lymphocytes in 90 % of adults [1, 2]. EBV is oncogenic in the development of Burkitt lymphoma, immunosuppression-related lymphoma, Hodgkin’s lymphoma, and nasopharyngeal carcinoma. Recently, EBV-associated gastric carcinomas were found to have a prevalence of 8.7 % and they tend to be located in the gastric cardia and body [2]. Primary gastric lymphoepithelioma-like carcinoma (LLC) is rare and associated with latent EBV infection [2–4]. We report a case of EBVassociated primary gastric LLC diagnosed and completely removed by endoscopic mucosal resection (EMR).

The EBV immunoevasins vIL-10 and BNLF2a protect newly infected B cells from immune recognition and elimination.

Lifelong persistence of Epstein-Barr virus (EBV) in infected hosts is mainly owed to the virus' pronounced abilities to evade immune responses of its human host. Active immune evasion mechanisms reduce the immunogenicity of infected cells and are known to be of major importance during lytic infection. The EBV genes BCRF1 and BNLF2a encode the viral homologue of IL-10 (vIL-10) and an inhibitor of the transporter associated with antigen processing (TAP), respectively. Both are known immunoevasins in EBV's lytic phase. Here we describe that BCRF1 and BNLF2a are functionally expressed instantly upon infection of primary B cells. Using EBV mutants deficient in BCRF1 and BNLF2a, we show that both factors contribute to evading EBV-specific immune responses during the earliest phase of infection. vIL-10 impairs NK cell mediated killing of infected B cells, interferes with CD4+ T-cell activity, and modulates cytokine responses, while BNLF2a reduces antigen presentation and recognition of newly infected cells by EBV-specific CD8+ T cells. Together, both factors significantly diminish the immunogenicity of EBV-infected cells during the initial, pre-latent phase of infection and may improve the establishment of a latent EBV infection in vivo.

Recurrent Inflammatory Myofibroblastic Tumor of the Heart

A previously healthy 15-year-old boy was admitted after 6 months of intermittent fevers, night sweats, and 9 kg weight loss. A systolic murmur was appreciated at the left upper sternal border. Laboratory data revealed elevated white blood cells (11 800/μL), erythrocyte sedimentation rate (75 mm/h), and C-reactive protein (10.5 mg/dL). The results of blood cultures were negative. Imaging demonstrated a hypermetabolic 2×2×4.5 cm pedunculated mass in the right ventricle, suggestive of a neoplasm with overlying thrombus (Figures 1A, 2A and 2B and online-only Data Supplement Movie I). Figure 1. Cardiac magnetic resonance imaging demonstrating the initial IMT originating from the interventricular septum near the right ventricular outflow tract ( A ) and the recurrent IMT near the tricuspid annulus ( B ). IMT indicates inflammatory myofibroblastic tumor. Figure 2. Transthoracic echocardiography before and after resection of the initial inflammatory myofibroblastic tumor. A and B , Preoperative images showed a large echogenic mass in the apex of the right ventricle extending superiorly through the right ventricular outflow tract. C and D , Postoperative echocardiography revealed no evidence of residual tumor. A4C indicates apical 4-chamber view; Ao, aorta; LA, left atrium; LV, left ventricle; PA, pulmonary artery; PSAX, parasternal short axis view; RA, right atrium; RV, right ventricle; RVOT, right ventricular outflow tract; Pre-op, preoperative; and Post-op, postoperative. Arrow denotes the tumor. At surgery, the mass was found to originate from the midportion of the interventricular septum and was covered with thrombus. …

