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Epstein-Barr virus (EBV) infection of B cells is associated with increased multiple sclerosis (MS) susceptibility. Recently, we found that CXCR3-expressing B cells preferentially infiltrate the central nervous system of MS patients. In chronic virus-infected mice, these types of B cells are sustained and show increased antiviral responsiveness. How EBV persistence in B cells influences their development remains unclear. First, we analyzed ex vivo B-cell subsets from MS patients who received autologous bone marrow transplantation (n=9), which is often accompanied by EBV reactivation. The frequencies of non-class-switched and class-switched memory B cells were reduced at 3-7 months, while only class-switched B cells returned back to baseline at 24-36 months post-transplantation. At these time points, EBV DNA load positively correlated to the frequency of CXCR3+ , and not CXCR4+ or CXCR5+ , class-switched B cells. Second, for CXCR3+ memory B cells trapped within the blood of MS patients treated with natalizumab (anti-VLA-4 antibody n=15), latent EBV infection corresponded to enhanced in vitro formation of anti-EBNA1 IgG-secreting plasma cells under germinal center-like conditions. These findings imply that EBV persistence in B cells potentiates brain-homing and antibody-producing CXCR3+ subsets in MS. This article is protected by copyright. All rights reserved.

Epstein-Barr virus (EBV)–associated T- and natural killer (NK)–cell malignancies, such as extranodal NK-/T-cell lymphoma (ENKTL), exhibit high chemoresistance and, accordingly, such patients have a poor prognosis. The rare nature of such cancers and nonmalignant T/NK lymphoproliferative disorders, such as chronic active EBV (CAEBV), has limited our understanding of the pathogenesis of these diseases. Here, we characterize a panel of ENKTL- and CAEBV-derived cell lines that had been established from human tumors to be used as preclinical models of these diseases. These cell lines were interleukin-2 dependent and found to carry EBV in a latency II gene-expression pattern. All cell lines demonstrated resistance to cell death induction by DNA damage–inducing agents, the current standard of care for patients with these malignancies. This resistance was not correlated with the function of the multidrug efflux pump, P-glycoprotein. However, apoptotic cell death could be consistently induced following treatment with A-1331852, a BH3-mimetic drug that specifically inhibits the prosurvival protein BCL-XL. A-1331852–induced apoptosis was most efficacious when prosurvival MCL-1 was additionally targeted, either by BH3-mimetics or genetic deletion. Xenograft models established from the ENKTL cell line SNK6 provided evidence that A-1331852 treatment could be therapeutically beneficial in vivo. The data here suggest that therapeutic targeting of BCL-XL would be effective for patients with EBV-driven T/NK proliferative diseases, however, MCL-1 could be a potential resistance factor.

Epstein–Barr virus (EBV), which is ubiquitous in the adult population, is causally associated with human malignancies. Like many infectious agents, EBV has evolved strategies to block host cell death, including through expression of viral homologues of cellular BCL-2 pro-survival proteins (vBCL-2s), such as BHRF1. Small molecule inhibitors of the cellular pro-survival BCL-2 family proteins, termed ‘BH3-mimetics’, have entered clinical trials for blood cancers with the BCL-2 inhibitor venetoclax already approved for treatment of therapy refractory chronic lymphocytic leukaemia and acute myeloid leukaemia in the elderly. The generation of BH3-mimetics that could specifically target vBCL-2 proteins may be an attractive therapeutic option for virus-associated cancers, since these drugs would be expected to only kill virally infected cells with only minimal side effects on normal healthy tissues. To achieve this, a better understanding of the contribution of vBCL-2 proteins to tumorigenesis and insights into their biochemical functions is needed. In the context of Burkitt lymphoma (BL), BHRF1 expression conferred strong resistance to diverse apoptotic stimuli. Furthermore, BHRF1 expression in mouse haematopoietic stem and progenitor cells accelerated MYC-induced lymphoma development in a model of BL. BHRF1 interacts with the cellular pro-apoptotic BCL-2 proteins, BIM, BID, PUMA and BAK, but its capability to inhibit apoptosis could not be mapped solely to one of these interactions, suggesting plasticity is a key feature of BHRF1. Site-directed mutagenesis revealed a site in BHRF1 that was critical for its interaction with PUMA and blocking DNA-damage-induced apoptosis, identifying a potentially therapeutically targetable vulnerability in BHRF1.

The EBV is known to persist in memory B cells, but it remains unclear how this affects cell numbers and humoral immunity. We here studied EBV persistence in memory B cell subsets and consequences on B cell memory in young children. EBV genome loads were quantified in 6 memory B cell subsets in EBV+ adults. The effects of EBV infection on memory B cell numbers and vaccination responses were studied longitudinally in children within the Generation R population cohort between 14 mo and 6 yr of age. EBV genomes were more numerous in CD27+IgG+, CD27+IgA+, and CD27−IgA+ memory B cells than in IgM-only, natural effector, and CD27−IgG+ B cells. The blood counts of IgM-only, CD27+IgA+, CD27−IgG+, and CD27+IgG+ memory B cells were significantly lower in EBV+ children than in uninfected controls at 14 mo of age—the age when these cells peak in numbers. At 6 yr, all of these memory B cell counts had normalized, as had plasma IgG levels to previous primary measles and booster tetanus vaccinations. In conclusion, EBV persists predominantly in Ig class-switched memory B cells, even when derived from T cell-independent responses (CD27−IgA+), and EBV infection results in a transient depletion of these cells in young children.

Kaposi's sarcoma-associated herpesvirus (KSHV) has tropism for B lymphocytes, in which it establishes latency, and can also cause lymphoproliferative disorders of these cells manifesting as primary effusion lymphoma (PEL) and multicentric Castleman disease (MCD). T cell immunity is vital for the control of KSHV infection and disease; however, few models of B lymphocyte infection exist to study immune recognition of such cells. Here, we developed a model of B lymphocyte infection with KSHV in which infected tonsillar B lymphocytes were expanded by providing mitogenic stimuli and then challenged with KSHV-specific CD4 + T cells. The infected cells expressed viral proteins found in PELs, namely, LANA and viral IRF3 (vIRF3), albeit at lower levels, with similar patterns of gene expression for the major latency, viral interleukin 6 (vIL-6), and vIRF3 transcripts. Despite low-level expression of open reading frame 50 (ORF50), transcripts for the immune evasion genes K3 and K5 were detected, with some downregulation of cell surface-expressed CD86 and ICAM. The vast majority of infected lymphocytes expressed IgM heavy chains with Igλ light chains, recapitulating the features seen in infected cells in MCD. We assessed the ability of the infected lymphocytes to be targeted by a panel of major histocompatibility complex (MHC) class II-matched CD4 + T cells and found that LANA-specific T cells restricted to different epitopes recognized these infected cells. Given that at least some KSHV latent antigens are thought to be poor targets for CD8 + T cells, we suggest that CD4 + T cells are potentially important effectors for the in vivo control of KSHV-infected B lymphocytes. IMPORTANCE KSHV establishes a latent reservoir within B lymphocytes, but few models exist to study KSHV-infected B cells other than the transformed PEL cell lines, which have likely accrued mutations during the transformation process. We developed a model of KSHV-infected primary B lymphocytes that recapitulates features seen in PEL and MCD by gene expression and cell phenotype analysis, allowing the study of T cell recognition of these cells. Challenge of KSHV-infected B cells with CD4 + T cells specific for LANA, a protein expressed in all KSHV-infected cells and malignancies in vivo , showed that these effectors could efficiently recognize such targets. Given that the virus expresses immune evasion genes or uses proteins with intrinsic properties, such as LANA, that minimize epitope recognition by CD8 + T cells, CD4 + T cell immunity to KSHV may be important for maintaining the virus-host balance.

