Epstein-Barr Virus (EBV) latent infection is associated with several human malignancies and is a causal agent of lymphoproliferative diseases during immunosuppression. While inhibitors of herpesvirus DNA polymerases, like gancyclovir, reduce EBV lytic cycle infection, these treatments have limited efficacy for treating latent infection. EBNA1 is an EBV-encoded DNA-binding protein required for viral genome maintenance during latent infection.
Here, we report the identification of a new class of small molecules that inhibit EBNA1 DNA binding activity. These compounds were identified by virtual screening of 90,000 low molecular mass compounds using computational docking programs with the solved crystal structure of EBNA1. Four structurally related compounds were found to inhibit EBNA1-DNA binding in biochemical assays with purified EBNA1 protein. Compounds had a range of 20–100 µM inhibition of EBNA1 in fluorescence polarization assays and were further validated for inhibition using electrophoresis mobility shift assays. These compounds exhibited no significant inhibition of an unrelated DNA binding protein. Three of these compounds inhibited EBNA1 transcription activation function in cell-based assays and reduced EBV genome copy number when incubated with a Burkitt lymphoma cell line.
These experiments provide a proof-of-principle that virtual screening can be used to identify specific inhibitors of EBNA1 that may have potential for treatment of EBV latent infection.
Latent Epstein-Barr virus (EBV) infection causes human lymphomas and carcinomas. EBV usually persists as an episome in malignant cells. EBV episome persistence, replication, and gene expression are dependent on EBNA1 binding to multiple cognate sites in oriP. To search for inhibitors of EBNA1- and oriP-dependent episome maintenance or transcription, a library of 40,550 small molecules was screened for compounds that inhibit EBNA1- and oriP-dependent transcription and do not inhibit EBNA1- and oriP-independent transcription. This screening identified roscovitine, a selective inhibitor of cyclin-dependent kinase 1 (CDK1), CDK2, CDK5, and CDK7. Based on motif predictions of EBNA1 serine 393 as a CDK phosphorylation site and 486RALL489 and 580KDLVM584 as potential cyclin binding domains, we hypothesized that cyclin binding to EBNA1 may enable CDK1, -2, -5, or -7 to phosphorylate serine 393. We found that Escherichia coli-expressed EBNA1 amino acids 387 to 641 were phosphorylated in vitro by CDK1-, -2-, -5-, and -7/cyclin complexes and serine 393 phosphorylation was roscovitine inhibited. Further, S393A mutation abrogated phosphorylation. S393A mutant EBNA1 was deficient in supporting EBNA1- and oriP-dependent transcription and episome persistence, and roscovitine had little further effect on the diminished S393A mutant EBNA1-mediated transcription or episome persistence. Immunoprecipitated FLAG-EBNA1 was phosphorylated in vitro, and roscovitine inhibited this phosphorylation. Moreover, roscovitine decreased nuclear EBNA1 and often increased cytoplasmic EBNA1, whereas S393A mutant EBNA1 was localized equally in the nucleus and cytoplasm and was unaffected by roscovitine treatment. These data indicate that roscovitine effects are serine 393 specific and that serine 393 is important in EBNA1- and oriPCp-dependent transcription and episome persistence.
The Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA-3C) regulates virus and cell genes and is essential for EBV-mediated transformation of primary B lymphocytes. EBNA-3C associates with the cellular DNA sequence-specific transcription factors RBP-Jκ and PU.1 and coactivates the EBV LMP1 promoter with EBNA-2 in BL2 and Raji cells under conditions of restrictive growth. We now find that EBNA-3C is similar to EBNA-LP in coactivating the LMP1 promoter with EBNA-2 in non-EBV-infected Burkitt lymphoma cells under conditions of maximal cell growth, whereas the EBV Cp promoter is repressed under the same conditions. EBNA-3A and EBNA-3B coactivation are at most 40% that of EBNA-3C. The RBP-Jκ binding sites of EBNA-2 and the LMP1 promoter are not required for EBNA-3C coactivation, whereas the PU.1 site in the LMP1 promoter is required for EBNA-2-mediated activation and EBNA-3C coactivation. EBNA-3C amino acids (aa) 365 to 545, including most of the previously identified repression domain (M. Bain, R. J. Watson, P. J. Farrell, and M. J. Allday, J. Virol. 70:2481–2489, 1996), are necessary and sufficient for coactivation with wild-type EBNA-2. EBNA-3C can also coactivate with the EBNA-2 acidic activating domain; this activation does not require aa 343 to 545. These data indicate that there are at least two mechanisms by which EBNA-3C coactivates the LMP1 promoter with EBNA-2. Of the proteins that interact with EBNA-3C in a yeast two-hybrid screen, only the ubiquitin-like proteins SUMO-1 and SUMO-3/hSMT3B map to aa 365 to 545, implicating these molecules in EBNA-3C coactivation. In addition, SUMO-1 associates at a high level with EBNA-3C in lymphoblasts. Promoter coactivation by EBNA-3C is likely to be important in ensuring adequate levels of LMP1, while inhibition of the EBNA-Cp promoter under the same conditions prevents uncontrolled up-regulation of EBNA expression from a positive-feedback loop.
