EBV-encoded Nuclear Antigen Research Articles

Herpesviruses and the microbiome

The focus of this article will be to examine the role of common herpesviruses as a component of the microbiome of atopic patients and to review clinical observations suggesting that atopic patients might be predisposed to more severe and atypical herpes-related illness because their immune response is biased toward a TH2 cytokine profile. Human populations are infected with 8 herpesviruses, including herpes simplex virus HSV1 and HSV2 (also termed HHV1 and HHV2), varicella zoster virus (VZV or HHV3), EBV (HHV4), cytomegalovirus (HHV5), HHV6, HHV7, and Kaposi sarcoma-associated herpesvirus (termed KSV or HHV8). Herpesviruses are highly adapted to lifelong infection of their human hosts and thus can be considered a component of the human "microbiome" in addition to their role in illness triggered by primary infection. HSV1 and HSV2 infection and reactivation can present with more severe cutaneous symptoms termed eczema herpeticum in the atopic population, similar to the more severe eczema vaccinatum, and drug reaction with eosinophilia and systemic symptoms syndrome (DRESS) is associated with reactivation of HSV6 and possibly other herpesviruses in both atopic and nonatopic patients. In this review evidence is reviewed that primary infection with herpesviruses may have an atypical presentation in the atopic patient and conversely that childhood infection might alter the atopic phenotype. Reactivation of latent herpesviruses can directly alter host cytokine profiles through viral expression of cytokine-like proteins, such as IL-10 (EBV) or IL-6 (cytomegalovirus and HHV8), viral encoded and secreted siRNA and microRNAs, and modulation of expression of host transcription pathways, such as nuclear factor κB. Physicians caring for allergic and atopic populations should be aware of common and uncommon presentations of herpes-related disease in atopic patients to provide accurate diagnosis and avoid unnecessary laboratory testing or incorrect diagnosis of other conditions, such as drug allergy or autoimmune disease. Antiviral therapy and vaccines should be administered promptly when indicated clinically.

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

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

Epstein-Barr Virus Immortalization of Human B-Cells Leads to Stabilization of Hypoxia-Induced Factor 1 Alpha, Congruent with the Warburg Effect

BackgroundEpstein-Barr virus (EBV) encodes six nuclear transformation-associated proteins that induce extensive changes in cellular gene expression and signaling and induce B-cell transformation. The role of HIF1A in EBV-induced B-cell immortalization has not been previously studied.Methods and FindingsUsing Western blotting and Q-PCR, we found that HIF1A protein is stabilized in EBV-transformed lymphoblastoid cells. Western blotting, GST pulldown assays, and immunoprecipitation showed that EBV-encoded nuclear antigens EBNA-5 and EBNA-3 bind to prolylhydroxylases 1 and 2, respectively, thus inhibiting HIF1A hydroxylation and degradation. Immunostaining and Q-PCR showed that the stabilized HIF1A translocates to the nucleus, forms a heterodimer with ARNT, and transactivates several genes involved in aerobic glycolysis. Using biochemical assays and Q-PCR, we also found that lymphoblastoid cells produce high levels of lactate, lactate dehydrogenase and pyruvate.ConclusionsOur data suggest that activation of the aerobic glycolytic pathway, corresponding to the Warburg effect, occurs in EBV-transformed lymphoblastoid cells, in contrast to mitogen-activated B-cells.

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.

EBNA3B-deficient EBV promotes B cell lymphomagenesis in humanized mice and is found in human tumors.