BACKGROUND: Oesophageal adenocarcinoma is the sixth most common cause of cancer death worldwide and Barrett's oesophagus is the biggest risk factor. We aimed to evaluate the efficacy of high-dose esomeprazole proton-pump inhibitor (PPI) and aspirin for improving outcomes in patients with Barrett's oesophagus.METHODS: The Aspirin and Esomeprazole Chemoprevention in Barrett's metaplasia Trial had a 2 × 2 factorial design and was done at 84 centres in the UK and one in Canada. Patients with Barrett's oesophagus of 1 cm or more were randomised 1:1:1:1 using a computer-generated schedule held in a central trials unit to receive high-dose (40 mg twice-daily) or low-dose (20 mg once-daily) PPI, with or without aspirin (300 mg per day in the UK, 325 mg per day in Canada) for at least 8 years, in an unblinded manner. Reporting pathologists were masked to treatment allocation. The primary composite endpoint was time to all-cause mortality, oesophageal adenocarcinoma, or high-grade dysplasia, which was analysed with accelerated failure time modelling adjusted for minimisation factors (age, Barrett's oesophagus length, intestinal metaplasia) in all patients in the intention-to-treat population. This trial is registered with EudraCT, number 2004-003836-77.FINDINGS: Between March 10, 2005, and March 1, 2009, 2557 patients were recruited. 705 patients were assigned to low-dose PPI and no aspirin, 704 to high-dose PPI and no aspirin, 571 to low-dose PPI and aspirin, and 577 to high-dose PPI and aspirin. Median follow-up and treatment duration was 8·9 years (IQR 8·2-9·8), and we collected 20 095 follow-up years and 99·9% of planned data. 313 primary events occurred. High-dose PPI (139 events in 1270 patients) was superior to low-dose PPI (174 events in 1265 patients; time ratio [TR] 1·27, 95% CI 1·01-1·58, p=0·038). Aspirin (127 events in 1138 patients) was not significantly better than no aspirin (154 events in 1142 patients; TR 1·24, 0·98-1·57, p=0·068). If patients using non-steroidal anti-inflammatory drugs were censored at the time of first use, aspirin was significantly better than no aspirin (TR 1·29, 1·01-1·66, p=0·043; n=2236). Combining high-dose PPI with aspirin had the strongest effect compared with low-dose PPI without aspirin (TR 1·59, 1·14-2·23, p=0·0068). The numbers needed to treat were 34 for PPI and 43 for aspirin. Only 28 (1%) participants reported study-treatment-related serious adverse events.INTERPRETATION: High-dose PPI and aspirin chemoprevention therapy, especially in combination, significantly and safely improved outcomes in patients with Barrett's oesophagus.FUNDING: Cancer Research UK, AstraZeneca, Wellcome Trust, and Health Technology Assessment.

Epstein-Barr virus (EBV) infection of B cells leads to the sequential activation of two viral promoters, Wp and Cp, resulting in the expression of six EBV nuclear antigens (EBNAs) and the viral Bcl2 homologue BHRF1. The viral transactivator EBNA2 is required for this switch from Wp to Cp usage during the initial stages of infection. EBNA2-dependent Cp transcription is mediated by the EBNA2 response element (E2RE), a region that contains at least two binding sites for cellular factors; one of these sites, CBF1, interacts with RBP-JK, which then recruits EBNA2 to the transcription initiation complex. Here we demonstrate that the B cell-specific transcription factor BSAP/Pax5 binds to a second site, CBF2, in the E2RE. Deletion of the E2RE in the context of a recombinant virus greatly diminished levels of Cp-initiated transcripts during the initial stages of infection but did not affect the levels of Wp-initiated transcripts or EBNA mRNAs. Consistent with this finding, viruses deleted for the E2RE were not markedly impaired in their ability to induce B cell transformation in vitro . In contrast, a larger deletion of the entire Cp region did reduce EBNA mRNA levels early after infection and subsequently almost completely ablated lymphoblastoid cell line (LCL) outgrowth. Notably, however, rare LCLs could be established following infection with Cp-deleted viruses, and these were indistinguishable from wild-type-derived LCLs in terms of steady-state EBV gene transcription. These data indicate that, unlike Wp, Cp is dispensable for the virus' growth-transforming activity. IMPORTANCE Epstein-Barr virus (EBV), a B lymphotropic herpesvirus etiologically linked to several B cell malignancies, efficiently induces B cell proliferation leading to the outgrowth of lymphoblastoid cell lines (LCLs). The initial stages of this growth-transforming infection are characterized by the sequential activation of two viral promoters, Wp and Cp, both of which appear to be preferentially active in target B cells. In this work, we have investigated the importance of Cp activity in initiating B cell proliferation and maintaining LCL growth. Using recombinant viruses, we demonstrate that while Cp is not essential for LCL outgrowth in vitro , it enhances transformation efficiency by >100-fold. We also show that Cp, like Wp, interacts with the B cell-specific activator protein BSAP/Pax5. We suggest that EBV has evolved this two-promoter system to ensure efficient colonization of the host B cell system in vivo .

Epstein–Barr virus (EBV), originally discovered through its association with Burkitt lymphoma, is now aetiologically linked to a remarkably wide range of lymphoproliferative lesions and malignant lymphomas of B-, T- and NK-cell origin. Some occur as rare accidents of virus persistence in the B lymphoid system, while others arise as a result of viral entry into unnatural target cells. The early finding that EBV is a potent B-cell growth transforming agent hinted at a simple oncogenic mechanism by which this virus could promote lymphomagenesis. In reality, the pathogenesis of EBV-associated lymphomas involves a complex interplay between different patterns of viral gene expression and cellular genetic changes. Here we review recent developments in our understanding of EBV-associated lymphomagenesis in both the immunocompetent and immunocompromised host. This article is part of the themed issue ‘Human oncogenic viruses’.

While the association of Epstein–Barr virus (EBV) with Burkitt lymphoma (BL) has long been recognised, the precise role of the virus in BL pathogenesis is not fully resolved. EBV can be lost spontaneously from some BL cell lines, and these EBV-loss lymphoma cells reportedly have a survival disadvantage. Here we have generated an extensive panel of EBV-loss clones from multiple BL backgrounds and examined their phenotype comparing them to their isogenic EBV-positive counterparts. We report that, while loss of EBV from BL cells is rare, it is consistently associated with an enhanced predisposition to undergo apoptosis and reduced tumorigenicity in vivo. Importantly, reinfection of EBV-loss clones with EBV, but surprisingly not transduction with individual BL-associated latent viral genes, restored protection from apoptosis. Expression profiling and functional analysis of apoptosis-related proteins and transcripts in BL cells revealed that EBV inhibits the upregulation of the proapoptotic BH3-only proteins, BIM and PUMA. We conclude that latent EBV genes cooperatively enhance the survival of BL cells by suppression of the intrinsic apoptosis pathway signalling via inhibition of the potent apoptosis initiators, BIM and PUMA.