EBV-regulated global changes in mRNA isoform usage

Approximately 90% of the global adult population is latently infected with Epstein-Barr Virus (EBV). Latent EBV infections are normally asymptomatic due to a robust cytotoxic T cell response. However, in the event of immunosuppression, as observed in HIV/AIDS patients, these latent infections can lead to B-cell lymphomas. In vitro EBV has the capacity to transform primary B-cells into immortalized Lymphoblastoid Cell Lines (LCLs). In order to assess changes in both overall mRNA abundance and mRNA isoform usage, we queried resting, primary human B cells and LCLs using Human Exon (HuEx) and conventional Affymetrix U133 arrays. Using a novel computational algorithm, SplicerEX, we identified 433 genes whose mRNAs undergo changes in alternative isoform usage during the transformation from primary B-cells to LCLs. Isoform changes were largely orthogonal from expression changes as only ~1/3 of mRNA isoform changes were also changed at the level of overall abundance. Isoform changes were classified into alternative 5’ initiation, internal exclusion/inclusion of exons, 3’ terminal exon choice, and 3’UTR alterations. The most striking mRNA isoform change was 3’UTR shortening, accounting for ~25% of all changes. Gene ontology analysis of mRNA isoform changes revealed a strong enrichment for nucleic acid binding proteins, including splicing and transcription factors. We have confirmed a subset of the predictions made by SplicerEX using isoform-specific RT-PCR. Importantly, many mRNA isoform changes observed were in fact regulated by EBV latent infection, not just proliferation per se, as they were also observed in the conversion of EBV-negative Burkitt’s lymphoma cells (BL41) to latency III expressing BL41/B95-8 cells. Our preliminary results further indicate that two transcription factors, the E2 family member TCF4 and the plasma cell differentiation factor XBP1, are both regulated by EBV at the level of alternative isoform usage. These proteins both impact activation of the BZLF1 lytic promoter and our data thus suggest a novel mechanism by which EBV maintains latent infection in immortalized B cells. These data may point to new approaches in regulating the latent/lytic switch crucial to the pathogenesis of EBV-associated AIDS lymphomas.

Poor prognosis of Hodgkin variant of Richter transformation in chronic lymphocytic leukaemia treated with cladribine ‐ Response to Jamroziak et al

We appreciate the thoughtful comments of Jamroziak and colleagues regarding our study of Hodgkin lymphoma as Richter transformation (Hodgkin-RT) in patients with chronic lymphocytic leukaemia (CLL) (Bockorny et al, 2012). Mirroring our pooled analysis that showed a decreased survival in Hodgkin-RT patients previously treated with fludarabine (0.7 ± 0.2 years), their retrospective analysis demonstrated similarly unsatisfactory outcomes in Hodgkin-RT patients treated with cladribine-based regimens for CLL. Of note, they showed a survival of only 0.34 years (95% confidence interval 0–1.08) in a small cohort of Hodgkin-RT patients whose CLL had previously been treated with cladribine (n = 6). A plausible explanation for this dismal outcome is the marked and prolonged T-lymphocyte depletion caused by the purine nucleoside analogues (PNAs), which amplifies the intrinsic immunosuppression of CLL and possibly leads to reactivation of a latent Epstein-Barr virus infection (Cheson et al, 1999; Dasanu, 2008). In addition, Jamroziak et al hypothesized that the first-line chemotherapy commonly used to treat Hodgkin transformation in CLL might be suboptimal against the PNA-exposed clones. In our cohort, the most commonly employed regimens for Hodgkin-RT therapy were ABVD (adriamycin, bleomycin, vinblastine, and dacarbazine) and CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone), with no statistical difference in overall survival between the two groups. As of today, the best therapeutic approach for Hodgkin-RT remains to be clarified (Tsimberidou et al, 2006). Rituximab with or without chemotherapy remains an attractive option for Hodgkin-RT cases with CD20+ Reed-Sternberg cells or marrow/ nodal histologies composed of a mix of Hodgkin lymphoma and CLL. In conclusion, the outcome of the Hodgkin variant of Richter syndrome in CLL, especially when previously exposed to PNAs, should not be viewed as favourable when compared to the classic RT (as diffuse large B-cell lymphoma). Selection of a more intense first-line chemotherapy regimen, with or without targeted antibody therapy may improve the general outcome. Studies aiming to clarify the molecular basis of Hodgkin-RT along with the discovery of innovative therapeutic approaches for this rare variant of RT are awaited. The authors have no competing interests.

