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The regulation of transcription from Epstein-Barr virus promoters is known to involve the association of the host CCCTC-binding factor (CTCF) protein. This control involves direct binding of CTCF across the EBV genome and the formation of three-dimensional loops between virus promoters and enhancers. We sought to address how the deletion of a CTCF binding site upstream of the C-promoter (Cp) affected viral transcription in infected lymphoblastoid cell lines (LCLs) and how binding of the EBV trans-activating protein EBNA2 was changed across this promoter. Transcript level from Cp was up-regulated with CTCF binding site deletion, and transcription from other promoters (Wp and Qp) was decreased, while transcript levels were largely unchanged by independent mutation of a Cp-RBPJκ binding site. In turn, expression of EBNA2 protein was also increased, likely driven by increases in polycistronic EBNA2-encoding transcripts. Finally, Cp up-regulation was associated with an 8-fold increase in EBNA2 enrichment across Cp, concomitant with increased association of the associated cellular factor RBPJκ, probably due to a more accessible three-dimensional chromatin conformation upstream of Cp. Overall, the data presented here confirm that binding of CTCF directly upstream of Cp is important for the regulation of transcription from this and other EBV promoters.

The oncogenic Epstein Barr virus (EBV) infects the majority of the human population and usually persists within its host for life without symptoms. The EBV oncoproteins nuclear antigen 3A (EBNA3A) and 3C (EBNA3C) are required for B cell transformation in vitro and are expressed in EBV associated immunoblastic lymphomas in vivo. In order to address the necessity of EBNA3A and EBNA3C for persistent EBV infection in vivo, we infected NOD-scid γcnull mice with reconstituted human immune system components (huNSG mice) with recombinant EBV mutants devoid of EBNA3A or EBNA3C expression. These EBV mutants established latent infection in secondary lymphoid organs of infected huNSG mice for at least 3 months, but did not cause tumor formation. Low level viral persistence in the absence of EBNA3A or EBNA3C seemed to be supported primarily by proliferation with the expression of early latent EBV gene products transitioning into absent viral protein expression without elevated lytic replication. In vitro, EBNA3A and EBNA3C deficient EBV infected B cells could be rescued from apoptosis through CD40 stimulation, mimicking T cell help in secondary lymphoid tissues. Thus, even in the absence of the oncogenes EBNA3A and 3C, EBV can access a latent gene expression pattern that is reminiscent of EBV persistence in healthy virus carriers without prior expression of its whole growth transforming program., Author summary Epstein Barr virus (EBV) was discovered 54 years ago as the first human candidate tumor virus. In vitro, EBV can readily transform human B cells into indefinitely growing cell lines. In this transformation process, multiple EBV proteins play an important role, such as the two oncogenes EBNA3A and especially EBNA3C. To address the necessity of these oncogenes in vivo, we investigated EBNA3A and 3C deficient EBV for their persistence in mice with reconstituted human immune system components (huNSG mice), an in vivo model for EBV infection, associated tumorigenesis and immune control. Here, we show that EBV devoid of EBNA3A or EBNA3C was able to establish latent infection in vivo. The persistence was characterized through proliferation with the expression of early latent EBV gene products transitioning into absent viral protein expression, but without tumor formation. Furthermore, we were able to rescue in vitro infected B cells with EBNA3A or EBNA3C deficient EBV from apoptosis by mimicking T cell help. This might allow the virus to access the gene expression pattern that is thought to also mediate persistence in healthy EBV carriers and does not seem to require prior expression of all growth transforming viral proteins.

Mature human B cells infected by Epstein-Barr virus (EBV) become activated, grow, and proliferate. If the cells are infected ex vivo, they are transformed into continuously proliferating lymphoblastoid cell lines (LCLs) that carry EBV DNA as extra-chromosomal episomes, express 9 latency-associated EBV proteins, and phenotypically resemble antigen-activated B-blasts. In vivo similar B-blasts can differentiate to become memory B cells (MBC), in which EBV persistence is established. Three related latency-associated viral proteins EBNA3A, EBNA3B, and EBNA3C are transcription factors that regulate a multitude of cellular genes. EBNA3B is not necessary to establish LCLs, but EBNA3A and EBNA3C are required to sustain proliferation, in part, by repressing the expression of tumour suppressor genes. Here we show, using EBV-recombinants in which both EBNA3A and EBNA3C can be conditionally inactivated or using virus completely lacking the EBNA3 gene locus, that—after a phase of rapid proliferation—infected primary B cells express elevated levels of factors associated with plasma cell (PC) differentiation. These include the cyclin-dependent kinase inhibitor (CDKI) p18INK4c, the master transcriptional regulator of PC differentiation B lymphocyte-induced maturation protein-1 (BLIMP-1), and the cell surface antigens CD38 and CD138/Syndecan-1. Chromatin immunoprecipitation sequencing (ChIP-seq) and chromatin immunoprecipitation quantitative PCR (ChIP-qPCR) indicate that in LCLs inhibition of CDKN2C (p18INK4c) and PRDM1 (BLIMP-1) transcription results from direct binding of EBNA3A and EBNA3C to regulatory elements at these loci, producing stable reprogramming. Consistent with the binding of EBNA3A and/or EBNA3C leading to irreversible epigenetic changes, cells become committed to a B-blast fate, Author summary Epstein-Barr virus (EBV) infection can cause several types of cancer associated with its major target in humans, the mature B cell. Furthermore, EBV is one of the most potent transforming agents ever identified, producing—in vitro—‘immortal’ B lymphoblastoid cell lines (LCLs) with outstanding reliability. However, the near-symbiotic relationship between EBV and its natural host (>95% of human adults are asymptomatically infected) provides a powerful argument that this gamma-herpesvirus did not primarily evolve to be a harmful tumour-causing virus. Consistent with this, we show here that 2 of the potentially oncogenic viral proteins (EBNA3A and EBNA3C) have evolved not to facilitate oncogenic progression but to block plasma cell differentiation in EBV-activated B cells. Specifically, they act to interrupt the gene regulation network that drives activated B cells to become terminally differentiated, quiescent plasma cells, thus allowing for sustained regeneration of virally infected B cells. EBNA3A and EBNA3C achieve this by epigenetically inhibiting expression of cellular genes essential for the differentiation pathway; these include the cyclin-dependent kinase inhibitor p18INK4c and the transcription factor BLIMP-1. This favours the establishment of EBV latency in long-lived memory B cells and therefore helps maintain a ubiquitous, generally asymptomatic infection in human populations.

The human tumor viruses Epstein-Barr virus (EBV) and Kaposi sarcoma-associated herpesvirus (KSHV) establish persistent infections in B cells. KSHV is linked to primary effusion lymphoma (PEL), and 90% of PELs also contain EBV. Studies on persistent KSHV infection in vivo and the role of EBV co-infection in PEL development have been hampered by the absence of small animal models. We developed mice reconstituted with human immune system components as a model for KSHV infection and find that EBV/KSHV dual infection enhanced KSHV persistence and tumorigenesis. Dual-infected cells displayed a plasma cell-like gene expression pattern similar to PELs. KSHV persisted in EBV-transformed B cells and was associated with lytic EBV gene expression, resulting in increased tumor formation. Evidence of elevated lytic EBV replication was also found in EBV/KSHV dually infected lymphoproliferative disorders in humans. Our data suggest that KSHV augments EBV-associated tumorigenesis via stimulation of lytic EBV replication.