Epstein-Barr virus (EBV) is typically acquired asymptomatically in childhood. In contrast, infection later in life often leads to infectious mononucleosis (IM), a febrile illness characterized by anti-EBV IgM antibody positivity, high loads of circulating latently infected B cells, and a marked lymphocytosis caused by hyperexpansion of EBV-specific CD8+ T cells plus a milder expansion of CD56dim NKG2A+ KIR− natural killer (NK) cells. How the two situations compare is unclear due to the paucity of studies on clinically silent infection. Here we describe five prospectively studied patients with asymptomatic infections identified in a seroepidemiologic survey of university entrants. In each case, the key blood sample had high cell-associated viral loads without a marked CD8 lymphocytosis or NK cell disturbance like those seen in patients during the acute phase of IM. Two of the cases with the highest viral loads showed a coincident expansion of activated EBV-specific CD8+ T cells, but overall CD8+ T cell numbers were either unaffected or only mildly increased. Two cases with slightly lower loads, in whom serology suggests the infection may have been caught earlier in the course of infection, also showed no T or NK cell expansion at the time. Interestingly, in another case with a higher viral load, in which T and NK cell responses were undetectable in the primary blood sample in which infection was detected, EBV-specific T cell responses did not appear until several months later, by which time the viral loads in the blood had already fallen. Thus, some patients with asymptomatic primary infections have very high circulating viral loads similar to those in patients during the acute phase of IM and a cell-mediated immune response that is qualitatively similar to that in IM patients but of a lower magnitude. However, other patients may have quite different immune responses that ultimately could reveal novel mechanisms of host control. IMPORTANCE Epstein-Barr virus (EBV) is transmitted orally, replicates in the throat, and then invades the B lymphocyte pool through a growth-transforming latent infection. While primary infection in childhood is usually asymptomatic, delayed infection is associated with infectious mononucleosis (IM), a febrile illness in which patients have high circulating viral loads and an exaggerated virus-induced immune response involving both CD8+ T cells and natural killer (NK) cells. Here we show that in five cases of asymptomatic infection, viral loads in the blood were as high as those in patients during the acute phase of IM, whereas the cell-mediated responses, even when they resembled those in patients during the acute phase of IM in timing and quality, were never as exaggerated. We infer that IM symptoms arise as a consequence not of the virus infection per se but of the hyperactivated immune response. Interestingly, there were idiosyncratic differences among asymptomatic cases in the relationship between the viral load and the response kinetics, emphasizing how much there is still to learn about primary EBV infection.

The Epstein-Barr virus (EBV) establishes a lifelong latent infection in humans. EBV infection of primary B cells causes cell activation and proliferation, a process driven by the viral latency III gene expression program, which includes EBV nuclear proteins (EBNAs), latent membrane proteins, and untranslated RNAs, including microRNAs. Some latently infected cells enter the long-lived memory B-cell compartment and express only EBNA1 transiently (Lat I) or no EBV protein at all (Lat 0). Targeting the molecular machinery that controls B-cell fate decisions, including the Bcl-2 family of apoptosis-regulating proteins, is crucial to the EBV cycle of infection. Here, we show that BIK (also known as NBK ), which encodes a proapoptotic “sensitizer” protein, is repressed by the EBNA2-driven Lat III program but not the Lat I program. BIK repression occurred soon after infection of primary B cells by EBV but not by a recombinant EBV in which the EBNA2 gene had been knocked out. Ectopic BIK induced apoptosis in Lat III cells by a mechanism dependent on its BH3 domain and the activation of caspases. We show that EBNA2 represses BIK in EBV-negative B-cell lymphoma-derived cell lines and that this host-virus interaction can inhibit the proapoptotic effect of transforming growth factor β1 (TGF-β1), a key physiological mediator of B-cell homeostasis. Reduced levels of TGF-β1-associated regulatory SMAD proteins were bound to the BIK promoter in response to EBV Lat III or ectopic EBNA2. These data are evidence of an additional mechanism used by EBV to promote B-cell survival, namely, the transcriptional repression of the BH3-only sensitizer BIK . IMPORTANCE Over 90% of adult humans are infected with the Epstein-Barr virus (EBV). EBV establishes a lifelong silent infection, with its DNA residing in small numbers of blood B cells that are a reservoir from which low-level virus reactivation and shedding in saliva intermittently occur. Importantly, EBV DNA is found in some B-cell-derived tumors in which viral genes play a key role in tumor cell emergence and progression. Here, we report for the first time that EBV can shut off a B-cell gene called BIK . When activated by a molecular signal called transforming growth factor β1 (TGF-β1), BIK plays an important role in killing unwanted B cells, including those infected by viruses. We describe the key EBV–B-cell molecular interactions that lead to BIK shutoff. These findings further our knowledge of how EBV prevents the death of its host cell during infection. They are also relevant to certain posttransplant lymphomas where unregulated cell growth is caused by EBV genes.

Disruption of T cell memory during severe immune suppression results in reactivation of chronic viral infections, such as Epstein Barr virus (EBV) and Cytomegalovirus (CMV). How different subsets of memory T cells contribute to the protective immunity against these viruses remains poorly defined. In this study we examined the compartmentalization of virus-specific, tissue resident memory CD8+ T cells in human lymphoid organs. This revealed two distinct populations of memory CD8+ T cells, that were CD69+CD103+ and CD69+CD103—, and were retained within the spleen and tonsils in the absence of recent T cell stimulation. These two types of memory cells were distinct not only in their phenotype and transcriptional profile, but also in their anatomical localization within tonsils and spleen. The EBV-specific, but not CMV-specific, CD8+ memory T cells preferentially accumulated in the tonsils and acquired a phenotype that ensured their retention at the epithelial sites where EBV replicates. In vitro studies revealed that the cytokine IL-15 can potentiate the retention of circulating effector memory CD8+ T cells by down-regulating the expression of sphingosine-1-phosphate receptor, required for T cell exit from tissues, and its transcriptional activator, Kruppel-like factor 2 (KLF2). Within the tonsils the expression of IL-15 was detected in regions where CD8+ T cells localized, further supporting a role for this cytokine in T cell retention. Together this study provides evidence for the compartmentalization of distinct types of resident memory T cells that could contribute to the long-term protection against persisting viral infections., Author Summary Some viruses have the capacity to establish chronic infections in humans. How different T cell populations effectively control these infections has not been clear. Continuous circulation of memory T cells was thought to be crucial for effective immune surveillance against such infections. Recent studies in mice however, have shown that non-circulating tissue resident memory populations can also contribute to protective immunity. In this study we have examined the distribution, localization and characteristics of Epstein-Barr virus and Cytomegalovirus-specific T cells in different human tissues. This showed that virus-specific T cells were differentially distributed in different tissues and there was preferential accumulation of EBV-specific resident memory T cells at sites where EBV reactivates. In vitro studies showed that IL-15 and TGF-β could cooperate to extinguish tissue exit signals in T cells and therefore potentiate their retention within tissues. IL-15 expression was also detected in areas where T cells aggregated within the tissue. Together our study provides insight into how distinct memory T cells are compartmentalized in tissues to maintain long-term protection against persisting viral infections.