The ability of Epstein-Barr virus (EBV) latent infection nuclear protein EBNA3C to activate transcription of two EBNA2-responsive genes and to inhibit EBNA2 activation of transcription in transient-transfection assays appears to be due to its ability to interact with RBPJkappa, a cell protein that links EBNA2 to its response elements. We now show that EBNA3A and EBNA3B expressed in non-EBV-infected Burkitt tumor lymphoblasts are similar to EBNA3C in binding to glutathione S-transferase-RBPJkappa in vitro and in coimmunoprecipitating from cell lysates with antibody to RBPJkappa. EBNA3A and EBNA3B can also inhibit the interaction of RBPJkappa with cognate DNA in vitro. Although EBNA3 open reading frames are each close to 1,000 codons long, EBNA3A amino acids 1 to 138, EBNA3B amino acids 1 to 311, and EBNA3C amino acids 1 to 183 are sufficient for RBPJkappa interaction, while EBNA3B amino acids I to 109 have less or no binding. The RBPJkappa interacting domains overlap with the most highly conserved domain (amino acids 90 to 320) among the EBNA3 proteins. Thus, the EBNA3 gene family appears to have evolved to differentially regulate promoters with RBPJkappa binding sites. EBNA2, EBNA3A, and EBNA3C are important in EBV transformation of primary human B lymphocytes. Their interaction with RBPJkappa links EBV transformation to the notch signaling pathway and the effects of activated notch in T-cell leukemogenesis.
The Epstein-Barr virus (EBV) nuclear antigen EBNA-1 plays an integral role in the maintenance of latency in EBV-infected B lymphocytes. EBNA-1 binds to sequences within the plasmid origin of replication (oriP). It is essential for the replication of the latent episomal form of EBV DNA and may also regulate the expression of the EBNA group of latency gene products. We have used sequence-specific DNA-binding assays to purify EBNA-1 away from nonspecific DNA-binding proteins in a B-lymphocyte cell extract. The availability of this eucaryotic protein has allowed an examination of the interaction of EBNA-1 with its specific DNA-binding sites and an evaluation of possible roles for the different binding loci within the EBV genome. DNA filter binding assays and DNase I footprinting experiments showed that the intact Raji EBNA-1 protein recognized the two binding site loci in oriP and the BamHI-Q locus and no other sites in the EBV genome. Competition filter binding experiments with monomer and multimer region I consensus binding sites indicated that cooperative interactions between binding sites have relatively little impact on EBNA-1 binding to region I. An analysis of the binding parameters of the Raji EBNA-1 to the three naturally occurring binding loci revealed that the affinity of EBNA-1 for the three loci differed. The affinity for the sites in region I of oriP was greater than the affinity for the dyad symmetry sites (region II) of oriP, while the physically distant region III locus showed the lowest affinity. This arrangement may provide a mechanism whereby EBNA-1 can lowest affinity. This arrangement may provide a mechanism whereby EBNA-1 can mediate differing regulatory functions through differential binding to its recognition sequence.
Epstein-Barr Virus (EBV) is an oncogenic γ-herpesvirus that capably establishes both latent and lytic modes of infection in host cells and causes malignant diseases in humans. Nuclear antigen 2 (EBNA2)-mediated transcription of both cellular and viral genes is essential for the establishment and maintenance of the EBV latency program in B lymphocytes. Here, we employed a protein affinity pull-down and LC-MS/MS analysis to identify nucleophosmin (NPM1) as one of the cellular proteins bound to EBNA2. Additionally, the specific domains that are responsible for protein-protein interactions were characterized as EBNA2 residues 300 to 360 and the oligomerization domain (OD) of NPM1. As in c-MYC, dramatic NPM1 expression was induced in EBV positively infected B cells after three days of viral infection, and both EBNA2 and EBNALP were implicated in the transactivation of the NPM1 promoter. Depletion of NPM1 with the lentivirus-expressed short-hairpin RNAs (shRNAs) effectively abrogated EBNA2-dependent transcription and transformation outgrowth of lymphoblastoid cells. Notably, the ATP-bound state of NPM1 was required to induce assembly of a protein complex containing EBNA2, RBP-Jκ, and NPM1 by stabilizing the interaction of EBNA2 with RBP-Jκ. In a NPM1-knockdown cell line, we demonstrated that an EBNA2-mediated transcription defect was fully restored by the ectopic expression of NPM1. Our findings highlight the essential role of NPM1 in chaperoning EBNA2 onto the latency-associated membrane protein 1 (LMP1) promoters, which is coordinated with the subsequent activation of transcriptional cascades through RBP-Jκ during EBV infection. These data advance our understanding of EBV pathology and further imply that NPM1 can be exploited as a therapeutic target for EBV-associated diseases.