Epstein-Barr virus (EBV) persistently infects more than 90% of the human population and is etiologically linked to several B cell malignancies, including Burkitt lymphoma (BL), Hodgkin lymphoma (HL), and diffuse large B cell lymphoma (DLBCL). Despite its growth transforming properties, most immune-competent individuals control EBV infection throughout their lives. EBV encodes various oncogenes, and of the 6 latency-associated EBV-encoded nuclear antigens, only EBNA3B is completely dispensable for B cell transformation in vitro. Here, we report that infection with EBV lacking EBNA3B leads to aggressive, immune-evading monomorphic DLBCL-like tumors in NOD/SCID/γc-/- mice with reconstituted human immune system components. Infection with EBNA3B-knockout EBV (EBNA3BKO) induced expansion of EBV-specific T cells that failed to infiltrate the tumors. EBNA3BKO-infected B cells expanded more rapidly and secreted less T cell-chemoattractant CXCL10, reducing T cell recruitment in vitro and T cell-mediated killing in vivo. B cell lines from 2 EBV-positive human lymphomas encoding truncated EBNA3B exhibited gene expression profiles and phenotypic characteristics similar to those of tumor-derived lines from the humanized mice, including reduced CXCL10 secretion. Screening EBV-positive DLBCL, HL, and BL human samples identified additional EBNA3B mutations. Thus, EBNA3B is a virus-encoded tumor suppressor whose inactivation promotes immune evasion and virus-driven lymphomagenesis.

Analysis of protein–protein interactions in a complex environment: capture of an analyte–receptor complex with standard additions of the receptor (CARSAR) approach

Traditional methods of analytical chemistry to detect an interaction between certain proteins in multicomponent mixtures (e.g. cell lysates, etc.) have limitations. This is due to difficulties in identification of a specific signal of an analyte (a molecule to be detected) against the background. In the present work, we propose the new analytical protocol for transducer-based sensors with a restricted sensitive area. It uses a combination of analyte-receptor complex precipitation with serial additions of the receptor (CARSAR). To test this new analytical strategy, we used a surface plasmon resonance technique to confirm an interaction between the Epstein-Barr virus-encoded nuclear antigen 6 and the mitochondrial ribosomal protein MRPS18-2.

The Replisome Pausing Factor Timeless Is Required for Episomal Maintenance of Latent Epstein-Barr Virus

The Epstein-Barr virus (EBV) genome is maintained as an extrachromosomal episome during latent infection of B lymphocytes. Episomal maintenance is conferred by the interaction of the EBV-encoded nuclear antigen 1 (EBNA1) with a tandem array of high-affinity binding sites, referred to as the family of repeats (FR), located within the viral origin of plasmid replication (OriP). How this nucleoprotein array confers episomal maintenance is not completely understood. Previous studies have shown that DNA replication forks pause and terminate with high frequency at OriP. We now show that cellular DNA replication fork pausing and protection factors Timeless (Tim) and Tipin (Timeless-interacting protein) accumulate at OriP during S phase of the cell cycle. Depletion of Tim inhibits OriP-dependent DNA replication and causes a complete loss of the closed-circular form of EBV episomes in latently infected B lymphocytes. Tim depletion also led to the accumulation of double-strand breaks at the OriP region. These findings demonstrate that Tim is essential for sustaining the episomal forms of EBV DNA in latently infected cells and suggest that DNA replication fork protection is integrally linked to the mechanism of plasmid maintenance.

Epstein-Barr Virus and Breast Cancer: Lack of Evidence for an Association in Iranian Women

Controversies regarding the role of Epstein-Barr virus (EBV) in breast cancer and lack of published literature in this regard in Iran, prompted us to assess EBV presence in 100 breast carcinoma and 42 control biopsies obtained from Iranian women. Breast carcinoma cases were comprised of 81 invasive ductal carcinoma NOS, 9 invasive lobular carcinoma, 1 apocrine carcinoma, 2 cribriform carcinoma, 2 papillary carcinoma and 5 mucinous carcinoma. Control biopsies consisted of 13 fibroadenoma, 9 benign epithelial proliferation (adenosis and sclerosing adenosis), 9 usual ductal hyperplasia, 4 atypical ductal hyperplasia, 4 non-proliferative fibrocystic changes and 3 normal breast tissue. To identify EBV-infected cells we applied immunohistochemical analysis, using monoclonal antibody against Epstein-Barr virus-encoded nuclear antigen 2 (EBNA-2) and latent membrane protein 1 (LMP-1). Further, polymerase chain reaction (PCR) was used to amplify EBV DNA, with primers that cover the EBV encoded RNA (EBER) and BamHIW regions. EBNA-2 and LMP-1 immunohistochemistry were negative in all breast cancer and control specimens. Using PCR, none of the 100 breast cancer samples or the 42 control specimens showed detectable EBV DNA. These results indicate that EBV may not play a significant role in the etiology of breast cancer in Iranian women.