The EBV is known to persist in memory B cells, but it remains unclear how this affects cell numbers and humoral immunity. We here studied EBV persistence in memory B cell subsets and consequences on B cell memory in young children. EBV genome loads were quantified in 6 memory B cell subsets in EBV

B-lymphocyte-induced maturation protein 1 (BLIMP1) exists as two major isoforms, α and β, which arise from alternate promoters. Inactivation of the full length BLIMP1α isoform is thought to contribute to B cell lymphomagenesis by blocking post-germinal centre (GC) B cell differentiation. In contrast, the shorter β isoform is functionally impaired and over-expressed in several haematological malignancies, including diffuse large B cell lymphomas (DLBCL). We have studied the influence on BLIMP1β expression of the Epstein-Barr virus (EBV), a human herpesvirus that is implicated in the pathogenesis of several GC-derived lymphomas, including a subset of DLBCL and Hodgkin’s lymphoma (HL). We show that BLIMP1β expression is increased following the EBV infection of normal human tonsillar GC B cells. We also show that this change in expression is accompanied by hypomethylation of the BLIMP1β-specific promoter. Furthermore, we confirmed previous reports that the BLIMP1β promoter is hypomethylated in DLBCL cell lines and show for the first time that BLIMP1β is hypomethylated in the Hodgkin/Reed-Sternberg (HRS) cells of HL. Our results provide evidence in support of a role for BLIMP1β in the pathogenesis of EBV-associated B cell lymphomas.

In patients with XLP, a primary immunodeficiency caused by mutations in SH2D1A, EBV infection can lead to somatic reversion of the disease-causing mutation selectively in effector memory CD8 T cells; reverted CD8 cells are better able to respond to and kill EBV-infected cells., Patients with the primary immunodeficiency X-linked lymphoproliferative disease (XLP), which is caused by mutations in SH2D1A, are highly susceptible to Epstein-Barr virus (EBV) infection. Nonetheless, some XLP patients demonstrate less severe clinical manifestations after primary infection. SH2D1A encodes the adaptor molecule SLAM-associated protein (SAP), which is expressed in T and natural killer cells and is required for cytotoxicity against B cells, the reservoir for EBV. It is not known why the clinical presentation of XLP is so variable. In this study, we report for the first time the occurrence of somatic reversion in XLP. Reverted SAP-expressing cells resided exclusively within the CD8+ T cell subset, displayed a CD45RA−CCR7− effector memory phenotype, and were maintained at a stable level over time. Importantly, revertant CD8+ SAP+ T cells, but not SAP− cells, proliferated in response to EBV and killed EBV-infected B cells. As somatic reversion correlated with EBV infection, we propose that the virus exerts a selective pressure on the reverted cells, resulting in their expansion in vivo and host protection against ongoing infection.

An important pathogenic event in Epstein-Barr virus (EBV)-associated lymphomas is the suppression of virus replication, which would otherwise lead to cell death. Because virus replication in B cells is intimately linked to their differentiation toward plasma cells, we asked whether the physiologic signals that drive normal B-cell differentiation are absent in EBV-transformed cells. We focused on BLIMP1α, a transcription factor that is required for plasma cell differentiation and that is inactivated in diffuse large B-cell lymphomas. We show that BLIMP1α expression is down-regulated after EBV infection of primary germinal center B cells and that the EBV oncogene, latent membrane protein-1 (LMP-1), is alone capable of inducing this down-regulation in these cells. Furthermore, the down-regulation of BLIMP1α by LMP-1 was accompanied by a partial disruption of the BLIMP1α transcriptional program, including the aberrant induction of MYC, the repression of which is required for terminal differentiation. Finally, we show that the ectopic expression of BLIMP1α in EBV-transformed cells can induce the viral lytic cycle. Our results suggest that LMP-1 expression in progenitor germinal center B cells could contribute to the pathogenesis of EBV-associated lymphomas by down-regulating BLIMP1α, in turn preventing plasma cell differentiation and induction of the viral lytic cycle.

Epstein-Barr virus (EBV) has been shown to encode at least 40 microRNAs (miRNAs), an important class of molecules that negatively regulate the expression of many genes through posttranscriptional mechanisms. Here, we have used real-time PCR assays to quantify the levels of EBV-encoded BHRF1 and BART miRNAs in latently infected cells and in cells induced into the lytic cycle. During latency, BHRF1 miRNAs were seen only in cells with detectable Cp- and/or Wp-initiated EBNA transcripts, while the BART miRNAs were expressed in all forms of latent infection. Surprisingly, levels of different BART miRNAs were found to vary up to 50-fold within a cell line. However, this variation could not be explained by differential miRNA turnover, as all EBV miRNAs appeared to be remarkably stable. Following entry into the virus lytic cycle, miR-BHRF1-2 and -1-3 were rapidly induced, coincident with the onset of lytic BHRF1 transcripts, while miR-BHRF1-1 expression was delayed until 48 h and correlated with the appearance of Cp/Wp-initiated EBNA transcripts. In contrast, levels of BART miRNAs were relatively unchanged during virus replication, despite dramatic increases in BART transcription. Finally, we show that BHRF1 and BART miRNAs were delayed relative to the induction of BHRF1 and BART transcripts in freshly infected primary B cell cultures. In summary, our data show that changes in BHRF1 and BART transcription are not necessarily reflected in altered miRNA levels, suggesting that miRNA maturation is a key step in regulating steady-state levels of EBV miRNAs.

Therapeutic targeting of virus-encoded proteins using cellular immunotherapy has proved successful for Epstein-Barr virus (EBV)–associated posttransplant lymphoproliferative disease. However, the more limited repertoire and immunogenicity of EBV-encoded proteins in other malignancies such as Hodgkin lymphoma and extranodal natural killer (NK)/T lymphoma has been more challenging to target. The immunosubdominant latent membrane protein 2 (LMP2) is considered the optimal target in such Latency II tumors, although data relating to its expression in T/NK malignancies are limited. In addressing the validity of LMP2 as an immunotherapeutic target we found that LMP2-specific effector CD8+ T cells recognized and killed EBV-positive NK- and T-cell tumor lines, despite an apparent absence of LMP2A protein and barely detectable levels of LMP2 transcripts from the conventional LMP2A and LMP2B promoters. We resolved this paradox by identifying in these lines a novel LMP2 mRNA, initiated from within the EBV terminal repeats and containing downstream, epitope-encoding exons. This same mRNA was also highly expressed in primary (extra-nodal) NK/T lymphoma tissue, with virtually undetectable levels of conventional LMP2A/B transcripts. Expression of this novel transcript in T/NK-cell lymphoproliferative diseases validates LMP2 as an attractive target for cellular immunotherapy and implicates this truncated LMP2 protein in NK- and T-cell lymphomagenesis. This study is registered at clinicaltrials.gov as NCT00062868.

Epstein-Barr virus was originally identified in the tumour cells of a Burkitt's lymphoma, and was the first virus to be associated with the pathogenesis of a human cancer. Studies on the relationship of EBV with Burkitt's lymphoma have revealed important general principles that are relevant to other virus-associated cancers. In addition, the impact of such studies on the knowledge of EBV biology has been enormous. Here, we review some of the key historical observations arising from studies on Burkitt's lymphoma that have informed our understanding of EBV, and we summarise the current hypotheses regarding the role of EBV in the pathogenesis of Burkitt's lymphoma.

Epstein-Barr virus (EBV) persists in the immune host by preferentially colonizing the isotype-switched (IgD−CD27+) memory B-cell pool. In one scenario, this is achieved through virus infection of naive (IgD+CD27−) B cells and their differentiation into memory via germinal center (GC) transit; in another, EBV avoids GC transit and infects memory B cells directly. We report 2 findings consistent with this latter view. First, we examined circulating non–isotype-switched (IgD+CD27+) memory cells, a population that much evidence suggests is GC-independent in origin. Whereas isotype-switched memory had the highest viral loads by quantitative polymerase chain reaction, EBV was detectable in the nonswitched memory pool both in infectious mononucleosis (IM) patients undergoing primary infection and in most long-term virus carriers. Second, we examined colonization by EBV of B-cell subsets sorted from a unique collection of IM tonsillar cell suspensions. Here viral loads were concentrated in B cells with the CD38 marker of GC origin but lacking other GC markers CD10 and CD77. These findings, supported by histologic evidence, suggest that EBV infection in IM tonsils involves extrafollicular B cells expressing CD38 as an activation antigen and not as a marker of ectopic GC activity.