Abstract 2037: Cyclin D1 overexpression supports stable EBV infection in nasopharyngeal epithelial cells

Abstract Poorly or undifferentiated nasopharyngeal carcinoma (NPC) is regularly associated with Epstein-Barr virus (EBV) infection. EBV infection of premalignant nasopharyngeal epithelial cells has been postulated to play an important role in the pathogenesis of nasopharyngeal carcinoma. We have previously reported that genetic alterations could be detected in premalignant nasopharyngeal epithelial tissues. It is not clear if these genetic alterations may associate with the establishment of EBV infection. In this study, we have examined the role of cyclin D1 in supporting EBV infection in nasopharyngeal epithelial cells. While cyclin D1 is commonly expressed in nasopharyngeal carcinoma tissues we found that overexpression of cycline D1 is readily detected in dysplastic nasopharyngeal epithelial closely associated with EBV infection. Furthermore, using a panel of immortalized nasopharyngeal epithelial cells, we demonstrated that overexpression of cyclin D1 or CKD4 supports EBV infection and result in the establishment of stable EBV infected nasopharyngeal cell lines. We also observed that EBV infection induced growth inhibition and senescence in nasopharyngeal epithelial cells, which may be the underlying reason for low detection rate of EBV infection in healthy nasopharyngeal epithelial tissues. However, cyclin D1 overexpression suppressed senescence and differentiation in the immortalized nasopharyngeal epithelial cells, suggesting cyclin D1 may play an important role in supporting EBV infection in these cells. We postulated that the altered expression of cyclin D1 or CDK4 in premalignant nasopharyngeal epithelial cells may over-ride the growth suppression effects of EBV infection and support EBV latent infection in premalignant nasopharyngeal epithelial cells. Acknowledgement: This study is sponsored by grant support received from the Hong Kong Research Grant Council (Grant number: GRF780911 and AoE /M-06/08) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2037. doi:1538-7445.AM2012-2037

Proteomic Profiling of EBNA1-Host Protein Interactions in Latent and Lytic Epstein-Barr Virus Infections

The Epstein-Barr nuclear antigen 1 (EBNA1) protein of Epstein-Barr virus (EBV) is expressed in both latent and lytic modes of EBV infection and contributes to EBV-associated cancers. Using a proteomics approach, we profiled EBNA1-host protein interactions in nasopharyngeal and gastric carcinoma cells in the context of latent and lytic EBV infection. We identified several interactions that occur in both modes of infection, including a previously unreported interaction with nucleophosmin and RNA-mediated interactions with several heterogeneous ribonucleoproteins (hnRNPs) and La protein.

The extract of Chrysanthemum indicum Linne inhibits EBV LMP1-induced NF-κB activation and the viability of EBV-transformed lymphoblastoid cell lines

Epstein-Barr virus (EBV) latent infection transforms B lymphocytes into proliferating lymphoblastoid cell lines (LCLs). EBV latent infection membrane protein 1 (LMP1) is required for EBV-mediated B lymphocyte transformation, and LMP1-induced NF-κB activation is essential for LCL survival. To identify a novel inhibitor candidate for LMP1-induced NF-κB activation, crude ethanol extracts of medicinal plants were screened for the potential NF-κB inhibitory activity. Seventy percent ethanol extract of Chrysanthemum indicum Linne extract (CIE) strongly reduced LMP1-induced NF-κB activation. In addition, CIE inhibited LMP1-induced IKKα or IKKβ activation. Interestingly, CIE treatment rapidly reduced LCL viability without exhibiting any adverse effects on the viability of human foreskin fibroblasts (HFF), EBV negative Burkitt’s lymphoma cell lines (BL41) or HeLa cells.Taken together, CIE has potent inhibitory effect on EBV LMP1-induced NF-κB activation and EBV-transformed LCL viability.

Can latent Epstein-Barr virus infection in the central nervous system contribute to neuroinflammation?

Can latent Epstein-Barr virus infection in the central nervous system contribute to neuroinflammation?