Latent Epstein-Barr virus (EBV) infection is causally linked to several human cancers. EBV expresses viral oncogenes that promote cell growth and inhibit the apoptotic response to uncontrolled proliferation. The EBV oncoprotein LMP1 constitutively activates NFκB and is critical for survival of EBV-immortalized B cells. However, during early infection EBV induces rapid B cell proliferation with low levels of LMP1 and little apoptosis. Therefore, we sought to define the mechanism of survival in the absence of LMP1/NFκB early after infection. We used BH3 profiling to query mitochondrial regulation of apoptosis and defined a transition from uninfected B cells (BCL-2) to early-infected (MCL-1/BCL-2) and immortalized cells (BFL-1). This dynamic change in B cell survival mechanisms is unique to virus-infected cells and relies on regulation of MCL-1 mitochondrial localization and BFL-1 transcription by the viral EBNA3A protein. This study defines a new role for EBNA3A in the suppression of apoptosis with implications for EBV lymphomagenesis. DOI: http://dx.doi.org/10.7554/eLife.22509.001, eLife digest Over 90% of adults around the world are infected with the Epstein-Barr virus. Like other closely related viruses, such as those that cause chicken pox and cold sores, an infection lasts for the rest of the person’s life, although the virus generally remains in a latent or dormant state. However, under certain conditions the latent viruses can cause cancers to develop; in fact, it is estimated that such infections are responsible for nearly 2% of all cancer deaths worldwide. One way that healthy human cells prevent cancer is by triggering their own death in a process called apoptosis. The Epstein-Barr virus can block apoptosis, therefore making the cells more likely to become cancerous. Previous research identified one protein in the Epstein-Barr virus that promotes cancer by preventing infected cells from dying as normal. However, even in the absence of this protein, Epstein-Barr virus-infected cells remain resistant to apoptosis. This suggests that the virus has another way of blocking cell death. Price et al. have now used a technique that stresses living cells in a way that reveals which proteins prevent apoptosis to study human cells infected with the Epstein-Barr virus. This revealed that soon after infection, the virus could force the human cell to produce MCL-1, a protein that prevents cell death. Later, the Epstein-Barr virus enlisted a second human protein called BFL-1, which makes the infected cell further resistant to apoptosis. Price et al. discovered that a protein in the Epstein-Barr virus called EBNA3A controls the production of the MCL-1 and BFL-1 proteins. In the future, developing therapies that target these proteins may lead to new treatments for cancers caused by the Epstein-Barr virus. Such treatments would be likely to have fewer side effects for patients than traditional chemotherapies. DOI: http://dx.doi.org/10.7554/eLife.22509.002

ChIP-seq performed on lymphoblastoid cell lines (LCLs), expressing epitope-tagged EBNA3A, EBNA3B or EBNA3C from EBV-recombinants, revealed important principles of EBNA3 binding to chromatin. When combined with global chromatin looping data, EBNA3-bound loci were found to have a singular character, each directly associating with either EBNA3-repressed or EBNA3-activated genes, but not with both. EBNA3A and EBNA3C showed significant association with repressed and activated genes. Significant direct association for EBNA3B loci could only be shown with EBNA3B-repressed genes. A comparison of EBNA3 binding sites with known transcription factor binding sites in LCL GM12878 revealed substantial co-localization of EBNA3s with RUNX3—a protein induced by EBV during B cell transformation. The beta-subunit of core binding factor (CBFβ), that heterodimerizes with RUNX3, could co-immunoprecipitate robustly EBNA3B and EBNA3C, but only weakly EBNA3A. Depletion of either RUNX3 or CBFβ with lentivirus-delivered shRNA impaired epitope-tagged EBNA3B and EBNA3C binding at multiple regulated gene loci, indicating a requirement for CBF heterodimers in EBNA3 recruitment during target-gene regulation. ShRNA-mediated depletion of CBFβ in an EBNA3C-conditional LCL confirmed the role of CBF in the regulation of EBNA3C-induced and -repressed genes. These results reveal an important role for RUNX3/CBF during B cell transformation and EBV latency that was hitherto unexplored.

Allday and collaborators demonstrate that the EBV transcription factor and oncoprotein EBNA3C directly induces the expression of AID and somatic mutations in B cells, providing a mechanism linking infection and lymphoma induction., Activation-induced cytidine deaminase (AID), the enzyme responsible for induction of sequence variation in immunoglobulins (Igs) during the process of somatic hypermutation (SHM) and also Ig class switching, can have a potent mutator phenotype in the development of lymphoma. Using various Epstein-Barr virus (EBV) recombinants, we provide definitive evidence that the viral nuclear protein EBNA3C is essential in EBV-infected primary B cells for the induction of AID mRNA and protein. Using lymphoblastoid cell lines (LCLs) established with EBV recombinants conditional for EBNA3C function, this was confirmed, and it was shown that transactivation of the AID gene (AICDA) is associated with EBNA3C binding to highly conserved regulatory elements located proximal to and upstream of the AICDA transcription start site. EBNA3C binding initiated epigenetic changes to chromatin at specific sites across the AICDA locus. Deep sequencing of cDNA corresponding to the IgH V-D-J region from the conditional LCL was used to formally show that SHM is activated by functional EBNA3C and induction of AID. These data, showing the direct targeting and induction of functional AID by EBNA3C, suggest a novel role for EBV in the etiology of B cell cancers, including endemic Burkitt lymphoma.

Detailed analyses of the chromatin around the BIM promoter has revealed that latent Epstein–Barr virus (EBV) triggers the recruitment of polycomb repressive complex 2 (PRC2) core subunits and the trimethylation of histone H3 lysine 27 (H3K27me3) at this locus. The recruitment is absolutely dependent on nuclear proteins EBNA3A and EBNA3C; what is more, epitope-tagged EBNA3C could be shown bound near the transcription start site (TSS). EBV induces no consistent changes in the steady-state expression of PRC2 components, but lentivirus delivery of shRNAs against PRC2 and PRC1 subunits disrupted EBV repression of BIM. The activation mark H3K4me3 is largely unaltered at this locus irrespective of H3K27me3 status, suggesting the establishment of a ‘bivalent’ chromatin domain. Consistent with the ‘poised’ nature of these domains, RNA polymerase II (Pol II) occupancy was not altered by EBV at the BIM TSS, but analysis of phospho-serine 5 on Pol II indicated that EBNA3A and EBNA3C together inhibit initiation of BIM transcripts. B cell lines carrying EBV encoding a conditional EBNA3C-oestrogen receptor-fusion revealed that this epigenetic repression of BIM was reversible, but took more than 3 weeks from when EBNA3C was inactivated.