Increased Peripheral T Cell Responses to EBV-Infected Cells with Frequent Detection of EBV-DNA In Plasma and Viral mRNA In Peripheral B-Cells In Immunocompetent EBV-Positive Diffuse Large B-Cell Lymphoma Patients

AbstractAbstract 4163 Introduction:It is well known that immunocompromised patients(pts) such as post-transplantation or HIV infection are at increased risk of EBV-associated B-cell lymphoproliferaitive disorders. However, the immunological status of immunocompetent EBV-positive diffuse large B-cell Lymphoma (DLBCL) has not been well elucidated. For example, EBV-positive DLBCL in the elderly is defined as an EBV-positive clonal B-cell lymphoid proliferation occurring in pts more than 50 years of age and without any known immunodeficiency in WHO classification 2008, accounting for 5 – 10% of DLBCL in Japan. This disease is speculated to be related to the immunological deterioration accompanying the aging process. We conducted a multi-center prospective study to assess EBV status and the T cell response to EBV of peripheral blood (PB) in EBV-positive DLBCL in comparison with EBV-negative DLBCL pts and normal old-healthy persons (old-HPs). Patients and Methods:Fifteen newly-diagnosed pts with EBV-positive DLBCL, 8 pts with EBV-negative DLBCL, and 16 old-HPs were enrolled. Median ages of pts with EBV-positive DLBCL, pts with EBV-negative DLBCL, and old-HPs were 74.5 years (range 29–82 years), 71 years (48-79), and 65 years (60-74), respectively. Among the 15 pts with EBV-positive DLBCL, 2 were pyothorax-associated lymphoma, 2 were treated with methotrexate for rheumatoid arthritis, and the remaining 11 pts were without past histories predisposing to immunodeficiency. The diagnosis of EBV-positive DLBCL was made by positive signals for in situ hybridization using EBV-encoded small RNA (EBER) on paraffin section. To analyze T cell reactivity to autologous EBV-infected B-cell lymphoblastoid cell lines (LCL), CFSE-labeled peripheral blood mononuclear cells (PBMCs) were co-cultured with irradiated autologous LCL, and the division indices (DI) of CD4+ and CD8+ T cells were determined on day 5 (Cytometry 34:143, 1998). Percentage of proliferating CFSElow IFN-γ+CD4+ T cells and CFSElow IFN-γ+CD8+ T cells was assessed on day 7. EBV DNA load in PBMCs and plasma was determined by quantitative real-time PCR. CD19+ B cells were separated from of PBMCs by immunomagnetic sorting, and viral mRNA expression of B cells was quantified by one-step multiplex real-time RT-PCR. Results:(1) Plasma EBV-DNA was detectable in 13 of the 15 EBV-positive DLBCL pts but in none of the 8 EBV-negative DLBCL pts or the 16 old-HPs. Copy number of EBV-positive DLBCL was significantly higher than EBV-negative DLBCL (p<0.00001) or old-HPs (p=0.0001). EBV-DNA in PBMC was positive in 10 of the 15 EBV-positive DLBCL pts, 2 of 8 EBV-negative DLBCL pts, and 2 of 15 old-HPs. (2) EBER1 of PB B cells was detected in all 10 EBV-positive DLBCL pts, 4 of 7 EBV-negative DLBCL pts, and 7 of 14 old-HPs. BamHI A rightward transcripts (BARTs) of B-cells was detected in 8 of 10 EBV-positive DLBCL pts, 3 of 10 EBV-negative DLBCL pts, and 2 of 14 old-HPs. Latent membrane protein 2 (LMP2) of B cells was detected in 2 of 10 EBV-positive DLBCL pts, 2 of 14 old-HPs, and none of 5 EBV-negative DLBCL pts. LMP1 of B cells was detected in 1 of 10 EBV-positive DLBCL pts, none of 7 EBV-negative DLBCL pts, and none of 14 old-HPs. EBV-encoded nuclear antigen 1 (EBNA1) or EBNA2 were not detected in any of the pts nor old-HPs. (3) DI of CD4+ T cells for 5 days in EBV-positive DLBCL (median 1.41) was significantly higher than in old-HPs (median 0.53) (p=0.0009), and DI of CD8+ T cells of EBV-positive DLBCL (median 1.94) showed a higher tendency than old-HP (median 1.35). (4) Percentage of CFSE-low IFN-γ+CD4+ T cells in EBV-positive DLBCL (median 9.8%) was significantly higher than in old-HPs (median 2.2%) (p=0.002), and percentage of CFSE-low IFN-γ+CD8+ T cells in EBV-positive DLBCL (median 12.0%) was significantly higher than in old-HPs (median 6.6%)(p=0.025). Conclusions:Measurement of the plasma EBV-DNA copy number is a good indicator for the diagnosis of EBV-positive DLBCL, and the frequent detection of EBV viral mRNA of peripheral B-cells in EBV-positive DLBCL pts might be associated with greater viral load. Proliferative and INF-γ secreting responses of both peripheral CD4+ T cells and CD8+ T cells to EBV-infected cells were increased in EBV-positive DLBCL compared to old-HPs, which might be driven by an elevated viral load. These findings suggest that systemic immunological reaction to EBV might be intact in EBV-positive DLBCL. Disclosures:No relevant conflicts of interest to declare.