The transformation of B cells by Epstein-Barr virus (EBV), into lymphoblastoid cell lines (LCLs) results in the upregulation of STAT1, a key transcription factor in the interferon signalling pathway. Although the mechanism of EBV induction of STAT1 protein expression has been intensively studied, there has been little investigation into the function of STAT1 in EBV-transformed LCLs. In this study, we have implemented a novel strategy to investigate the functional role of STAT1 through the introduction of the simian virus 5 (SV5) V-protein into LCLs by retroviral gene transfer. The V-protein is a virally evolved STAT1 inhibitor that specifically targets STAT1 for proteasomal degradation. Using this in vitro model, we have shown that major histocompatibility complex (MHC) class I and class II molecules are downregulated at the cell surface following a reduction in STAT1 protein expression. With regards to MHC class I, the impairment of the antigen processing machinery renders the cells less recognized by the host EBV-specific immunosurveillance. In addition, downregulation of STAT1 increases the expression of LMP2A and lytic cycle antigens and results in a higher proportion of cells entering the lytic cycle. These results suggest that STAT1 is involved in maintaining the latency III viral program observed in transformed B cells and regulating immunorecognition by EBV-specific T cells.

Background. To investigate how intense Plasmodium falciparum infection predisposes to Epstein-Barr virus (EBV)-positive Burkitt lymphoma (BL) we analyzed the effect of acute malaria on existing EBV-host balance. Methods. EBV genome loads in peripheral blood mononuclear cells were assayed by quantitative polymerase chain reaction and EBV-specific CD8_ T cell responses were assayed by interferon-y enzyme-linked immunospot assay. Results. Gambian children from whom samples were obtained during an acute malaria attack and again up to 6 weeks later had extremely high viral loads reaching levels that in the United Kingdom are seen only in patients with infectious mononucleosis. Gambian control subjects (children and adults with no recent history of malaria) had lower median viral loads although they were still >10-fold above the median for healthy UK adults. Limited experiments with EBV epitope peptides (restricted through the HLA-B*3501 and HLA-B*5301 alleles) also suggested an impairment of virus-specific CD8+ T cell function in children with malaria but only during acute disease. Conclusions. Acute malaria is associated with sustained increase in EBV load and possibly a transient decrease in EBV-specific T cell surveillance. We infer that the unusually high set point of virus carriage in P. falciparum-challenged populations allied with the parasites capacity to act as a chronic B cell stimulus probably contributes to the pathogenesis of endemic BL.

Epstein-Barr virus (EBV) can infect various cell types but limits its classical growth-transforming function to B lymphocytes, the cells in which it persists in vivo. Transformation initiates with the activation of Wp, a promoter present as tandemly repeated copies in the viral genome. Assays with short Wp reporter constructs have identified two promoter-activating regions, one of which (UAS2) appears to be lineage independent, while the other (UAS1) was B-cell specific and contained two putative binding sites for the B-cell-specific activator protein BSAP/Pax5. To address the physiologic relevance of these findings, we first used chromosome immunoprecipitation assays and found that BSAP is indeed bound to Wp sequences on the EBV genome in transformed cells. Thereafter, we constructed recombinant EBVs carrying two Wp copies, both wild type, with UAS1 or UAS2 deleted, or mutated in the BSAP binding sites. All the viruses delivered their genomes to the B-cell nucleus equally well. However, the BSAP binding mutant (and the virus with UAS1 deleted) showed no detectable activity in B cells, whether measured by early Wp transcription, expression of EBV latent proteins, or outgrowth of transformed cells. This was a B-cell-specific defect since, on entry into epithelial cells, an environment where Wp is not the latent promoter of choice, all the Wp mutant viruses initiated infection as efficiently as wild-type virus. We infer that EBV ensures the B-cell specificity of its growth-transforming function by exploiting BSAP/Pax5 as a lineage-specific activator of the transforming program.

The Epstein-Barr virus (EBV) latent cycle promoter Wp, present in each tandemly arrayed copy of the BamHI W region in the EBV genome, drives expression of the EB viral nuclear antigens (EBNAs) at the initiation of virus-induced B-cell transformation. Thereafter, an alternative EBNA promoter, Cp, becomes dominant, Wp activity declines dramatically, and bisulfite sequencing of EBV-transformed lymphoblastoid cell lines (LCLs) shows extensive Wp methylation. Despite this, Wp is never completely silenced in LCLs. Here, using a combination of bisulfite sequencing and methylation-specific PCR, we show that in standard LCLs transformed with wild-type EBV isolates, some Wp copies always remain unmethylated, and in LCLs transformed with a recombinant EBV carrying just two BamHI W copies, Wp is completely unmethylated. Furthermore, we have analyzed rare LCLs, recently established using wild-type EBV isolates, and rare Burkitt lymphoma (BL) cell clones, recently established from tumors carrying EBNA2-deleted EBV genomes, which express EBNAs exclusively from Wp-initiated transcripts. Here, in sharp contrast to standard LCL and BL lines, all resident copies of Wp appear to be predominantly hypomethylated. Thus, studies of B cells with atypical patterns of Wp usage emphasize the strong correlation between the presence of unmethylated Wp sequences and promoter activity.

Epstein–Barr virus (EBV) infection and growth activation of human B cells is central to virus biology and disease pathogenesis, but is poorly understood in quantitative terms. Here, using virus at defined m.o.i., the different stages of this process at the single-cell level are followedin vitro. Virus binding to the B-cell surface, assayed by quantitative PCR, is highly efficient, particularly at the low m.o.i. values that most likely reflect physiologic eventsin vivo. However, only 10–15 % of bound virus genomes reach the cell nucleus, as visualized by sensitive fluorescencein situhybridization (FISH) assay; viral genomes acquired per cell nucleus range from 1 to >10, depending on the m.o.i. Thereafter, despite differences in initial genome load, almost all nuclear genome-positive cells then go on to express the virus-encoded nuclear antigen EBNA2, upregulate the cell activation antigen CD23 and transit the cell cycle. EBNA2-positive cells in the first cycle post-infection then grow out to lymphoblastoid cell lines (LCLs) just as efficiently as do cells limiting-diluted from already established LCLs. This study therefore identifies EBV genome delivery to the nucleus as a key rate-limiting step in B-cell transformation, and highlights the remarkable efficiency with which a single virus genome, having reached the nucleus, then drives the transformation programme.