Evaluation of: Nikitin PA, Yan CM, Forte E et al.: An ATM/Chk2-mediated DNA damage-responsive signaling pathway suppresses Epstein-Barr virus transformation of primary human B cells. Cell. Host Microbe 8(6), 510-522 (2010). Viruses have evolved elegant strategies to manipulate the host while the host counters with defense systems including the interferon response, apoptosis and the DNA damage response (DDR). Viruses have multiple strategies for manipulating the DDR and the same virus can even activate or inhibit the DDR at different stages of infection. Epstein-Barr virus (EBV) is implicated in several human cancers, including Burkitt's lymphoma, nasopharyngeal carcinoma, post-transplant lymphoproliferative disease and HIV-associated lymphomas. Although multiple viral proteins have been implicated in EBV-associated malignancies, the cellular pathways that control EBV-induced transformation and tumorigenesis remain incompletely understood. In this study, Nikitin et al. demonstrate that early EBV infection induces a cellular DDR that restricts virus-mediated transformation. The EBV-encoded EBNA3C protein subsequently attenuates this response to favor transformation and immortalization of host cells.

A defining characteristic of the aggressive B cell tumour Burkitt's lymphoma (BL) is a reciprocal chromosomal translocation that activates the Myc oncogene by juxtaposing it to one of the immunoglobulin gene loci. The consequences of activating Myc include cell growth and proliferation that can lead to lymphomagenesis; however, as part of a fail-safe mechanism that has evolved in metazoans to reduce the likelihood of neoplastic disease, activated oncogenes such as Myc may also induce cell death by apoptosis and/or an irreversible block to proliferation called senescence. For lymphoma to develop it is necessary that these latter processes are repressed. More than 95% of a subset of BL – known as endemic (e)BL because they are largely restricted to regions of equatorial Africa and similar geographical regions – carry latent Epstein–Barr virus (EBV) in the form of nuclear extra-chromosomal episomes. Although EBV is not generally regarded as a driving force of BL cell proliferation, it plays an important role in the pathogenesis of eBL. Latency-associated EBV gene products can inhibit a variety of pathways that lead to apoptosis and senescence; therefore EBV probably counteracts the proliferation-restricting activities of deregulated Myc and so facilitates the development of BL.

Marek's disease virus (MDV) is a lymphotropic alphaherpesvirus that induces fatal rapid-onset T-cell lymphomas in chickens, its natural host. The MDV-encoded nuclear oncoprotein Meq is essential for lymphomagenesis and acts as a regulator of transcription. Meq has structural features, including a basic domain adjacent to a leucine zipper motif (B-ZIP), that suggest it is related to the Jun/Fos family of transcription factors. Via the leucine zipper, Meq can form homodimers or heterodimerize with c-Jun. Meq/Meq homodimers are associated with transrepression, and Meq/Jun heterodimers can transactivate target genes carrying an AP-1-like binding site. In order to determine the role of the leucine zipper and of Meq dimerization in T lymphomagenesis, specific point mutations were engineered into the highly oncogenic RB-1B strain of MDV to produce virus completely lacking a functional Meq leucine zipper (RB-1B Meq BZIP/BZIP ) or virus encoding Meq that cannot homodimerize but can still bind to c-Jun and an AP-1-like site on DNA (RB-1B Meq Hom/Hom ). Both of these mutant viruses were capable of replication in cultured chicken embryo fibroblasts. However both mutations resulted in a complete loss of oncogenicity, since no lymphomas were produced up to 90 days postinfection in experimentally infected chicks. We conclude that the leucine zipper is necessary for the oncogenic activity of Meq and/or the efficient establishment of long-term MDV latency in T cells. Moreover, it appears that the ability to form homodimers is an absolute requirement and the ability to bind c-Jun alone is insufficient for the T-cell lymphomagenesis associated with virulent MDV.

Viral nuclear oncoproteins EBNA3A and EBNA3C are essential for the efficient immortalization of B cells by Epstein–Barr virus (EBV) in vitro and it is assumed that they play an essential role in viral persistence in the human host. In order to identify cellular genes regulated by EBNA3A expression, cDNA encoding EBNA3A was incorporated into a recombinant adenoviral vector. Microarray analysis of human diploid fibroblasts infected with either adenovirus EBNA3A or an empty control adenovirus consistently showed an EBNA3A-specific induction of mRNA corresponding to the chaperones Hsp70 and Hsp70B/B′ and co-chaperones Bag3 and DNAJA1/Hsp40. Analysis of infected fibroblasts by real-time quantitative RT-PCR and Western blotting confirmed that EBNA3A, but not EBNA3C, induced expression of Hsp70, Hsp70B/B′, Bag3 and DNAJA1/Hsp40. This was also confirmed in a stable, inducible expression system. EBNA3A activated transcription from the Hsp70B promoter, but not multimerized heat-shock elements in transient transfection assays, consistent with specific chaperone and co-chaperone upregulation. Co-immunoprecipitation experiments suggest that EBNA3A can form a complex with the chaperone/co-chaperone proteins in both adenovirus-infected cells and EBV-immortalized lymphoblastoid cell lines. Consistent with this, induction of EBNA3A resulted in redistribution of Hsp70 from the cytoplasm to the nucleus. EBNA3A therefore specifically induces (and then interacts with) all of the factors necessary for an active Hsp70 chaperone complex.

It is well established that Epstein-Barr virus nuclear antigen 3C (EBNA3C) can act as a potent repressor of gene expression, but little is known about the sequence of events occurring during the repression process. To explore further the role of EBNA3C in gene repression–particularly in relation to histone modifications and cell factors involved–the three host genes previously reported as most robustly repressed by EBNA3C were investigated. COBLL1, a gene of unknown function, is regulated by EBNA3C alone and the two co-regulated disintegrin/metalloproteases, ADAM28 and ADAMDEC1 have been described previously as targets of both EBNA3A and EBNA3C. For the first time, EBNA3C was here shown to be the main regulator of all three genes early after infection of primary B cells. Using various EBV-recombinants, repression over orders of magnitude was seen only when EBNA3C was expressed. Unexpectedly, full repression was not achieved until 30 days after infection. This was accurately reproduced in established LCLs carrying EBV-recombinants conditional for EBNA3C function, demonstrating the utility of the conditional system to replicate events early after infection. Using this system, detailed chromatin immunoprecipitation analysis revealed that the initial repression was associated with loss of activation-associated histone modifications (H3K9ac, H3K27ac and H3K4me3) and was independent of recruitment of polycomb proteins and deposition of the repressive H3K27me3 modification, which were only observed later in repression. Most remarkable, and in contrast to current models of RBPJ in repression, was the observation that this DNA-binding factor accumulated at the EBNA3C-binding sites only when EBNA3C was functional. Transient reporter assays indicated that repression of these genes was dependent on the interaction between EBNA3C and RBPJ. This was confirmed with a novel EBV-recombinant encoding a mutant of EBNA3C unable to bind RBPJ, by showing this virus was incapable of repressing COBLL1 or ADAM28/ADAMDEC1 in newly infected primary B cells., Author Summary The Epstein-Barr nuclear protein EBNA3C is a well-characterised repressor of host gene expression in B cells growth-transformed by EBV. It is also well established that EBNA3C can interact with the cellular factor RBPJ, a DNA-binding factor in the Notch signalling pathway conserved from worms to humans. However, prior to this study, little was known about the role of the interaction between these two proteins during the repression of host genes. We therefore chose three genes–the expression of which is very robustly repressed by EBNA3C –to explore the molecular interactions involved. Hitherto these genes had not been shown to require RBPJ for EBNA3C-mediated repression. We have described the sequence of events during repression and challenge a widely held assumption that if a protein interacts with RBPJ it would be recruited to DNA because of the intrinsic capacity of RBPJ to bind specific sequences. We show that interaction with RBPJ is essential for the repression of all three genes during the infection of B cells by EBV, but that RBPJ itself is only recruited to the genes when EBNA3C is functional. These data suggest an unexpectedly complex interaction of multiple proteins when EBNA3C prevents the expression of cellular genes.