Epstein-Barr virus-encoded EBNA1 enhances RNA polymerase III-dependent EBER expression through induction of EBER-associated cellular transcription factors

BackgroundEpstein-Barr Virus (EBV)-encoded RNAs (EBERs) are non-polyadenylated RNA molecules transcribed from the EBV genome by RNA polymerase III (pol III). EBERs are the most abundant viral latent gene products, although the precise mechanisms by which EBV is able to achieve such high levels of EBER expression are not fully understood. Previously EBV has been demonstrated to induce transcription factors associated with EBER expression, including pol III transcription factors and ATF-2. We have recently demonstrated that EBV-encoded nuclear antigen-1 (EBNA1) induces cellular transcription factors, and given these findings, we investigated the role of EBNA1 in induction of EBER-associated transcription factors.ResultsOur data confirm that in epithelial cells EBNA1 can enhance cellular pol III transcription. Transient expression of EBNA1 in Ad/AH cells stably expressing the EBERs led to induction of both EBER1 and EBER2 and conversely, expression of a dominant negative EBNA1 led to reduced EBER expression in EBV-infected Ad/AH cells. EBNA1 can induce transcription factors used by EBER genes, including TFIIIC, ATF-2 and c-Myc. A variant chromatin precipitation procedure showed that EBNA1 is associated with the promoters of these genes but not with the promoters of pol III-transcribed genes, including the EBERs themselves. Using shRNA knock-down, we confirm the significance of both ATF-2 and c-Myc in EBER expression. Further, functional induction of a c-Myc fusion protein led to increased EBER expression, providing c-Myc binding sites upstream of EBER1 were intact. In vivo studies confirm elevated levels of the 102 kD subunit of TFIIIC in the tumour cells of EBV-positive nasopharyngeal carcinoma biopsies.ConclusionsOur findings reveal that EBNA1 is able to enhance EBER expression through induction of cellular transcription factors and add to the repertoire of EBNA1's transcription-regulatory properties.