Epstein-Barr Virus (EBV) infects human B cells to establish a permanent infection in hosts, which can cause diseases ranging from infectious mononucleosis to a broad spectrum of human malignancies. The conversion of human primary B cells into indefinitely proliferating lymphoblastoid cell lines (LCLs) by in vitro EBV infection provides a suitable model for virus-mediated cellular transformation. Epstein-Barr nuclear antigen (EBNA) 2-mediated transcription is essential for the establishment and maintenance of EBV latent infection. In this report, we have extensively explored the mechanism by which EBNA2 activates the latency-specific LMP1 promoter to establish a permanent infection in B cells. We have identified and characterized the protein-protein interaction of EBNA2 with the nuclear shuttle protein nucleophosmin (NPM1) in vivo and in vitro. In particular, we have determined that the expression of NPM1 is promptly induced upon EBV infection and that EBNA2 has a role in activating NPM1 gene expression. Furthermore, we have shown that oligomerized NPM1 is charged by ATP and binds to EBNA2, which is crucial for its ability to stabilize its interaction with the DNA binding protein RBP-Jκ, which is in turn essential for supporting the transcriptional cascades of EBV latent infection. Our findings provide striking evidence to illustrate a new model for understanding EBV pathology.
Importance of the field
Epstein-Barr virus (EBV) is a ubiquitious human herpesvirus that is causally associated with endemic forms of Burkitt’s lymphoma (BL), nasopharyngeal carcinoma, and lymphoproliferative disease in immunosuppressed individuals. On a global scale, EBV infects over 90% of the adult population and is responsible for ~1% of all human cancers. To date, there is no efficacious drug or therapy for the treatment of EBV infection and EBV-related diseases.
Areas covered in this review
In this review, we discuss the existing anti-EBV inhibitors and those under development. We discuss the value of different molecular targets, including EBV lytic DNA replication enzymes, as well as proteins that are expressed exclusively during latent infection, like EBNA1 and LMP1. Since the atomic structure of the EBNA1 DNA binding domain has been described, it is an attractive target for in silico methods of drug design and small molecule screening. We discuss the use of computational methods that can greatly facilitate the development of novel inhibitors and how in silico screening methods can be applied to target proteins with known structures, like EBNA1, to treat EBV infection and disease.
What the reader will gain
The reader will be familiarized with the problems in targeting of EBV for inhibition by small molecules and how computational methods can greatly facilitate this process.
Take home message
Despite the impressive efficacy of nucleoside analogues for the treatment of herpesvirus lytic infection, there remain few effective treatments for latent infections. Since EBV-latent infection persists within and contributes to the formation of EBV-associated cancers, targeting EBV latent proteins is an unmet medical need. High throughput in silico screening can accelerate the process of drug discovery for novel and selective agents that inhibit EBV latent infection and associated disease.
Epstein-Barr virus (EBV); DNA polymerase; LMP1; EBNA1; computational screening
The nuclear antigen 3 family genes (EBNA-3, EBNA-4, and EBNA-6) of Epstein-Barr virus (EBV) are important for EBV-induced immortalization and survival of B lymphocytes. However, little is known about how the expression of these genes is regulated. Each of the EBNA-3, EBNA-4, and EBNA-6 genes consists of two exons separated by a small intron. Reverse transcriptase PCR assays revealed that the vast majority of the EBNA-3, EBNA-4, and EBNA-6 mRNA, expressed in transfected and EBV-infected B cells, retained intron sequences. Northern blot and S1 protection assays confirmed that most of the EBNA-3 mRNA contained intron. Examination of deletion mutants of EBNA-3 indicated that the EBNA-3 protein was not necessary for intron retention and that there was no splicing silencing element encoded in the EBNA-3 mRNA. Cell fractionation and RNA gradient analysis revealed that the unspliced EBNA 3 family mRNAs were transported into the cytoplasm and associated with the polysomes. However, Western blot analysis of FLAG-epitope tagged EBNA-3 gave no indication of the presence of splice variant protein forms of EBNA-3. In contrast, transiently transfected cells expressing EBNA-3 revealed a sixfold increase in EBNA-3 protein expression from the genomic EBNA-3 gene compared to EBNA-3 cDNA. These data show that the intronic sequences can influence EBNA-3 protein expression and suggest that intron retention may provide a means for the fine-tuning of expression of the individual EBNA 3 family genes.