Epstein-Barr virus (EBV) contributes to the development of several human cancers including the endemic form of Burkitt's lymphoma (BL). In culture, EBV induces the continuous proliferation of primary B cells as lymphoblastoid cell lines (LCLs) and if EBV-negative BL-derived cells are infected with EBV, latency-associated viral factors confer resistance to various inducers of apoptosis. Nuclear proteins EBNA3A and EBNA3C (but not EBNA3B) are necessary to establish LCLs and their expression may be involved in the resistance of BL cells to cytotoxic agents. We have therefore created recombinant EBVs from which each of the EBNA3 genes has been independently deleted, and revertant viruses in which the genes have been re-introduced into the viral genome. Infection of EBV-negative BL cells with this panel of EBVs and challenge with various cytotoxic drugs showed that EBNA3A and EBNA3C cooperate as the main determinants of both drug resistance and the downregulation of the proapoptotic Bcl-2-family member Bcl-2-interacting mediator of cell death (Bim). The regulation of Bim is predominantly at the level of RNA, with little evidence of post-translational Bim stabilization by EBV. In the absence of Bim, EBNA3A and EBNA3C appear to provide no survival advantage. The level of Bim is a critical regulator of B cell survival and reduced expression is a major determinant of lymphoproliferative disease in mice and humans; moreover, Bim is uniquely important in the pathogenesis of BL. By targeting this tumour-suppressor for repression, EBV significantly increases the likelihood of B lymphomagenesis in general, and BL in particular. Our results may also explain the selection pressure that gives rise to a subset of BL that retain expression of the EBNA3 proteins.

Epstein-Barr virus (EBV) infects most of the world's population and is causally associated with several human cancers, but little is known about how EBV genetic variation might influence infection or EBV-associated disease. There are currently no published wild-type EBV genome sequences from a healthy individual and very few genomes from EBV-associated diseases. We have sequenced 71 geographically distinct EBV strains from cell lines, multiple types of primary tumor, and blood samples and the first EBV genome from the saliva of a healthy carrier. We show that the established genome map of EBV accurately represents all strains sequenced, but novel deletions are present in a few isolates. We have increased the number of type 2 EBV genomes sequenced from one to 12 and establish that the type 1/type 2 classification is a major feature of EBV genome variation, defined almost exclusively by variation of EBNA2 and EBNA3 genes, but geographic variation is also present. Single nucleotide polymorphism (SNP) density varies substantially across all known open reading frames and is highest in latency-associated genes. Some T-cell epitope sequences in EBNA3 genes show extensive variation across strains, and we identify codons under positive selection, both important considerations for the development of vaccines and T-cell therapy. We also provide new evidence for recombination between strains, which provides a further mechanism for the generation of diversity. Our results provide the first global view of EBV sequence variation and demonstrate an effective method for sequencing large numbers of genomes to further understand the genetics of EBV infection. IMPORTANCE Most people in the world are infected by Epstein-Barr virus (EBV), and it causes several human diseases, which occur at very different rates in different parts of the world and are linked to host immune system variation. Natural variation in EBV DNA sequence may be important for normal infection and for causing disease. Here we used rapid, cost-effective sequencing to determine 71 new EBV sequences from different sample types and locations worldwide. We showed geographic variation in EBV genomes and identified the most variable parts of the genome. We identified protein sequences that seem to have been selected by the host immune system and detected variability in known immune epitopes. This gives the first overview of EBV genome variation, important for designing vaccines and immune therapy for EBV, and provides techniques to investigate relationships between viral sequence variation and EBV-associated diseases.

CtBP is a corepressor of transcription that acts by inhibiting coactivators and recruiting to promoter control elements (via interactions with DNA-binding transcription factors) a complex of proteins that modify histones, repress transcription and silence gene expression. There are two highly homologous genes, CtBP1 and CtBP2 that encode CtBP. In addition, a CtBP1-related protein has been described that has 11 fewer amino acids at its N-terminus than CtBP1. This variant of CtBP1--known as CtBP3 or BARS [Brefeldin A (BFA) ribosylated substrate]--plays a critical role in the fragmentation of the Golgi complex at the onset of mitosis. Although there are some reports of CtBP with a cytoplasmic distribution after transfection, it is unclear how in normal cells a nuclear corepressor regulates the behavior of the Golgi complex at the onset of mitosis. Using polyclonal rabbit antibodies that were raised against a human CtBP1-GST fusion-protein, here we show by immunofluorescence laser-scanning confocal microscopy that in mitotic cells a species of CtBP becomes associated with the centrosomes at the onset of prophase and then throughout mitosis. The association can be seen in both cycling mitotic cells and nocodazole-arrested cells. The interaction was confirmed by coimmunoprecipitation and centrosome isolation. Since centrosomes are considered to be the organising centre for Golgi morphogenesis, the interaction demonstrated here may explain how a nuclear corepressor of transcription can exert a regulatory effect on the Golgi complex at a specific stage of the cell cycle.

Marek’s disease virus (MDV) is an oncogenic herpesvirus that induces fatal T cell lymphomas in chickens. With more than 20 billion doses of vaccine used annually, vaccination constitutes the cornerstone of Marek’s disease control. Despite the success of vaccination, evolution of virulence among MDV strains continues to threaten the effectiveness of the current Marek’s disease vaccines. MDV-encoded protein MEQ (MDV EcoRI Q) probably acts as a transcription factor and is considered to be the major MDV oncoprotein. MEQ sequence shows a Pro-Leu-Asp-Leu-Ser (PLDLS) motif known to bind C-terminal-binding protein (CtBP), a highly conserved cellular transcriptional corepressor with roles in the regulation of development, proliferation, and apoptosis. Here we show that MEQ can physically and functionally interact with CtBP through this motif and that this interaction is critical for oncogenesis because mutations in the CtBP-interaction domain completely abolished oncogenicity. This direct role for MEQ–CtBP interaction in MDV oncogenicity highlights the convergent evolution of molecular mechanisms of neoplastic transformation by herpesviruses because Epstein–Barr virus oncoproteins EBNA 3A and 3C also interact with CtBP. We also demonstrate that the nononcogenic MDV generated by mutagenesis of the CtBP-interaction domain of MEQ has the potential to be an improved vaccine against virulent MDV infection. Engineering MDV with precisely defined attenuating mutations, therefore, represents an effective strategy for generating new vaccines against this major poultry disease.