Epstein-Barr virus (EBV) genotypes among human immunodeficiency virus (HIV)-related B-cell Lymphomas and B-cell post-transplant lymphoproliferative disorders (B-PTLD) - late-onset lymphomas, especially in the HIV setting, are associated with type-B-EBV

We investigated 26 B-cell post-transplant lymphoproliferative disorders (B-PTLD) and 15 human immunodeficiency virus-related aggressive B-cell lymphomas (HIV-BCL) from England that were associated with Epstein-Barr virus (EBV) for the polymorphic sequences of the EBV-encoded nuclear antigen 3C (EBNA3C) gene to distinguish the two different EBV strains. Type-A-EBV was identified in 92% of B-PTLDS and in 53% of HIV-BCL (P = 0.003). Among HIV-BCL, patients associated with type-B-EBV had been HIV positive for significantly longer when compared to those associated with type-A (P = 0.037) although there were no correlations with ethnicity, CD4 cell counts or plasma HIV viral load.

The enhanced transcriptional activity of the V-val subtype of Epstein-Barr virus nuclear antigen 1 in epithelial cell lines

The Epstein-Barr virus encoded nuclear antigen 1 (EBNA1) is required for the replication and maintenance of the episomal EBV genome and for the transactivation of viral gene expression. EBNA1 has been classified into five subtypes, among which the V-val subtype was reported to be associated with nasopharyngeal carcinoma (NPC). Here we report a higher transcriptional activity of the V-val subtype of EBNA1 than for the prototype derived from B95.8 cells to transactivate FR-containing luciferase plasmid, which was mainly a consequence of the mutations in the carboxy-terminus of EBNA1. This interpretation was further supported by the finding that the variant form of EBNA1 has a higher binding affinity for the FR sequence than the prototype by electrophoretic mobility shift assays. The functional advantage of the V-val EBNA1 investigated in this study may contribute to the oncogenesis of NPC.

Intrathecal immune responses to EBV in early MS

EBV has been consistently associated with MS, but its signature in the CNS has rarely been examined. In this study, we assessed EBV-specific humoral and cellular immune responses in the cerebrospinal fluid (CSF) of patients with early MS, other inflammatory neurological diseases (OIND) and non-inflammatory neurological diseases (NIND). The neurotropic herpesvirus CMV served as a control. Virus-specific humoral immune responses were assessed in 123 consecutive patients and the intrathecal recruitment of virus-specific antibodies was expressed as antibody indexes. Cellular immune responses tested in the blood of 55/123 patients were positive in 46/55. The CD8(+) CTL responses of these 46 patients were assessed in the blood and CSF using a CFSE-based CTL assay. We found that viral capsid antigen and EBV-encoded nuclear antigen-1, but not CMV IgG antibody indexes, were increased in early MS as compared with OIND and NIND patients. There was also intrathecal enrichment in EBV-, but not CMV-specific, CD8(+) CTL in early MS patients. By contrast, OIND and NIND patients did not recruit EBV- nor CMV-specific CD8(+) CTL in the CSF. Our data, showing a high EBV-, but not CMV-specific intrathecal immune response, strengthen the association between EBV and MS, in particular at the onset of the disease.

Elevated Epstein–Barr virus‐encoded nuclear antigen‐1 immune responses predict conversion to multiple sclerosis

The aims of the study were to determine the immune responses to candidate viral triggers of multiple sclerosis (MS) in patients with clinically isolated syndromes (CISs), and to evaluate their potential value in predicting conversion to MS. Immune responses to Epstein-Barr virus (EBV), human herpesvirus 6, cytomegalovirus (HCMV), and measles were determined in a cohort of 147 CIS patients with a mean follow-up of 7 years and compared with 50 demographically matched controls. Compared with controls, CIS patients showed increased humoral (p < 0.0001) and cellular (p = 0.007) immune responses to the EBV-encoded nuclear antigen-1 (EBNA1), but not to other EBV-derived proteins. Immunoglobulin G (IgG) responses to other virus antigens and frequencies of T cells specific for HCMV and influenza virus gene products were unchanged in CIS patients. EBNA1 was the only viral antigen with which immune responses correlated with number of T2 lesions (p = 0.006) and number of Barkhof criteria (p=0.001) at baseline, and with number of T2 lesions (p = 0.012 at both 1 and 5 years), presence of new T2 lesions (p = 0.003 and p = 0.028 at 1 and 5 years), and Expanded Disability Status Scale score (p = 0.015 and p = 0.010 at 1 and 5 years) during follow-up. In a univariate Cox regression model, increased EBNA1-specific IgG responses predicted conversion to MS based on McDonald criteria (hazard ratio [95% confidence interval], 2.2 [1.2-4.3]; p = 0.003). Our results indicate that elevated immune responses toward EBNA1 are selectively increased in CIS patients and suggest that EBNA1-specific IgG titers could be used as a prognostic marker for disease conversion and disability progression.