Epstein-Barr virus (EBV), a human B-lymphotropic herpesvirus with cell growth-transforming ability, is linked to three different B-cell malignancies, endemic Burkitt's lymphoma (BL), Hodgkin's disease, and posttransplant lymphoproliferative disease (PTLD) (36). We know relatively little about the role played by the virus in these different tumor contexts, except for those oligoclonal or polyclonal PTLD lesions which arise in the setting of profound T-cell impairment, classically in early posttransplant patients. These lesions express the same spectrum of EBV latent-cycle proteins (53) as do B cells transformed by EBV to permanent lymphoblastoid cell lines (LCLs) in vitro (19). This latency III form of infection is characterized by BamHI C promoter (Cp) and to a lesser extent BamHI W promoter (Wp) activity, leading to expression of EBV nuclear antigen EBNA1, -2, -3A, -3B, -3C, and -LP, and by activation of EBNA2-responsive promoters elsewhere in the viral genome, leading to expression of latent membrane proteins LMP1 and LMP2. The LCL-like nature of viral gene expression in PTLDs strongly suggests that EBV is the principal, perhaps the sole, driving force behind these lymphoproliferations. The situation is quite different in endemic BL, where EBV's role clearly complements that of the principal cellular genetic change, namely, activation of the c-myc oncogene by translocation to an immunoglobulin (Ig) gene locus (26). BL tumor cells carry a transformation-competent wild-type EBV genome but display a latency I form of infection in which the nuclear antigen EBNA1 is selectively expressed from the BamHI Q promoter while all latency III-associated promoters are silent (39, 42, 43). The highly restricted nature of EBV gene expression in BL raises questions as to what extent, if at all, the virus contributes to the malignant cell phenotype; most interest in this regard has focused on the potential role of either the EBNA1 protein or the noncoding EBER RNAs in mediating partial protection from the strong proapoptotic effects associated with c-myc-driven cell growth (23, 24, 40, 41). In recent work we identified a subset of BL tumors which did not display the typical latency I form of infection. Instead the tumor cells expressed five nuclear antigens, namely, EBNA1, -3A, -3B, and -3C and a truncated (W1W2 repeat domain only) EBNA-LP, in the absence of EBNA2 and of the LMPs (18). This was associated with transcription exclusively from Wp and was hence termed “Wp-restricted latency.” Interestingly, all three BL cell lines of this type carried a transformation-competent wild-type genome and also a transformation-defective genome in which a deletion had removed the EBNA2 gene and the Y1Y2 unique exons of EBNA-LP. These findings raised a number of questions. What is the origin of the EBNA2-deleted virus? What is the virus genome content of individual tumor cells? Are Wp transcripts derived from both or just from one of these genomes? Is the presence of an EBNA2-deleted genome consistently linked to Wp restriction? Does the broadened pattern of viral antigen expression in Wp-restricted latency alter the cell phenotype in ways that might be important in tumor pathogenesis? Here we have addressed these questions by establishing single-cell clones from three Wp-restricted BL lines, Sal-, Oku-, and Ava-BL, and determining their viral genome contents and patterns of latent gene expression. In addition, we asked whether in vitro infection of EBV-negative BL cell lines with a recombinant EBNA2-deleted virus strain established a Wp-restricted form of latency. Finally, we compared BL lines showing Wp-restricted latency with classical latency I BL lines for their susceptibility to apoptosis.

Epstein-Barr virus (EBV) strains from the highly HLA-A11-positive Chinese population are predominantly type 1 and show a variety of sequence changes (relative to the contemporary Caucasian prototype strain B95.8) in the nuclear antigen EBNA3B sequences encoding two immunodominant HLA-A11 epitopes, here called IVT and AVF. This has been interpreted by some as evidence of immune selection and by others as random genetic drift. To study epitope variation in a broader genomic context, we sequenced the whole of EBNA3B and parts of the EBNA2, 3A, and 3C genes from each of 31 Chinese EBV isolates. At each locus, type 1 viruses showed

The Epstein–Barr virus (EBV) has recently been associated with hepatocellular carcinoma (HCC) arising in Japanese patients. We analyzed 82 cases of HCC from Germany and the U.K. for the presence of EBV DNA and viral gene products within tumor cells. Initial screening of whole sections using quantitative (Q)-PCR detected EBV DNA in 9/58 U.K. cases and in 9/24 German cases; in positive cases viral load was very low, ranging between 1.4 and 49.1 copies of the EBV genome/1000 cell equivalents, compared to much higher values for EBV-positive Hodgkin’s disease and nasopharyngeal carcinoma controls (range, 714–3259/1000 cells). EBV DNA was not detected in the tumor cells of any of the Q-PCR-positive cases either by Q-PCR of pure tumor cell populations isolated by laser capture microdissection or by isotopic in situ hybridization. Furthermore, none of the German or U.K. HCC tumors tested positive for EBER or EBNAI expression in tumor cells. Our results provide strong evidence that HCCs from the U.K. or Germany are not associated with EBV.

The morphology of the pathognomonic Hodgkin and Reed-Sternberg cells (HRS) of Hodgkin lymphoma was described over a century ago, yet it was only relatively recently that the B-cell origin of these cells was identified. In a proportion of cases, HRS cells harbour monoclonal forms of the B lymphotropic Epstein-Barr virus (EBV). This review summarises current knowledge of the pathogenesis of Hodgkin lymphoma with a particular emphasis on the contribution of EBV.

Epstein-Barr virus (EBV) is etiologically linked to endemic Burkitt lymphoma (BL), but its contribution to lymphomagenesis, versus that of the chromosomal translocation leading to c-myc gene deregulation, remains unclear. The virus's growth-transforming (Latency III) program of gene expression is extinguished in tumor cells, and only a single viral protein, the EBV nuclear antigen (EBNA)1, is expressed via the alternative Latency I program. It is not known if BL arises from a B-cell subset in which EBV naturally adopts a Latency I infection or if a clone with limited antigen expression has been selected from an EBV-transformed Latency III progenitor pool. Here we identify a subset of BL tumors in which the Latency III-associated EBNA promoter Wp is active and most EBNAs are expressed, but where a gene deletion has specifically abrogated the expression of EBNA2. This implies that BL can be selected from a Latency III progenitor and that the principal selection pressure is for downregulation of the c-Myc antagonist EBNA2.

In 1964, a new herpesvirus, Epstein-Barr virus (EBV), was discovered in cultured tumor cells derived from a Burkitt lymphoma (BL) biopsy taken from an African patient. This was a momentous event that reinvigorated research into viruses as a possible cause of human cancers. Subsequent studies demonstrated that EBV was a potent growth-transforming agent for primary B cells, and that all cases of BL carried characteristic chromosomal translocations resulting in constitutive activation of the c-MYC oncogene. These results hinted at simple oncogenic mechanisms that would make Burkitt lymphoma paradigmatic for cancers with viral etiology. In reality, the pathogenesis of this tumor is rather complicated with regard to both the contribution of the virus and the involvement of cellular oncogenes. Here, we review the current understanding of the roles of EBV and c-MYC in the pathogenesis of BL and the implications for new therapeutic strategies to treat this lymphoma.

We have validated a flexible, high-throughput and relatively inexpensive RT-QPCR array platform for absolute quantification of Epstein–Barr virus transcripts in different latent and lytic infection states. Several novel observations are reported. First, during infection of normal B cells, Wp-initiated latent gene transcripts remain far more abundant following activation of the Cp promoter than was hitherto suspected. Second, EBNA1 transcript levels are remarkably low in all forms of latency, typically ranging from 1 to 10 transcripts per cell. EBNA3A, -3B and -3C transcripts are likewise very low in Latency III, typically at levels similar to or less than EBNA1 transcripts. Thirdly, a subset of lytic gene transcripts is detectable in Burkitt lymphoma lines at low levels, including: BILF1, which has oncogenic properties, and the poorly characterized LF1, LF2 and LF3 genes. Analysis of seven African BL biopsies confirmed this transcription profile but additionally revealed significant expression of LMP2 transcripts., Highlights • We have validated a QPCR array platform to quantify Epstein–Barr virus transcripts. • Detection of 45 viral transcripts starting from ng amounts of RNA. • EBNA1 transcript levels are remarkably low in all forms of latency. • BILF1, LF1, LF2 and LF3 transcripts are detectable in Burkitt lymphoma lines. • Significant levels of LMP2 transcripts were also detected in BL biopsies.