The Polo-like kinases (Plks) are a highly conserved family of protein kinases that function in regulation of cell cycle and DNA damage-induced checkpoints. Evidence of a tumor suppressor function for the Plks in human neoplasia is lacking. Here, we report that Snk/Plk2 is transcriptionally down-regulated in B-cell neoplasms. Silencing occurs with very high frequency in Burkitt lymphoma (BL) but is also detected in B-cell neoplasms of other types and is associated with aberrant cytosine methylation in the CpG island located at the 5' end of the SNK/PLK2 gene. Silencing is specific to malignant B cells because SNK/PLK2 was unmethylated (and expressed) in primary B lymphocytes, in EBV-immortalized B lymphoblastoid cell lines (LCLs), and in adenocarcinomas (of the breast) and squamous-cell carcinomas (of the head and neck). Expression of Snk/Plk2 in BL cell lines was restored by demethylating agents. The related PLK1 and PLK3 (FNK/PRK) genes were overexpressed in BL cell lines lacking Snk/Plk2 expression, consistent with functional degeneracy among the Plk family. Ectopic expression of Snk/Plk2 in BL cells resulted in apoptosis, a potential mechanistic basis underlying the strong selective pressure for abrogation of Snk/Plk2 function in B-cell neoplasia.

A yeast two-hybrid screen using EBNA3C as bait revealed an interaction between this Epstein–Barr virus (EBV)-encoded nuclear protein and the C8 (α7) subunit of the human 20S proteasome. The interaction was confirmed by glutathione S-transferase (GST) pull-down experiments and these also revealed that the related proteins EBNA3A and EBNA3B can bind similarly to C8/α7. The interaction between these viral proteins and GST–C8/α7 was shown to be significantly more robust than the previously reported interaction between C8/α7 and the cyclin-dependent kinase inhibitor p21WAF1/CIP1. Co-immunoprecipitation of the EBNA3 proteins with C8/α7 was also demonstrated after transfection of expression vectors into B cells. Consistent with this ability to bind directly to an α-subunit of the 20S proteasome, EBNAs 3A, 3B and 3C were all degraded in vitro by purified 20S proteasomes. However, surprisingly, no sign of proteasome-mediated turnover of these latent viral proteins in EBV-immortalized B cells could be detected, even in the presence of gamma interferon. In actively proliferating lymphoblastoid cell lines, EBNAs 3A, 3B and 3C appear to be remarkably stable, with no evidence of either de novo synthesis or proteasome-mediated degradation.

Human B cells isolated from peripheral blood were activated and induced to proliferate by either Epstein–Barr virus (EBV) or the T cell-derived mitogens CD40 ligand (CD40L) plus interleukin (IL)-4. Although both populations initially proliferated as B-blasts, significant differences were revealed over a longer period. EBV infection resulted in continuously proliferating lymphoblastoid cell lines (LCLs), whereas most of the CD40L/IL-4-stimulated B cells had a finite proliferative lifespan of 3–4 weeks. Cell cycle analysis, trypan blue staining and Western blot analysis for cleavage of poly(ADP-ribose) polymerase (PARP) all demonstrated that the decrease in proliferation in CD40L/IL-4-stimulated B cells is not due to cell death. Instead, these cells arrest, accumulate in G0/G1and undergo alterations in cell surface marker expression, cellular morphology and immunoglobulin production, all consistent with plasmacytoid differentiation. In contrast, B cells infected with EBV continued to proliferate and retained a blast-like phenotype. Differences in both cytokine production and the expression of cell cycle regulators were identified between the two B-cell populations, which might contribute to the differentiation of the CD40L/IL-4-stimulated B cells and suggest potential mechanisms by which EBV may overcome this. The study has also identified a window of opportunity during which a comparison of isogenic populations of EBV- and mitogen-driven B blasts can be made.

Epstein-Barr virus (EBV) is involved in the pathogenesis of several B cell lymphoproliferations, but the precise contribution it makes to the aetiology of each remains unclear. In vitro, the virus has potent growth transforming activity and efficiently induces the continuous proliferation of normal human B cells. A comparison of EBV-infected primary B cells with an isogenic population induced to proliferate by CD40-ligand (CD40L) and IL4 has revealed that EBV can override - by a novel mechanism - the p53/pRb-mediated G1 checkpoint activated in normal B cells by a genotoxic stress. In cells responding to cisplatin, although p53 is stabilized and activated, EBV latent gene expression appears to inhibit the accumulation of newly synthesized p21(WAF1/CIP1) and the downregulation of cyclin D2 that occur in the normal cells. Consequently, in the EBV-infected cells, CDK2 remains active, hyperphosphorylation of pRb is maintained and the replication of damaged DNA can occur. Under conditions of severe genomic stress, this absence of p21(WAF1/CIP1) function can result in apoptosis; however, when damage is less sustained, genomic instability may arise and this in turn could contribute to the development of a variety of EBV-associated B cell malignancies.

Expression of the lytic cycle genes of Epstain-Barr virus (EBV) is induced in type I Burkitt's lymphoma-derived cells by treatment with phorbol esters (e.g., phorbol myristate acetate [PMA]), anti-immunoglobulin, or the cytokine transforming growth factor β (TGF-β). Concomitantly, all these agents induce apoptosis as judged by a sub-G1fluorescence-activated cell sorter (FACS) profile, proteolytic cleavage of poly(ADP-ribose) polymerase (PARP) and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining. However, caspase activation is not required for induction of the lytic cycle since the latter is not blocked by the caspase inhibitor ZVAD. Furthermore, not all agents that induce apoptosis in these cultures (for example, cisplatin and ceramide) induce the EBV lytic programme. Although it is closely associated with the lytic cycle, apoptosis is neither necessary nor sufficient for its activation. Multiparameter FACS analysis of cultures treated with PMA, anti-Ig, or TGF-β revealed BZLF1-expressing cells distributed in different phases of the cell cycle according to which inducer was used. However, BZLF1-positive cells did not appear to undergo apoptosis and accumulate with a sub-G1DNA content, irrespective of the inducer used. This result, which suggests that lytic gene expression is protective, was confirmed and extended by immunofluorescence staining doubled with TUNEL analysis. BZLF1- and also gp350-expressing cells were almost always shown to be negative for TUNEL staining. Similar experiments using EBV-positive and -negative subclones of Akata BL cells carrying an episomal BZLF1 reporter plasmid confirmed that protection from apoptosis was associated with the presence of the EBV genome. Finally, treatment with phosphonoacetic acid or acyclovir prior to induction with PMA, anti-Ig, or TGF-β blocked the protective effect in Mutu-I cells. These data suggest that a late gene product(s) may be particularly important for protection against caspase activity and cell death.