E2F1, ARID3A/Bright and Oct-2 factors bind to the Epstein–Barr virus C promoter, EBNA1 and oriP, participating in long-distance promoter–enhancer interactions

The Epstein-Barr virus (EBV) C promoter (Cp) regulates several genes required for B-cell proliferation in latent EBV infection. The family of repeats (FR) region of the latent origin of plasmid replication (oriP) functions as an Epstein-Barr nuclear antigen 1 (EBNA1)-dependent distant enhancer of Cp activity, and the enhancer-promoter interaction is mediated by a higher-order multi-protein complex containing several copies of EBNA1. Using DNA-affinity purification with a 170 bp region of the Cp in combination with mass spectrometry, we identified the cell cycle-regulatory protein E2F1, the E2F-binding protein ARID3A, and the B-cell-specific transcription factor Oct-2 as components of this multi-protein complex. Binding of the three factors to the FR region of oriP was determined by DNA-affinity and immunoblot analysis. Co-immunoprecipitation and proximity ligation analysis revealed that the three factors, E2F1, ARID3A and Oct-2, interact with each other as well as with EBNA1 in the nuclei of EBV-positive cells. Using the chromatin immunoprecipitation assay, we showed that E2F1 and Oct-2 interacted with the FR part of oriP and the Cp, but the ARID3A interaction was, however, only detected at the Cp. Our findings support the hypothesis that EBNA1 initiates transcription at the Cp via interactions between multiple EBNA1 homodimers and cellular transcription factors in a large molecular machinery that forms a dynamic interaction between Cp and FR.

ASK1 is Involved in EBV LMP1-induced NF-κB Activation

Epstein-Barr virus (EBV) latent infection transforms B lymphocytes into proliferating lymphoblastoid cell lines (LCLs). EBV latent infection membrane protein 1 (LMP1) is required for EBV-mediated B lymphocyte transformation, and LMP1-induced NF-κB activation is essential for LCL survival. Previously, it was reported that the level of reactive oxygen species (ROS) and the expression of apoptosis signal-regulating kinase 1 (ASK1) are elevated in EBV-positive Burkitt’s lymphoma (BL) cells, the potential role of ASK1 in LMP1-induced NF-κB activation was thus investigated in this study. In EBV-positive BL cells, ASK1 was highly expressed and activated. In addition, TRAF6-ASK1 interaction was significantly increased in EBV-positive BL cells. Interestingly, the expression of LMP1 alone facilitated ASK1 activation. The expression of a dominant negative ASK1 mutant (ASK1KM) strongly blocked LMP1-induced NF-κB activation. Furthermore, LMP1-induced NF-κB activation was significantly reduced in ASK1 knock out (ASK1-/-) mouse embryonic fibroblasts (MEFs). Taken together, these results demonstrate that ASK1 is activated by LMP1 and is critical for LMP1-induced NF-κB activation.

Association of innate immune activation with latent Epstein-Barr virus in active MS lesions

To determine whether the activation of innate immune responses, which can be elicited by pathogenic and endogenous triggers, is associated with the presence of Epstein-Barr virus (EBV) infection in the multiple sclerosis (MS) brain. White matter postmortem MS (n = 10) and control tissue (n = 11) was analyzed for the expression of the proinflammatory cytokine interferon α (IFNα) by immunohistochemistry and for EBV by using the highly sensitive method of EBV-encoded RNA (EBER) in situ hybridization. We detected overexpression of IFNα in active areas of white matter MS lesions but not in inactive MS lesions, normal-appearing white matter, or normal brains. The presence of IFNα in macrophages and microglia (expressing human leukocyte antigen class II) is suggestive of local production as part of an acute inflammatory process. Interestingly, EBERs were also specifically detected in areas where IFNα was overexpressed in these preselected active MS lesions. EBER+ cells were also found in CNS lymphoma and stroke cases, but were absent in other control brains. We next addressed a potential mechanism, e.g., the role of EBERs in eliciting IFNα production, and transfected EBERs into human embryonic kidney (HEK) cells. We used HEK cells that stably expressed Toll-like receptor-3, which recognizes double-stranded RNAs, associated with many viral infections. EBERs elicited IFNα production in vitro. These findings suggest that latent EBV infection may contribute to the inflammatory milieu in active MS lesions by activating innate immune responses, e.g., IFNα production. Unraveling the underlying mechanisms may help in uncovering causal pathways and developing better treatment strategies for MS and other neuroinflammatory diseases.