The Synthesis of Truncated Polypeptides for Immune Surveillance and Viral Evasion

BackgroundCytotoxic T cells detect intracellular pathogens by surveying peptide loaded MHC class I molecules (pMHC I) on the cell surface. Effective immune surveillance also requires infected cells to present pMHC I promptly before viral progeny can escape. Rapid pMHC I presentation apparently occurs because infected cells can synthesize and present peptides from antigenic precursors called defective ribosomal products (DRiPs). The molecular characteristics of DRiPs are not known.Methodology/Principal FindingsHere, using a novel method for detecting antigenic precursors and proteolytic intermediates, we tracked the synthesis and processing of Epstein-Barr Virus encoded nuclear antigen 1 (EBNA1). We find that ribosomes initiated translation appropriately, but rapidly produced DRiPs representing ∼120 amino acid truncated EBNA1 polypeptides by premature termination. Moreover, specific sequences in EBNA1 mRNA strongly inhibited the generation of truncated DRiPs and pMHC I presentation.SignificanceOur results reveal the first characterization of virus DRiPs as truncated translation products. Furthermore, production of EBNA1-derived DRiPs is down-regulated in cells, possibly limiting the antigenicity of EBNA1.

Notch1, Notch2, and Epstein-Barr virus–encoded nuclear antigen 2 signaling differentially affects proliferation and survival of Epstein-Barr virus–infected B cells

AbstractThe canonical mode of transcriptional activation by both the Epstein-Barr viral protein, Epstein-Barr virus–encoded nuclear antigen 2 (EBNA2), and an activated Notch receptor (Notch-IC) requires their recruitment to RBPJ, suggesting that EBNA2 uses the Notch pathway to achieve B-cell immortalization. To gain further insight into the biologic equivalence between Notch-IC and EBNA2, we performed a genome-wide expression analysis, revealing that Notch-IC and EBNA2 exhibit profound differences in the regulation of target genes. Whereas Notch-IC is more potent in regulating genes associated with differentiation and development, EBNA2 is more potent in inducing viral and cellular genes involved in proliferation, survival, and chemotaxis. Because both EBNA2 and Notch-IC induced the expression of cell cycle–associated genes, we analyzed whether Notch1-IC or Notch2-IC can replace EBNA2 in B-cell immortalization. Although Notch-IC could drive quiescent B cells into the cell cycle, B-cell immortalization was not maintained, partially due to an increased apoptosis rate in Notch-IC–expressing cells. Expression analysis revealed that both EBNA2 and Notch-IC induced the expression of proapoptotic genes, but only in EBNA2-expressing cells were antiapoptotic genes strongly up-regulated. These findings suggest that Notch signaling in B cells and B-cell lymphomas is only compatible with proliferation if pathways leading to antiapototic signals are active.

Nucleoside diphosphate kinase/Nm23 and Epstein-Barr virus.

Nm23-H1 was discovered as the first metastasis suppressor gene about 20 years ago. Since then, extensive work has contributed to understanding its role in various cellular signaling pathways. Its association with a range of human cancers as well as its ability to regulate cell cycle and suppress metastasis has been explored. We have determined that the EBV-encoded nuclear antigens, EBNA3C and EBNA1, required for EBV-mediated lymphoproliferation and for maintenance EBV genome extrachromosomally in dividing mammalian cells, respectively, target and disrupt the physiological role of Nm23-H1 in the context of cell proliferation and cell migration. This review will focus on the interaction of Nm23-H1 with the Epstein-Barr virus nuclear antigens, EBNA3C and EBNA1 and the functional significance of this interaction as it relates to EBV pathogenesis.