The EBNA1 protein of Epstein-Barr virus (EBV) plays essential roles in enabling the replication and persistence of EBV genomes in latently infected cells and activating EBV latent gene expression, in all cases by binding to specific recognition sites in the latent origin of replication, oriP. Here we show that EBNA1 binding to its recognition sites in vitro is greatly stimulated by binding to the cellular deubiquitylating enzyme, USP7, and that USP7 can form a ternary complex with DNA-bound EBNA1. Consistent with the in vitro effects, the assembly of EBNA1 on oriP elements in human cells was decreased by USP7 silencing, whereas assembly of an EBNA1 mutant defective in USP7 binding was unaffected. USP7 affinity column profiling identified a complex between USP7 and human GMP synthetase (GMPS), which was shown to stimulate the ability of USP7 to cleave monoubiquitin from histone H2B in vitro. Accordingly, silencing of USP7 in human cells resulted in a consistent increase in the level of monoubquitylated H2B. The USP7-GMPS complex formed a quaternary complex with DNA-bound EBNA1 in vitro and, in EBV infected cells, was preferentially detected at the oriP functional element, FR, along with EBNA1. Down-regulation of USP7 reduced the level of GMPS at the FR, increased the level of monoubiquitylated H2B in this region of the origin and decreased the ability of EBNA1, but not an EBNA1 USP7-binding mutant, to activate transcription from the FR. The results indicate that USP7 can stimulate EBNA1-DNA interactions and that EBNA1 can alter histone modification at oriP through recruitment of USP7.
Epstein-Barr virus (EBV) infections persist for the lifetime of the host largely due to the actions of the EBNA1 viral protein. EBNA1 enables the replication and stable persistence of EBV genomes and activates the expression of other EBV genes by binding to specific DNA sequences in the EBV genome. We have shown that the cellular protein USP7 stimulates EBNA1 binding to its DNA sequences and that EBNA1 recruits USP7 to the EBV genome, which in turn recruits another cellular protein GMP synthetase. The complex of USP7 and GMP synthetase then functions to alter the chromatin structure at a region of the EBV genome that controls EBV persistence. These changes to the EBV genome are likely important for enabling the persistence of EBV genomes in infected cells.
EBNA-3A, -3B, and -3C are three latent infection nuclear proteins important for Epstein-Barr virus (EBV)-induced B-cell immortalization and the immune response to EBV infection. All three are hypothesized to function as transcriptional transactivators, but little is known about their precise mechanism of action or their role in EBV pathogenesis. We have cloned and studied the three EBNA-3 homologues from a closely related lymphocryptovirus (LCV) which naturally infects rhesus monkeys. The rhesus LCV EBNA-3A, -3B, and -3C homologues have 37, 40, and 36% amino acid identity with the EBV genes, respectively. Function, as measured by in vitro assays, also appears to be conserved with the EBV genes, since the rhesus LCV EBNA-3s can interact with the transcription factor RBP-Jκ and the rhesus LCV EBNA-3C encodes a Q/P-rich domain with transcriptional activation properties. In order to better understand the relationship between these EBV and rhesus LCV latent infection genes, we asked if the rhesus LCV EBNA-3 locus could be recombined into the EBV genome and if it could substitute for the EBV EBNA-3s when assayed for human B-cell immortalization. Recombination between the EBV genome and rhesus LCV DNA was reasonably efficient. However, these studies suggest that the rhesus LCV EBNA-3 locus was not completely interchangeable with the EBV EBNA-3 locus for B-cell immortalization and that at least one determinant of the species restriction for LCV-induced B-cell immortalization maps to the EBNA-3 locus. The overall conservation of EBNA-3 structure and function between EBV and rhesus LCV indicates that rhesus LCV infection of rhesus monkeys can provide an important animal model for studying the role of the EBNA-3 genes in LCV pathogenesis.
Epstein-Barr virus (EBV) causes a persistent infection in human B cells by establishing specific transcription programs to control B cell activation and differentiation. Transcriptional reprogramming of EBV infected B cells is predominantly driven by the action of EBV nuclear antigens, among them the transcriptional repressor EBNA3A. By comparing gene expression profiles of wt and EBNA3A negative EBV infected B cells, we have previously identified a broad array of cellular genes controlled by EBNA3A. We now find that genes repressed by EBNA3A in these cells are significantly enriched for the repressive histone mark H3K27me3, which is installed by Polycomb group (PcG) proteins. This PcG-controlled subset of genes also carries H3K27me3 marks in a variety of other tissues, suggesting that the commitment to PcG silencing is an intrinsic feature of these gene loci that can be used by EBNA3A. In addition, EBNA3A targets frequently reside in co-regulated gene clusters. To study the mechanism of gene repression by EBNA3A and to evaluate the relative contribution of PcG proteins during this process, we have selected the genomic neighbors CXCL10 and CXCL9 as a model for co-repressed and PcG-controlled genes. We show that EBNA3A binds to CBF1 occupied intergenic enhancers located between CXCL10 and CXCL9 and displaces the transactivator EBNA2. This impairs enhancer activity, resulting in a rapid transcriptional shut-down of both genes in a CBF1-dependent manner and initiation of a delayed gain of H3K27me3 marks covering an extended chromatin domain. H3K27me3 marks increase gradually and are maintained by EBNA3A. Our study provides direct evidence that repression by EBNA3A requires CBF1 and that EBNA3A and EBNA2 compete for access to CBF1 at identical genomic sites. Most importantly, our results demonstrate that transcriptional silencing by EBNA3A precedes the appearance of repressive PcG marks and indicate that both events are triggered by loss of enhancer activity.