Two Epstein-Barr virus latent cycle promoters for nuclear antigen expression, Wp and Cp, are activated sequentially during virus-induced transformation of B cells to B lymphoblastoid cell lines (LCLs) in vitro. Previously published restriction enzyme studies have indicated hypomethylation of CpG dinucleotides in the Wp and Cp regions of the viral genome in established LCLs, whereas these same regions appeared to be hypermethylated in Burkitt's lymphoma cells, where Wp and Cp are inactive. Here, using the more sensitive technique of bisulfite genomic sequencing, we reexamined the situation in established LCLs with the typical pattern of dominant Cp usage; surprisingly, this showed substantial methylation in the 400-bp regulatory region upstream of the Wp start site. This was not an artifact of long-term in vitro passage, since, in cultures of recently infected B cells, we found progressive methylation of Wp (but not Cp) regulatory sequences occurring between 7 and 21 days postinfection, coincident with the period in which dominant nuclear antigen promoter usage switches from Wp to Cp. Furthermore, in the equivalent in vivo situation, i.e., in the circulating B cells of acute infectious mononucleosis patients undergoing primary EBV infection, we again frequently observed selective methylation of Wp but not Cp sequences. An effector role for methylation in Wp silencing was supported by methylation cassette assays of Wp reporter constructs and by bandshift assays, where the binding of two sets of transcription factors important for Wp activation in B cells, BSAP/Pax5 and CREB/ATF proteins, was shown to be blocked by methylation of their binding sites.

The Epstein-Barr virus (EBV) nuclear antigen EBNA1 is the only viral protein detectably expressed in virus genome-positive Burkitt’s lymphoma (BL); recent work has suggested that viral strains with particular EBNA1 sequence changes are preferentially associated with this tumor and that, within a patient, the tumor-associated variant may have arisen de novo as a rare mutant of the dominant preexisting EBV strain (K. Bhatia, A. Raj, M. J. Gutierrez, J. G. Judde, G. Spangler, H. Venkatesh, and I. T. Magrath, Oncogene 13:177–181, 1996). In the present work we first study 12 BL patients and show that the virus strain in the tumor is identical in EBNA1 sequence and that it is matched at several other polymorphic loci to the dominant strain rescued in vitro from the patient’s normal circulating B cells. We then analyze BL-associated virus strains from three different geographic areas (East Africa, Europe, and New Guinea) alongside virus isolates from geographically matched control donors by using sequence changes in two separate regions of the EBNA1 gene (N-terminal codons 1 to 60 and C-terminal codons 460 to 510) to identify the EBNA1 subtype of each virus. Different geographic areas displayed different spectra of EBNA1 subtypes, with only limited overlap between them; even type 2 virus strains, which tended to be more homogeneous than their type 1 counterparts, showed geographic differences at the EBNA1 locus. Most importantly, within any one area the EBNA1 subtypes associated with BL were also found to be prevalent in the general population. We therefore find no evidence that Burkitt lymphomagenesis involves a selection for EBV strains with particular EBNA1 sequence changes.

The Epstein-Barr virus (EBV)-encoded leader protein EBNA-LP is made up of several 66-amino-acid repeats (the W1W2 domains) linked to a unique 45-amino-acid C-terminal sequence (the Y1Y2 domain). This protein is highly expressed along with a second nuclear antigen, EBNA-2, during the initial stages of virus-induced B-cell transformation. While EBNA-2's essential role in transformation as a transcriptional activatory is well documented, very little is known about EBNA-LP function except that recombinant viruses lacking the EBNA-LP Y1Y2 exons show reduced, but still detectable, transforming ability. This was taken as evidence that EBNA-LP plays an auxiliary role but is not essential for transformation. A recent study showed that EBNA-LP could cooperate with EBNA-2 in activating cyclin D2 transcription in resting B cells (A.J. Sinclair, L Palmero, G. Peters, and P.J. Farrell, EMBO J. 13:3321-3328, 1994). Here we report that EBNA-LP can also cooperate with EBNA-2 in up-regulating expression of the major EBV effector protein of B-cell transformation, latent membrane protein 1 (LMP1). In transient-transfection assays, EBNA-LP enhanced the level of EBNA-2-induced LMP1 expression by 5- to 10-fold in one Latency I Burkitt's lymphoma cell line, Eli-BL, and was absolutely required, along with EBNA-2, to induce LMP1 in a second line, Akata-BL. These changes in LMP1 protein expression appeared to be reflected at the transcriptional level. A study of EBNA-LP mutants showed that this cooperative function mapped to the W1W2 repeat domain rather than to Y1Y2. Because a Y1Y2-deleted form of EBNA-LP may therefore retain some aspects of wild-type function, the original data from virus recombinants leave open the possibility that EBNA-LP is actually an essential transforming gene.

At class II CRP-dependent promoters the DNA site for CRP overlaps the DNA site for RNA polymerase, covering the -35 region. Transcription activation at class II CRP- dependent promoters requires a contact between an activating region in the upstream subunit of the bound CRP dimer and a contact site in the C-terminal domain of the alpha-subunit of RNA polymerase. Transcription activation is suppressed by amino acid substitutions in the activating region, but activation can be restored by second site substitutions at K52 or E96. These substitutions identify two separate regions on the surface of CRP that appear to be able to interact with RNA polymerase specifically at class II promoters. Using the method of 'oriented heterodimers' we show that these alternative activating regions are functional in the downstream subunit of the bound CRP dimer.

In Epstein-Barr virus (EBV)-transformed B lymphoblastoid and many Burkitt lymphoma cell lines, the EBV EBNA-1 protein is one of six viral nuclear antigens expressed from a common transcription unit under the control of one of two promoters, Wp or Cp. In contrast, EBNA-1 is the only EBV nuclear antigen expressed in Burkitt and other EBV-positive tumors. We previously identified a promoter of EBNA-1 transcription, designated Fp, in early-passage Mutu Burkitt tumor cells, and this promoter is also active in long-term Mutu and Akata Burkitt cell lines which maintain the exclusive expression of EBNA-1 characteristic of the tumor. However, transcription initiation within Fp reporter gene plasmids in EBV-negative cells occurs at positions 100 to 200 bases downstream of the Fp start site in the BamHI-Q restriction fragment. Here we demonstrate that transcription initiation within newly established Burkitt lymphoma cell lines is consistent with the transcription initiation we observed in reporter plasmids. Furthermore, previous observations of transcription from Fp to generate EBNA-1 transcripts can be attributed to lytic-cycle gene expression. These data, in conjunction with our previous characterization of promoter regulatory elements, define a fourth EBNA-1 promoter, Qp, that is active in latently infected Burkitt tumor cells.