Lessons from genome-scans – the example of lung cancer

Epstein–Barr virus (EBV) is a human lymphotropic gamma herpesvirus. It is ubiquitous in all human populations. Like other herpesviruses, its productive cycle leads to death of its host cell. Most frequently, however, the virus enters a non-lytic interaction with its main target, the B lymphocyte. This is associated with blast transformation and immortalization. The derived lymphoblastic cell lines (LCLs) are driven by the virus. They do not produce the lytic viral proteins except occasionally in a small minority of the cells. The immortalized cells express nine virally encoded proteins, six EBV-encoded nuclear antigens (EBNAs), and three latent membrane proteins (LMPs). The text summarizes the viral promoter usage, the protein expression patterns in the transformed cells and their phenotypic effects. This is followed by a brief summary of the main immune responses and the disease associations of the virus.

Identification of Intracellular Proteins Associated with the EBV-Encoded Nuclear Antigen 5 Using an Efficient TAP Procedure and FT-ICR Mass Spectrometry

Epstein-Barr virus nuclear antigen 5 (EBNA5) is one of the first viral proteins detected after primary EBV infection and has been shown to be required for efficient transformation of B lymphocytes. EBNA5 is a protein that has many suggested functions but the underlying biology remains to be clarified. To gain further insight into the biological roles of the proposed multifunctional EBNA5, we isolated EBNA5 containing protein complexes using a modified tandem affinity purification (TAP) method and identified the protein components by LC-MS/MS analysis of tryptic digests on a LTQ-FT-ICR mass spectrometer. The modified TAP tag contained a Protein A domain and a StrepTagII sequence separated by two Tobacco Etch Virus protease cleavage sites and was fused to the C-terminus of EBNA5. Our results confirmed the wide applicability of this two-step affinity purification strategy for purification of protein complexes in mammalian cells. A total of 147 novel putative EBNA5 interaction partners were identified, 37 of which were validated with LC-MS/MS in split-tag experiments or in co-immuno precipitates from HEK293 cell extracts. This subgroup included the Bcl2-associated Athanogene 2 (BAG2) co-chaperone involved in protein folding and renaturation, the 26S proteasome subunit 2 involved in regulation of ubiquitin/proteasome protein degradation, and the heterogeneous ribonucleoprotein M (hnRNP M) involved in pre-mRNA processing. These EBNA5 interactors were further verified by co-immunoprecipitations from cell extracts of three EBV-positive lymphoblastoid lines. The combination of the Hsp70, Hsc70, BAG2 and 26S proteasome subunit 2 interactors suggests that EBNA5 might have a functional relationship with protein quality control systems that recognize proteins with abnormal structures and either refold them to normal conformation or target them for degradation. Our study also confirms previously identified interactors including HA95, Hsp70, Hsc70, Hsp27, HAX-1, Prolyl 4-hydroxylase, S3a, and alpha- and beta-tubulin.

EBV-encoded EBNA-6 binds and targets MRS18-2 to the nucleus, resulting in the disruption of pRb-E2F1 complexes

Epstein-Barr virus (EBV), like other DNA tumor viruses, induces an S-phase in the natural host cell, the human B lymphocyte. This is linked with blast transformation. It is believed that the EBV-encoded nuclear antigen 6 (EBNA-6) is involved in the regulation of cell cycle entry. However, the possible mechanism of this regulation is not approached. In our current study, we found that EBNA-6 binds to a MRPS18-2 protein, and targets it to the nucleus. We found that MRPS18-2 binds to both hypo- and hyperphosphorylated forms of Rb protein specifically. This binding targets the small pocket of pRb, which is a site of interaction with E2F1. The MRPS18-2 competes with the binding of E2F1 to pRb, thereby raising the level of free E2F1. Our experimental data suggest that EBNA-6 may play a major role in the entry of EBV infected B cells into the S phase by binding to and raising the level of nuclear MRPS18-2, protein. This would inhibit pRb binding to E2F1 competitively and lift the block preventing S-phase entry.