Undifferentiated nasopharyngeal carcinoma (NPC) is an epithelial malignancy that is consistently associated with Epstein-Barr virus (EBV) but which very rarely has p53 gene mutations in primary tumours. Since the tumour suppressor p53 is mutated in most human cancers or the wild type protein is inactivated in a significant number of the remainder, here we have investigated cellular factors that could compromise p53 function in primary NPC. Twenty-five primary tumours were judged to carry only wild type p53 by SSCP analysis of all exons and sequence determination of exons 4-9. Only one tumour was found to express significant levels of hMdm2 and in 24/25 there were no detectable mutations or deletions in exons 1beta and 2 of the p14(ARF) gene. However, immunohistochemistry consistently revealed that all the tumour cells express substantial amounts of the p53-related protein p63. Semi-quantitative RT-PCR analysis of mRNA from tumour biopsies showed that the dominant species expressed was invariably the truncated deltaN-isotype. Since this can block p53-mediated transactivation, it is potentially a dominant-negative isoform. In normal nasopharyngeal epithelium the distribution of p63 was restricted to the proliferating basal and suprabasal layers. We suggest that deltaN-p63 is a good candidate as a suppressor of wild type p53 function in these tumours and also that it may prove to be a valuable diagnostic marker for undifferentiated NPC.

Several Epstein-Barr virus (EBV)-negative Burkitt lymphoma-derived cell lines (for example, BL41 and Ramos) are extremely sensitive to genotoxic drugs despite being functionally null for the tumor suppressor p53. They rapidly undergo apoptosis, largely from G(2)/M of the cell cycle. 5-bromo-2'-deoxyuridine labeling experiments showed that although the treated cells can pass through S phase, they are unable to complete cell division, suggesting that a G(2)/M checkpoint is activated. Surprisingly, latent infection of these genotoxin-sensitive cells with EBV protects them from both apoptosis and cell cycle arrest, allowing them to complete the division cycle. However, a comparison with EBV-immortalized B-lymphoblastoid cell lines (which have functional p53) showed that EBV does not block apoptosis per se but rather abrogates the activation of, or signalling from, the checkpoint in G(2)/M. Furthermore, analyses of BL41 and Ramos cells latently infected with P3HR1 mutant virus, which expresses only a subset of the latent viral genes, showed that LMP-1, the main antiapoptotic latent protein encoded by EBV, is not involved in the protection afforded here by viral infection. This conclusion was confirmed by analysis of clones of BL41 stably expressing LMP-1 from a transfected plasmid, which respond like the parental cell line. Although steady-state levels of Bcl-2 and related proteins varied between BL41 lines and clones, they did not change significantly during apoptosis, nor was the level of any of these anti- or proapoptotic proteins predictive of the outcome of treatment. We have demonstrated that a subset of EBV latent gene products can inactivate a cell cycle checkpoint for monitoring the fidelity and timing of cell division and therefore genomic integrity. This is likely to be important in EBV-associated growth transformation of B cells and perhaps tumorigenesis. Furthermore, this study suggests that EBV will be a unique tool for investigating the intimate relationship between cell cycle regulation and apoptosis.

EBNA3C can specifically repress the expression of reporter plasmids containing EBV Cp latency-associated promoter elements. Cp is normally the main promoter for EBNA mRNA initiation, so it appears that EBNA3C contributes to a negative autoregulatory control loop. By mutational analysis it was previously established that this repression is consistent with EBNA3C being targeted to Cp by binding the cellular sequence-specific DNA-binding protein CBF1 (also known as recombination signal-binding protein [RBP]-Jκ. Further analysis suggested that in vivo a corepressor interacts with EBNA3C in this DNA binding complex. Results presented here are all consistent with a component of such a corepressor exhibiting histone deacetylase activity. The drug trichostatin A, which specifically inhibits histone deacetylases, relieved two- to threefold the repression of Cp induced by EBNA3C in two different cell types. Moreover, repression of pTK-CAT-Cp4× by EBNA3C was specifically enhanced by cotransfection of an expression plasmid for human histone deacetylase-1 (HDAC1). Consistent with these functional assays, in vitro-translated HDAC1 bound to a glutathione S -transferase (GST) fusion protein including full-length EBNA3C, and in the reciprocal experiment EBNA3C bound to a GST fusion with the N terminus of HDAC1. Coimmunoprecipitations also revealed an EBNA3C-HDAC1 interaction in vivo, and GST-EBNA3C bound functional histone deacetylase enzyme activity from HeLa cell nuclear extracts. The region of EBNA3C involved in the interaction with HDAC1 appears to correspond to the region which is necessary for binding to CBF1/RBP-Jκ. A direct physical interaction between EBNA3C and HDAC1 was demonstrated with recombinant proteins purified from bacterial cells, and we therefore conclude that HDAC1 and CBF1/RBP-Jκ bind to the same or adjacent regions of EBNA3C. These data suggest that recruitment of histone deacetylase activity makes a significant contribution to the repression of transcription from Cp because EBNA3C bridges an interaction between CBF1/RBP-Jκ and HDAC1.

The status of p53 was investigated in breast tumours arising in germ-line carriers of mutant alleles of BRCA1 and BRCA2 and in a control series of sporadic breast tumours. p53 expression was detected in 20/26 (77%) BRCA1-, 10/22 (45%) BRCA2-associated and 25/72 (35%) grade-matched sporadic tumours. Analysis of p53 sequence revealed that the gene was mutant in 33/50 (66%) BRCA-associated tumours, whereas 7/20 (35%) sporadic grade-matched tumours contained p53 mutation (P

EBNA3C is a potent repressor of transcription when bound to DNA as a fusion with the DNA binding domain (DBD) of GALA. A survey of promoters has revealed that the wild-type, unfused EBNA3C can specifically repress expression from reporter plasmids containing the Epstein-Barr virus Cp latency-associated promoter. Repression of Cp activity required amino acids 207 to 368, which encompasses a region resembling a basic DBD adjacent to a leucine zipper DNA binding motif and a site which binds to the cellular factor CBF1/RBP-Jkappa. However, amino acids 207 to 368 are dispensable when the protein is bound to DNA as a fusion with the GAL4 DBD, thus implicating this region in DNA binding. Mutation of the CBF1/RBP-Jkappa binding site in EBNA3C abrogated repression, strongly suggesting that CBF1/RBP-Jkappa is necessary for targeting the viral protein to Cp. Consistent with this result, mutation of the EBNA2 response element (a CBF1/RBP-Jkappa binding site) in Cp also prevented significant repression. In addition, amino acids 346 to 543, which were previously defined as important for the repressor activity of the GAL4-EBNA3C fusion proteins, also appear to be necessary for the repression of Cp. Since repression by these fusions was not observed in all cell types, it seems likely that EBNA3C either depends on a corepressor which may interact with amino acids 346 to 543 or is modified in a cell-specific manner in order to repress. These data are consistent with EBNA3C contributing to the regulation of EBNA expression in latently infected B cells through CBF1/RBP-Jkappa and another factor, but this need not directly involve EBNA2. Finally, although it has been reported that EBNA3C can upregulate CD21 in some B cells, we were unable to demonstrate any effect of EBNA3C on reporter plasmids which contain the CD21 promoter.