The nucleotide polymorphisms within the Epstein–Barr virus C and Q promoters from nasopharyngeal carcinoma affect transcriptional activity in vitro

Epstein-Barr virus (EBV) is a ubiquitous human gamma herpesvirus that is associated with Burkitt's lymphoma (BL), gastric carcinoma, nasopharyngeal carcinoma (NPC), and NK/T-cell lymphoma. Two viral promoters, Cp and Qp, are important for EBV latent infection. The latency Cp, which is used in primary infection, drives expression of the full spectrum of EBV nuclear antigens. Qp is active in EBV-associated tumors and drives the latency I/II expression pattern. In this study, we determined nucleotides polymorphisms in the Cp and Qp promoter regions in peripheral blood mononuclear cells (PBMCs) from Cantonese healthy carriers and in biopsies of NPC, nasal NK/T lymphoma, BL, and gastric carcinoma. The sequence changes of -12G>T and +69 C>T in Cp and -197 G>A and +1 G>C in Qp were frequently identified in NPC. Transient transfection studies using luciferase gene reporters revealed a significant reduction (57.11%) in gene expression from the Cp +69T variant and increased expression (43.5%) from the Qp +1C variant compared to the prototype, suggesting that these sequence variations affect promoter activity. Our results indicate that the nucleotides polymorphisms in Cp and Qp occur frequently in NPC and might contribute to the oncogenesis of EBV.

Anti-N-methyl-D-aspartate receptor encephalitis with serum anti-thyroid antibodies and IgM antibodies against Epstein-Barr virus viral capsid antigen: a case report and one year follow-up

BackgroundAnti-N-methyl-D-aspartate receptor encephalitis is an increasingly common autoimmune disorder mediated by antibodies to certain subunit of the N-methyl-D-aspartate receptor. Recent literatures have described anti-thyroid and infectious serology in this encephalitis but without follow-up.Case presentationA 17-year-old Chinese female patient presented with psychiatric symptoms, memory deficits, behavioral problems and seizures. She then progressed through unresponsiveness, dyskinesias, autonomic instability and central hypoventilation during treatment. Her conventional blood work on admission showed high titers of IgG antibodies to thyroglobulin, thyroid peroxidase and IgM antibodies to Epstein-Barr virus viral capsid antigen. An immature ovarian teratoma was found and removal of the tumor resulted in a full recovery. The final diagnosis of anti-N-methyl-D-aspartate receptor encephalitis was made by the identification of anti-N-methyl-D-aspartate receptor antibodies in her cerebral spinal fluid. Pathology studies of the teratoma revealed N-methyl-D-aspartate receptor subunit 1 positive ectopic immature nervous tissue and Epstein-Barr virus latent infection. She was discharged with symptoms free, but titers of anti-thyroid peroxidase and anti-thyroglobulin antibodies remained elevated. One year after discharge, her serum remained positive for anti-thyroid peroxidase and anti-N-methyl-D-aspartate receptor antibodies, but negative for anti-thyroglobulin antibodies and IgM against Epstein-Barr virus viral capsid antigen.ConclusionsPersistent high titers of anti-thyroid peroxidase antibodies from admission to discharge and until one year later in this patient may suggest a propensity to autoimmunity in anti- N-methyl-D-aspartate receptor encephalitis and support the idea that neuronal and thyroid autoimmunities represent a pathogenic spectrum. Enduring anti-N-methyl-D-aspartate receptor antibodies from admission to one year follow-up but seroreversion of Epstein-Barr virus viral capsid antigen IgM may raise the important issue of elucidating the triggers and boosters of anti- N-methyl-D-aspartate receptor encephalitis.