Epstein-Barr virus (EBV) is a γ-herpesvirus which establishes a latent infection in human B cells and is associated with the pathogenesis of several types of cancer. Here, we report that cellular genes repressed by the EBV nuclear antigen 3A (EBNA3A) in EBV infected B cells frequently form contiguous clusters in the human genome and are committed to epigenetic silencing by Polycomb group (PcG) proteins. The chemokine genes CXCL10 and CXCL9 and their receptors on NK and T cells are critical weapons of the infected host to control herpesvirus infections. CXCL10 and CXCL9 are close neighbors within an extended PcG-controlled domain. We show that EBNA3A binds to intergenic enhancers located between CXCL10 and CXCL9 and displaces the transactivator EBNA2. This process impairs enhancer activity, resulting in a rapid transcriptional shut-down of both genes followed by a delayed gain of PcG histone marks. These PcG marks increase within the following weeks and are maintained by EBNA3A. Our results show that rapid transcriptional shut-down of distal genes and domain-wide PcG silencing is triggered by loss of enhancer activity and suggest that EBNA3A can reprogram the cellular genome in order to escape the immune surveillance of the host.
Latent infection by Epstein-Barr virus (EBV) requires both replication and maintenance of the viral genome. EBV nuclear antigen 1 (EBNA1) is a virus-encoded protein that is critical for the replication and maintenance of the genome during latency in proliferating cells. We have previously demonstrated that EBNA1 recruits the cellular origin recognition complex (ORC) through an RNA-dependent interaction with EBNA1 linking region 1 (LR1) and LR2. We now show that LR1 and LR2 bind to G-rich RNA that is predicted to form G-quadruplex structures. Several chemically distinct G-quadruplex-interacting drugs disrupted the interaction between EBNA1 and ORC. The G-quadruplex-interacting compound BRACO-19 inhibited EBNA1-dependent stimulation of viral DNA replication and preferentially blocked proliferation of EBV-positive cells relative to EBV-negative cell lines. BRACO-19 treatment also disrupted the ability of EBNA1 to tether to metaphase chromosomes, suggesting that maintenance function is also mediated through G-quadruplex recognition. These findings suggest that the EBNA1 replication and maintenance function uses a common G-quadruplex binding capacity of LR1 and LR2, which may be targetable by small-molecule inhibitors.
Epstein-Barr Nuclear Antigen 1 (EBNA1) is essential for Epstein-Barr virus to immortalize naïve B-cells. Upon binding a cluster of 20 cognate binding-sites termed the family of repeats, EBNA1 transactivates promoters for EBV genes that are required for immortalization. A small domain, termed UR1, that is 25 amino-acids in length, has been identified previously as essential for EBNA1 to activate transcription. In this study, we have elucidated how UR1 contributes to EBNA1's ability to transactivate. We show that zinc is necessary for EBNA1 to activate transcription, and that UR1 coordinates zinc through a pair of essential cysteines contained within it. UR1 dimerizes upon coordinating zinc, indicating that EBNA1 contains a second dimerization interface in its amino-terminus. There is a strong correlation between UR1-mediated dimerization and EBNA1's ability to transactivate cooperatively. Point mutants of EBNA1 that disrupt zinc coordination also prevent self-association, and do not activate transcription cooperatively. Further, we demonstrate that UR1 acts as a molecular sensor that regulates the ability of EBNA1 to activate transcription in response to changes in redox and oxygen partial pressure (pO2). Mild oxidative stress mimicking such environmental changes decreases EBNA1-dependent transcription in a lymphoblastoid cell-line. Coincident with a reduction in EBNA1-dependent transcription, reductions are observed in EBNA2 and LMP1 protein levels. Although these changes do not affect LCL survival, treated cells accumulate in G0/G1. These findings are discussed in the context of EBV latency in body compartments that differ strikingly in their pO2 and redox potential.
Epstein-Barr virus (EBV) infects human B-cells and immortalizes them. Immortalization results in diseases that range from infectious mononucleosis to malignancies such as lymphomas. During immortalization, EBV expresses a small number of viral genes that modulate cellular proliferation and differentiation. One of the genes expressed by EBV, Epstein-Barr nuclear antigen 1 (EBNA1), activates the expression of the other viral genes required for immortalization. In this report, we have explored the mechanism by which EBNA1 activates gene expression. We have determined that EBNA1 uses the micronutrient zinc to self-associate, and that self-association is necessary for it to activate gene expression. Further, we have determined that environmental conditions such as oxygen tension and oxidative stress modulate EBNA1's capacity to self-associate, and therefore to activate gene expression. The gene expression profile and proliferative phenotype of EBV-infected cells is known to vary in differing environmental niches in the human body, such as lymph nodes and in peripheral circulation. We interpret our results to postulate that these differences arise as a consequence of varying oxygen tension in these microenvironments on EBNA1's capacity to activate viral gene expression. Our findings can be exploited to devise novel therapeutics against EBV-associated diseases that target EBNA1 through oxidative stress.