The expression of Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA3C) is essential for the activation and immortalization of human B lymphocytes by EBV. EBNA3C consists of 992 amino acids and includes a potential bZIP motif and regions rich in acidic, proline, and glutamine residues. Thus, EBNA3C resembles several trans regulators of gene expression. It has recently been shown that a fragment of EBNA3C can activate reporter gene expression when fused to the DNA-binding domain of GAL4 (D. Marshall and C. Sample, J. Virol. 69:3624-3630,1995). Although EBNA3C binds DNA, a specific site for EBNA3C binding has not been identified; to test the ability of full-length EBNA3C to regulate transcription, EBNA3C (amino acids 11 to 992) was fused to the DNA-binding domain of GAL4. We show that this fusion protein does not transactivate but rather is a potent repressor of reporter gene expression. Repression is dependent on the dose of GAL4-EBNA3C and on the presence of GAL4-binding sites within reporter plasmids. Repression is not restricted to B cells nor is it species or promoter specific. Repression is independent of the location of the GAL4-binding sites relative to the transcription start site. A fragment of EBNA3C (amino acids 280 to 525) which represses expression in a manner which is nearly identical to that of the full-length protein has been identified; this fragment is rich in acidic and proline residues. A second, less potent repressor region located C terminal to amino acids 280 to 525 has also been identified; this domain is rich in proline and glutamine residues. We also show binding of EBNA3C, in vitro, to the TATA-binding protein component of TFIID, and this suggests a mechanism by which EBNA3C may communicate with the basal transcription complex.

Cisplatin treatment of Epstein-Barr virus-immortalized human B lymphoblastoid cell lines (LCLs) results in p53-mediated apoptosis which occurs largely in a population of cells at the G1/S boundary of the cell cycle. Cell cycle progression appears to be required for this apoptosis because arresting cells earlier in G1 inhibited apoptosis despite the accumulation of p53. Overexpression of wild-type p53 also induces apoptosis in an LCL. Therefore six mutant genes derived from Burkitt's lymphoma (BL) cells were assayed for their ability to induce apoptosis when similarly overexpressed. The same genes were analysed in transient transfection assays for their ability to transactivate appropriate reporter plasmids. A correlation between the ability of p53 to transactivate and induce apoptosis was revealed. The only mutant capable of transactivation also induced apoptosis. Further analysis of the BL lines in which p53 had been characterized showed that whereas some lines were essentially resistant to cisplatin, three were rapidly induced to undergo apoptosis. All three have a single p53 allele encoding a mutant which is incapable of transactivation or (for two tested) mediating apoptosis when expressed in an LCL. Cell cycle analysis revealed that this apparently p53-independent apoptosis did not follow G1 arrest but in fact occurred largely in cells distributed in the G2/M phase of the cell cycle. These data suggest the existence of a second checkpoint in the G2 or M phase which, in the absence of a functional p53, is the primary point of entry into the apoptosis programme following DNA damage.

To explore the role of p16INK4a as an intrinsic barrier to B cell transformation by EBV, we transformed primary B cells from an individual homozygous for a deletion in the CDKN2A locus encoding p16INK4a and p14ARF. Using recombinant EBV-BAC viruses expressing conditional EBNA3C (3CHT), we developed a system that allows inactivation of EBNA3C in lymphoblastoid cell lines (LCLs) lacking active p16INK4a protein but expressing a functional 14ARF-fusion protein (p14/p16). The INK4a locus is epigenetically repressed by EBNA3C – in cooperation with EBNA3A – despite the absence of functional p16INK4a. Although inactivation of EBNA3C in LCLs from normal B cells leads to an increase in p16INK4a and growth arrest, EBNA3C inactivation in the p16INK4a-null LCLs has no impact on the rate of proliferation, establishing that the repression of INK4a is a major function of EBNA3C in EBV-driven LCL proliferation. This conditional LCL system allowed us to use microarray analysis to identify and confirm genes regulated specifically by EBNA3C, independently of proliferation changes modulated by the p16INK4a-Rb-E2F axis. Infections of normal primary B cells with recombinant EBV-BAC virus from which EBNA3C is deleted or with 3CHT EBV in the absence of activating ligand 4-hydroxytamoxifen, revealed that EBNA3C is necessary to overcome an EBV-driven increase in p16INK4a expression and concomitant block to proliferation 2–4 weeks post-infection. If cells are p16INK4a-null, functional EBNA3C is dispensable for the outgrowth of LCLs., Author Summary Epstein-Barr virus (EBV) is a causative agent of several types of B cell lymphoma. In human B cells, EBV reduces protein levels of at least two tumour suppressors that would otherwise be activated in response to over-expressed oncogenes. These proteins are BIM, which induces cell death and p16INK4a, which prevents cell proliferation. Repression of both is via epigenetic methylation of histones and is dependent on expression of both EBNA3A and EBNA3C – two EBV proteins required for the transformation of normal B cells into lymphoblastoid cell lines (LCLs). In this report we have used EBV with a conditionally active EBNA3C – active only in the presence of 4-hydroxytamoxifen – together with B cells from an individual carrying a homozygous deletion of p16INK4a to confirm that regulation of p16INK4a expression is a major function of EBNA3C and demonstrate that if B cells lack p16INK4a, then EBNA3C is no longer required for EBV-driven proliferation of LCLs. Furthermore we show that early after the infection of normal B cells, EBV induces p16INK4a accumulation that – if unchecked by EBNA3C (and EBNA3A) – prevents LCL outgrowth. Formal proof that p16INK4a is the main target of EBNA3C comes with the production of p16-null LCLs that have never expressed functional EBNA3C.

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.

Meq is the major Marek's disease virus (MDV)-encoded oncoprotein and is essential for T-cell lymphomagenesis. Meq and several noncoding RNAs, including three microRNA (MiR) clusters, are expressed from the repeats of the MDV genome during latent infection of T cells. To investigate the state of the chromatin in this and flanking regions, we carried out chromatin immunoprecipitation (ChIP) analysis of covalent histone modifications and associated bound proteins. T-cell lines and a lymphoma were compared. The chromatin around the promoters for Meq and the noncoding RNAs in both cell lines and the lymphoma were associated with H3K9 acetylation and H3K4 trimethylation, which are marks of transcriptionally active chromatin. These correlated with bound Meq–c-Jun heterodimers. The only binding site for Meq homodimers is located at the lytic origin of replication (OriLyt), next to the lytic gene pp38 . This region lacked active marks and was associated with repressive histone modifications (H3K27 and H3K9 trimethylation). DNA CpG methylation was investigated using methylated DNA precipitation (MeDP). In cell lines, DNA methylation was abundant across the repeats but noticeably reduced or absent around the active promoters. In primary tumors, CpG methylation occurred less than 2 months after infection, focused within the ICP4 gene. These data suggest that nonrandom de novo DNA methylation occurs early in lymphomagenesis. In addition, the histone data indicate a role for Meq in the epigenetic regulation of the MDV genome repeats in transformed T cells and suggest that the OriLyt region and the Meq/ MiR region might be separated by chromatin boundary elements, and preliminary data on CTCF binding are consistent with this.