Epstein-Barr virus (EBV) is present in all cases of endemic Burkitt lymphoma (BL) but in few European/North American sporadic BLs. Gene expression arrays of sporadic tumors have defined a consensus BL profile within which tumors are classifiable as “molecular BL” (mBL). Where endemic BLs fall relative to this profile remains unclear, since they not only carry EBV but also display one of two different forms of virus latency. Here, we use early-passage BL cell lines from different tumors, and BL subclones from a single tumor, to compare EBV-negative cells with EBV-positive cells displaying either classical latency I EBV infection (where EBNA1 is the only EBV antigen expressed from the wild-type EBV genome) or Wp-restricted latency (where an EBNA2 gene-deleted virus genome broadens antigen expression to include the EBNA3A, -3B, and -3C proteins and BHRF1). Expression arrays show that both types of endemic BL fall within the mBL classification. However, while EBV-negative and latency I BLs show overlapping profiles, Wp-restricted BLs form a distinct subgroup, characterized by a detectable downregulation of the germinal center (GC)-associated marker Bcl6 and upregulation of genes marking early plasmacytoid differentiation, notably IRF4 and BLIMP1. Importantly, these same changes can be induced in EBV-negative or latency I BL cells by infection with an EBNA2-knockout virus. Thus, we infer that the distinct gene profile of Wp-restricted BLs does not reflect differences in the identity of the tumor progenitor cell per se but differences imposed on a common progenitor by broadened EBV gene expression.

Epstein-Barr virus (EBV) is strongly implicated in the pathogenesis of several human malignancies, but notably in all endemic and some sporadic cases of Burkitt's lymphoma (BL). Although the virus's contribution to the development of BL remains unclear, evidence now suggests that a common feature of all BL tumours is the expression of the TCL-1 oncogene. A recent report shows that TCL-1 expression in virus-positive BL tumour cells is dependent on the presence of EBV. This finding suggests that the ability of EBV to induce TCL-1 would circumvent the need for the additional genetic or epigenetic changes that lead to the constitutive expression of this oncogene in EBV-negative BL tumours.

Previous studies have reported that the tumour cells of nasopharyngeal carcinoma (NPC) exhibit recurrent chromosome abnormalities. These genetic changes are broadly assumed to lead to changes in gene expression which are important for the pathogenesis of this tumour. However, this assumption has yet to be formally tested at a global level. Therefore a genome wide analysis of chromosome copy number and gene expression was performed in tumour cells micro-dissected from the same NPC biopsies. Cellular tumour suppressor and tumour-promoting genes (TSG, TPG) and Epstein-Barr Virus (EBV)-encoded oncogenes were examined. The EBV-encoded genome maintenance protein EBNA1, along with the putative oncogenes LMP1, LMP2 and BARF1 were expressed in the majority of NPCs that were analysed. Significant downregulation of expression in an average of 76 cellular TSGs per tumour was found, whilst a per-tumour average of 88 significantly upregulated, TPGs occurred. The expression of around 60% of putative TPGs and TSGs was both up-and down-regulated in different types of cancer, suggesting that the simplistic classification of genes as TSGs or TPGs may not be entirely appropriate and that the concept of context-dependent onco-suppressors may be more extensive than previously recognised. No significant enrichment of TPGs within regions of frequent genomic gain was seen but TSGs were significantly enriched within regions of frequent genomic loss. It is suggested that loss of the FHIT gene may be a driver of NPC tumourigenesis. Notwithstanding the association of TSGs with regions of genomic loss, on a gene by gene basis and excepting homozygous deletions and high-level amplification, there is very little correlation between chromosomal copy number aberrations and expression levels of TSGs and TPGs in NPC.

Burkitt's lymphoma (BL), an EBV-associated tumour, occurs at high incidence in populations where malaria is holoendemic. Previous studies in one such population suggested that acute P.falciparum infection impairs EBV-specific T-cell surveillance, allowing expansion of EBV infected B-cells from which BL derives. We re-examined the situation in the same area, The Gambia, after a reduction in malaria endemicity. Cellular immune responses to EBV were measured in children with uncomplicated malaria before (day 0) and after treatment (day 28), comparing EBV genome loads in blood and EBV-specific CD8(+) T-cell numbers (assayed by MHC Class I tetramers and IFN(Gamma) ELISPOTS) with those seen in age- and sex-matched healthy controls. No significant changes were seen in EBV genome loads, percentage of EBV-specific CD8(+) T-cells and IFN(Gamma) producing T-cells in acute versus convalescent samples, nor any difference versus controls. Regression assays performed also no longer detected any impairment of EBV-specific T-cell surveillance. Acute uncomplicated malaria infection no longer alters EBV-specific immune responses in children in The Gambia. Given the recent decline in malaria incidence in that country, we hypothesise that gross disturbance of the EBV-host balance may be a specific effect of acute malaria only in children with a history of chronic/recurrent malaria challenge.

Summary The effects of a number of mutations in crp have been measured at different cyclic AMP receptor protein (CRP)-dependent Class II promoters, where the CRP-binding site is centred around 411/2 base pairs upstream from the transcription start point. The amino acid substitutions HL159 and TA158 result in reduced CRP-dependent activation, but the reduction varies from one Class II promoter to another. Deletions in the C-terminus of the RNA polymerase alpha subunit suppress the effects of HL159 and TA158. The role of the C-terminus of alpha at these promoters is assessed. Other changes at E58, K52 and E96 affect CRP activity specifically at Class II promoters and their role is discussed.

Transcription initiation at the Escherichia coli nirB promoter is induced by anaerobic growth and further increased by the presence of nitrite or nitrate in the growth medium. Expression from this promoter is totally dependent on the transcription factor, FNR, which binds between positions -52 and -30 upstream of the transcription startsite. The 20 base pairs from position -79 to -60 contain an inverted repeat of two 10-base sequence elements that are related to sequences at the NarL-binding site at the E. coli narG promoter. Comparison of these, and sequence elements at other promoters regulated by NarL, suggests a consensus NarL-binding sequence. Mutations in the putative NarL-binding site at the nirB promoter decrease FNR-dependent anaerobic induction, suggesting that NarL acts as a helper to FNR during transcription activation. These mutations also suppress induction by nitrite: single mutations at symmetry-related positions have similar effects, whilst double mutations have more severe effects, probably because two NarL subunits bind to the inverted repeat. Disruption of narL decreases nitrite induction of the nirB promoter whilst not suppressing induction by nitrate, suggesting that there may be a second nitrate-responsive factor. Nitrate induction was, however, suppressed by double mutations at symmetry-related positions in the NarL-binding site, suggesting that this putative second factor may bind to sequences similar to those recognized by NarL.

Epstein-Barr virus (EBV) is associated with malignant diseases of lymphoid and epithelial cell origin. The tropism of EBV is due to B-cell-restricted expression of CD21, the major receptor molecule for the virus. However, efficient infection of CD21 − epithelial cells can be achieved via transfer from EBV-coated B cells. We compare and contrast here the early events following in vitro infection of primary B cells and epithelial cells. Using sensitive, quantitative reverse transcription-PCR assays for several latent and lytic transcripts and two-color immunofluorescence staining to analyze expression at the single cell level, we confirmed and extended previous reports indicating that the two cell types support different patterns of transcription. Furthermore, whereas infection of B cells with one or two copies of EBV resulted in rapid amplification of the viral genome to >20 copies per cell, such amplification was not normally observed after infection of primary epithelial cells or undifferentiated epithelial lines. In epithelial cells, EBNA1 expression was detected in only ca. 40% of EBER + cells, and the EBV genome was subsequently lost during prolonged culture. One exception was that infection of AGS, a gastric carcinoma line, resulted in maintenance of EBNA1 expression and amplification of the EBV episome. In contrast to B cells, where amplification of the EBV episome occurred even with a replication-defective BZLF1-knockout virus, amplification in AGS cells was dependent upon early lytic cycle gene expression. These data highlight the influence of the host cell on the outcome of EBV infection with regard to genome expression, amplification, and maintenance.