EBNA3C, one of the Epstein-Barr virus (EBV)-encoded latent antigens, is essential for primary B-cell transformation. Cyclin D1, a key regulator of G1 to S phase progression, is tightly associated and aberrantly expressed in numerous human cancers. Previously, EBNA3C was shown to bind to Cyclin D1 in vitro along with Cyclin A and Cyclin E. In the present study, we provide evidence which demonstrates that EBNA3C forms a complex with Cyclin D1 in human cells. Detailed mapping experiments show that a small N-terminal region which lies between amino acids 130–160 of EBNA3C binds to two different sites of Cyclin D1- the N-terminal pRb binding domain (residues 1–50), and C-terminal domain (residues 171–240), known to regulate Cyclin D1 stability. Cyclin D1 is short-lived and ubiquitin-mediated proteasomal degradation has been targeted as a means of therapeutic intervention. Here, we show that EBNA3C stabilizes Cyclin D1 through inhibition of its poly-ubiquitination, and also increases its nuclear localization by blocking GSK3β activity. We further show that EBNA3C enhances the kinase activity of Cyclin D1/CDK6 which enables subsequent ubiquitination and degradation of pRb. EBNA3C together with Cyclin D1-CDK6 complex also efficiently nullifies the inhibitory effect of pRb on cell growth. Moreover, an sh-RNA based strategy for knock-down of both cyclin D1 and EBNA3C genes in EBV transformed lymphoblastoid cell lines (LCLs) shows a significant reduction in cell-growth. Based on these results, we propose that EBNA3C can stabilize as well as enhance the functional activity of Cyclin D1 thereby facilitating the G1-S transition in EBV transformed lymphoblastoid cell lines.
Epstein-Barr virus (EBV), a ubiquitous human herpesvirus, is linked to the development of multiple cancers, including lymphomas and epithelial carcinomas. EBNA3C, one of its essential latent antigens encoded by EBV, is expressed in EBV-associated lymphomas and contributes to aberrant cell growth after EBV infection. Cyclin D1 over-expression is associated with numerous cancers and is crucial for the transition from G1 to S phase in the mammalian cell-cycle. This study demonstrates that EBNA3C can enhance the functional activity of the Cyclin D1/CDK6 complex which in turn facilitates the G1 to S transition by neutralizing the growth inhibitory effects of pRb. Thus, manipulation of Cyclin D1 functions by EBNA3C provides a favorable environment to promote malignant transformation of EBV infected B-cells.
As an inhibitor of cyclin-dependent kinases, p16INK4A is an important tumour suppressor and inducer of cellular senescence that is often inactivated during the development of cancer by promoter DNA methylation. Using newly established lymphoblastoid cell lines (LCLs) expressing a conditional EBNA3C from recombinant EBV, we demonstrate that EBNA3C inactivation initiates chromatin remodelling that resets the epigenetic status of p16INK4A to permit transcriptional activation: the polycomb-associated repressive H3K27me3 histone modification is substantially reduced, while the activation-related mark H3K4me3 is modestly increased. Activation of EBNA3C reverses the distribution of these epigenetic marks, represses p16INK4A transcription and allows proliferation. LCLs lacking EBNA3A express relatively high levels of p16INK4A and have a similar pattern of histone modifications on p16INK4A as produced by the inactivation of EBNA3C. Since binding to the co-repressor of transcription CtBP has been linked to the oncogenic activity of EBNA3A and EBNA3C, we established LCLs with recombinant viruses encoding EBNA3A- and/or EBNA3C-mutants that no longer bind CtBP. These novel LCLs have revealed that the chromatin remodelling and epigenetic repression of p16INK4A requires the interaction of both EBNA3A and EBNA3C with CtBP. The repression of p16INK4A by latent EBV will not only overcome senescence in infected B cells, but may also pave the way for p16INK4A DNA methylation during B cell lymphomagenesis.
We previously showed that two Epstein-Barr virus latency-associated proteins—EBNA3A and EBNA3C—contribute to enhanced B cell survival by inhibiting the expression of the death-inducing protein BIM. This repression involves remodelling of the BIM gene promoter by polycomb proteins and DNA methylation within an unusually large CpG-island that flanks the transcription initiation site. Here we show that the same two proteins, EBNA3A and EBNA3C, functionally cooperate in the polycomb-mediated chromatin remodelling of another tumour suppressor gene, p16INK4A, that encodes a cyclin-dependent kinase inhibitor capable of blocking cell proliferation. Both EBV proteins can bind the highly conserved co-repressor of transcription CtBP, and these interactions appear to be required for the efficient repression of p16INK4A. Thus by utilising the polycomb system to induce the heritable repression of two major tumour suppressor genes—one that induces cell death (BIM) and one that induces growth arrest (p16INK4A)—EBV profoundly alters latently infected B cells and their progeny, making them significantly more prone to malignant transformation.