Transforming growth factor-beta (TGF-beta) potently induces apoptosis in Burkitt's lymphoma (BL) cell lines and in explanted primary human B lymphocytes. The physiological relevance and mechanism of TGF-beta-mediated apoptosis induction in these cells remains to be determined. Here we demonstrate the requirement for TGF-beta-mediated regulation of BIK and BCL-X(L) to activate an intrinsic apoptotic pathway in centroblastic BL cells. TGF-beta directly induced transcription of BIK and a consensus Smad-binding element identified in the BIK promoter recruits TGF-beta-activated Smad transcription factor complexes in vivo. TGF-beta also transcriptionally repressed expression of the apoptosis inhibitor BCL-X(L). Inhibition of BCL-X(L) sensitised BL cells to TGF-beta-induced apoptosis whereas overexpression of BCL-X(L) or suppression of BIK by shRNA, diminished TGF-beta-induced apoptosis. BIK and BCL-X(L) were also identified as TGF-beta target genes in purified normal human centroblast B cells and immunohistochemical analyses of tonsil tissue revealed widespread TGF-beta receptor-regulated Smad activation and a focal pattern of BIK expression. Furthermore, using a selective inhibitor of the TGF-beta receptor we provide evidence that autocrine TGF-beta signalling through ALK5 contributes to the default apoptotic programme in normal human centroblasts undergoing spontaneous apoptosis. Our data suggests that TGF-beta may act as a physiological mediator of human germinal centre homoeostasis by regulation of BIK and BCL-X(L).

Deletion of the terminal repeats (TR) from herpesvirus saimiri (HVS) renders it unable to produce infectious virus or generate plaques. However, a TR-deleted HVS bacterial artificial chromosome can form replication compartments. Complementation of this mutant shows that one copy of the TR, plus the right junction of the genome with the TR, is sufficient for efficient plaque formation and generation of infectious virus. Within the TR unit, the region around the cleavage site of the genome appears both necessary and sufficient for virus production. Analysis of episomes from productive cells indicates a propensity to amplify TR numbers during the lytic cycle.

Epstein-Barr virus (EBV) is associated with a number of human cancers, and latent EBV gene expression has been reported to interfere with cell cycle checkpoints and cell death pathways. Here we show that latent EBV can compromise the mitotic spindle assembly checkpoint and rescue Burkitt's lymphoma (BL)-derived cells from caspase-dependent cell death initiated in aberrant mitosis. This leads to unscheduled mitotic progression, resulting in polyploidy and multi- and/or micronucleation. The EBV latent genes responsible for this phenotype are expressed from the P3HR1 strain of virus and several viruses with similar genomic deletions that remove the EBNA2 gene. Although EBNA2 and the latent membrane proteins are not expressed, the EBNA3 proteins are present in these BL cells. Survival of the EBV-positive cells is not consistently associated with EBV lytic gene expression or with the genes that are expressed in EBV latency I BL cells (i.e., EBNA1, EBERs, and BARTs) but correlates with reduced expression of the cellular proapoptotic BH3-only protein Bim. These data suggest that a subset of latent EBV gene products may increase the likelihood of damaged DNA being inherited because of the impaired checkpoint and enhanced survival capacity. This could lead to greater genetic diversity in progeny cells and contribute to tumorigenesis. Furthermore, since it appears that this restricted latent EBV expression interferes with the responses of Burkitt's lymphoma-derived cells to cytotoxic drugs, the results of this study may have important therapeutic implications in the treatment of some BL.

To determine whether latent Epstein-Barr virus (EBV) modifies DNA damage responses in B lymphocytes, cells were treated with agents either producing DNA cross-links and adducts or generating double-strand breaks. The cyclin-dependent kinase inhibitor p21 WAF1 accumulated in mitogen-stimulated primary B cells following exposure to all genotoxins tested. In contrast, when proliferation was EBV driven, p21 WAF1 failed to accumulate after treatment with the DNA adduct-producing agents. The tumor suppressor p53 was stabilized and phosphorylated after all treatments, irrespective of whether latent EBV was present. This suggests that regulatory pathways upstream of p53 are unaffected by latent EBV but downstream effectors are altered if DNA adducts or distortions are involved.

Inactivation of retinoblastoma protein (Rb) plays a critical role in the development of human malignancies. It has been shown that Rb is degraded through a proteasome-dependent pathway, yet the mechanism is largely unclear. MDM2 is frequently found amplified and overexpressed in a variety of human tumors. In this study, we find that MDM2 promotes Rb degradation in a proteasome-dependent and ubiquitin-independent manner. We show that Rb, MDM2, and the C8 subunit of the 20S proteasome interact in vitro and in vivo and that MDM2 promotes Rb-C8 interaction. Expression of wild-type MDM2, but not the mutant MDM2 defective either in Rb interaction or in RING finger domain, promotes cell cycle S phase entry independent of p53. Furthermore, MDM2 ablation results in Rb accumulation and inhibition of DNA synthesis. Taken together, these findings demonstrate that MDM2 is a critical negative regulator for Rb and suggest that MDM2 overexpression contributes to cancer development by destabilizing Rb.

CtBP (carboxyl-terminal binding protein) has been shown to be a highly conserved co-repressor of transcription that is important in development, cell cycle regulation, and transformation. Viral proteins E1A and EBNA3C and all the various Drosophila and vertebrate transcription factors to which CtBP has been reported to bind contain a conserved "PXDLS" CtBP-interaction domain. Here we show that EBNA3A binds CtBP both in vitro and in vivo but that this interaction does not require a near consensus (98)PLDLR(102) motif in the NH(2) terminus of EBNA3A. However, further deletion and mutation analysis revealed that CtBP interacts with this viral protein through a cryptic, bipartite motif located in the COOH terminus of EBNA3A. The two components of this binding domain are similar to the canonical PXDLS motif but do not include the highly conserved, and normally critical, first proline residue. These nonconsensus sites, (857)ALDLS(861) and (886)VLDLS(890), synergize to produce very efficient binding to CtBP. Interaction with CtBP was shown to be important in the repression of transcription by EBNA3A and in the ability of EBNA3A to cooperate with activated Ras to immortalize and transform primary rat embryo fibroblasts. Similar bipartite sequences can be found in other viral and cellular proteins that can interact with CtBP, including the retinoblastoma-interacting protein-methyltransferase RIZ, the oncoprotein EVI1, and Marek's disease virus transforming protein Meq.

CtBP has been shown to be a highly conserved corepressor of transcription. E1A and all the various transcription factors to which CtBP binds contain a conserved PLDLS CtBP-interacting domain, and EBNA3C includes a PLDLS motif (amino acids [aa] 728 to 732). Here we show that EBNA3C binds to CtBP both in vitro and in vivo and that the interaction requires an intact PLDLS. The C terminus of EBNA3C (aa 580 to 992) has modest trans -repressor activity when it is fused to the DNA-binding domain of Gal4, and deletion or mutation of the PLDLS sequence ablates this and unmasks a transactivation function within the fragment. However, loss of the CtBP interaction motif had little effect on the ability of full-length EBNA3C to repress transcription. A striking correlation between CtBP binding and the capacity of EBNA3C to cooperate with (Ha-)Ras in the immortalization and transformation of primary rat embryo fibroblasts was also revealed.