The ability of distant cis-acting DNA elements to interact functionally has been proposed to be mediated by the interaction of proteins associated site specifically with those cis-acting elements. We have found that the DNA-linking regions of EBNA1 are essential for its contribution to both replication and transcription. The synthesis of plasmids containing the Epstein-Barr virus (EBV) origin of plasmid replication (oriP) can be mediated entirely by the cellular machinery; however, the replicated molecules are lost rapidly from proliferating cells. When EBNA1 is provided in trans, plasmids containing oriP (oriP plasmids) are synthesized during repeated S phases, and the newly formed daughter molecules are precisely segregated to the daughter cells. The contribution(s) of EBNA1 to the stable replication of oriP plasmids is therefore likely to be postsynthetic. In latently infected cells, EBNA1 also regulates the expression of multiple EBV promoters located as many as 10 kbp away. EBNA1 supports replication and transcription through binding to oriP; both the ability of EBNA1 to bind to DNA and the integrity of its binding sites in oriP are required. However, DNA binding by EBNA1 is not sufficient to support replication or transcription, indicating that an additional activity (or activities) is required. EBNA1 links DNAs to which it binds and can form a loop between the two subelements of oriP, the family of repeats and the region of dyad symmetry, each of which contains multiple binding sites for EBNA1. We have constructed a set of derivatives of EBNA1 which contain both, one, or neither of its linking regions in various contexts. Analyses of these derivatives demonstrate that the linking regions of EBNA1 are essential for its support of replication and transcription and that the ability of derivatives of EBNA1 to link DNAs correlates strongly with their support of these activities in cells. These findings indicate that protein-protein associations of the linking regions of EBNA1 underlie its long-range contributions to replication and transcription.
Epstein-Barr virus (EBV) latent antigen EBNA3C is implicated in B-cell immortalization and linked to several B-cell malignancies. Deregulation of H2AX can induce genomic instability with increased chromosomal aberrations, which ultimately leads to tumorigenesis. Here we demonstrated that EBNA3C can attenuate H2AX expression at the transcript and protein levels. A reduction of total H2AX levels was clearly observed upon infection of primary B cells with wild-type EBV but not with EBNA3C knockout recombinant EBV. H2AX also interacted with EBNA3C through its N-terminal domain (residues 1 to 100). Furthermore, H2AX mutated at Ser139 failed to interact with EBNA3C. Luciferase-based reporter assays also revealed that the binding domain of EBNA3C is sufficient for transcriptional inhibition of the H2AX promoter. EBNA3C also facilitated H2AX degradation through recruitment of components of the ubiquitin proteasome pathway. We further demonstrated that knockdown of H2AX in lymphoblastoid cell lines (LCLs) led to the upregulation of the Bub1 oncoprotein and downregulated expression of p53. Overall, our study provides additional insights into EBV-associated B-cell lymphomas, which are linked to the regulation of the DNA damage response system in infected cells. The importance of these insights are as follows: (i) EBNA3C downregulates H2AX expression at the protein and transcript levels in epithelial cells, B cells, and EBV-transformed LCLs, (ii) EBNA3C binds with wild-type H2AX but not with the Ser139 mutant of H2AX, (iii) the N terminus (residues 1 to 100) of EBNA3C is critical for binding to H2AX, (iv) localization of H2AX is predominantly nuclear in the presence of EBNA3C, and (v) H2AX knocked down in LCLs led to enhanced expression of Bub1 and downregulation of the tumor suppressor p53, which are both important for driving the oncogenic process.
Epstein-Barr virus (EBV)-negative Burkitt's lymphoma (BL) cell lines have been converted to EBV genome positivity by in vitro infection with the transforming EBV strain B95.8 and with the nontransforming mutant strain P3HR1, which has a deletion in the gene encoding the nuclear antigen EBNA2. These B95.8- and P3HR1-converted lines have been compared for their patterns of expression of EBV latent genes (i.e., those viral genes constitutively expressed in all EBV-transformed lines of normal B-cell origin) and for their recognition by EBV-specific cytotoxic T lymphocytes (CTLs), in an effort to identify which latent gene products provide target antigens for the T-cell response. B95.8-converted lines on several different EBV-negative BL-cell backgrounds all showed detectable expression of the nuclear antigens EBNA1, EBNA2, and EBNA3 and of the latent membrane protein (LMP); such converts were also clearly recognized by EBV-specific CTL preparations with restriction through selected human leukocyte antigen (HLA) class I antigens on the target cell surface. The corresponding P3HR1-converted lines (lacking an EBNA2 gene) expressed EBNA1 and EBNA3 but, surprisingly, showed no detectable LMP; furthermore, these converts were not recognized by EBV-specific CTLs. Such differences in T-cell recognition were not due to any differences in expression of the relevant HLA-restricting determinants between the two types of convert, as shown by binding of specific monoclonal antibodies and by the susceptibility of both B95.8 and P3HR1 converts to allospecific CTLs directed against these same HLA molecules. The results suggest that in the normal infectious cycle, EBNA2 may be required for subsequent expression of LMP and that both EBNA2 and LMP (but not EBNA1 or EBNA3) may provide target antigens for the EBV-specific T-cell response.
The Epstein-Barr Virus (EBV) Nuclear Antigen 1 (EBNA1) protein is required for the establishment of EBV latent infection in proliferating B-lymphocytes. EBNA1 is a multifunctional DNA-binding protein that stimulates DNA replication at the viral origin of plasmid replication (OriP), regulates transcription of viral and cellular genes, and tethers the viral episome to the cellular chromosome. EBNA1 also provides a survival function to B-lymphocytes, potentially through its ability to alter cellular gene expression. To better understand these various functions of EBNA1, we performed a genome-wide analysis of the viral and cellular DNA sites associated with EBNA1 protein in a latently infected Burkitt lymphoma B-cell line. Chromatin-immunoprecipitation (ChIP) combined with massively parallel deep-sequencing (ChIP-Seq) was used to identify cellular sites bound by EBNA1. Sites identified by ChIP-Seq were validated by conventional real-time PCR, and ChIP-Seq provided quantitative, high-resolution detection of the known EBNA1 binding sites on the EBV genome at OriP and Qp. We identified at least one cluster of unusually high-affinity EBNA1 binding sites on chromosome 11, between the divergent FAM55 D and FAM55B genes. A consensus for all cellular EBNA1 binding sites is distinct from those derived from the known viral binding sites, suggesting that some of these sites are indirectly bound by EBNA1. EBNA1 also bound close to the transcriptional start sites of a large number of cellular genes, including HDAC3, CDC7, and MAP3K1, which we show are positively regulated by EBNA1. EBNA1 binding sites were enriched in some repetitive elements, especially LINE 1 retrotransposons, and had weak correlations with histone modifications and ORC binding. We conclude that EBNA1 can interact with a large number of cellular genes and chromosomal loci in latently infected cells, but that these sites are likely to represent a complex ensemble of direct and indirect EBNA1 binding sites.
The Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA3C) is a virus-encoded latent antigen essential for primary B-cell transformation. In this report we demonstrate that although the carboxy terminus of EBNA3C predominantly regulates cyclin A-dependent kinase activity, the region of greatest affinity for cyclin A lies within the EBNA3 amino-terminal homology domain of EBNA3C. Detailed mapping studies employing both in vitro binding assays and coimmunoprecipitation experiments implicated a small region of EBNA3C, amino acids 130 to 159 within the EBNA3 homology domain, as having the greatest affinity for cyclin A. The EBNA3 homology domain has the highest degree of amino acid similarity (approximately 30%) between the EBNA3 proteins, and, indeed, EBNA3B, but not EBNA3A, showed binding activity with cyclin A. We also show that EBNA3C binds to the α1 helix of the highly conserved mammalian cyclin box, with cyclin A amino acids 206 to 226 required for strong binding to EBNA3C amino acids 130 to 159. Interestingly, EBNA3C also bound human cyclins D1 and E in vitro, although the affinity was approximately 30% of that seen for cyclin A. Previously it was demonstrated that full-length EBNA3C rescues p27-mediated suppression of cyclin A-dependent kinase activity (J. S. Knight and E. S. Robertson, J. Virol. 78:1981-1991, 2004). It was also demonstrated that the carboxy terminus of EBNA3C recapitulates this phenotype. Surprisingly, the amino terminus of EBNA3C with the highest affinity for cyclin A was unable to rescue p27 suppression of kinase activity and actually downregulates cyclin A activity when introduced into EBV-infected cells. The data presented here suggests that the amino terminus of EBNA3C may play an important role in recruiting cyclin A complexes, while the carboxy terminus of EBNA3C is necessary for the functional modulation of cyclin A complex kinase activity.
Epstein-Barr virus (EBV) establishes a life-long latent infection in humans. In proliferating latently infected cells, EBV genomes persist as multiple episomes that undergo one DNA replication event per cell cycle and remain attached to the mitotic chromosomes. EBV nuclear antigen 1 (EBNA-1) binding to the episome and cellular genome is essential to ensure proper episome replication and segregation. However, the nature and regulation of EBNA-1 interaction with chromatin has not been clearly elucidated. This activity has been suggested to involve EBNA-1 binding to DNA, duplex RNA, and/or proteins. EBNA-1 binding protein 2 (EBP2), a nucleolar protein, has been proposed to act as a docking protein for EBNA-1 on mitotic chromosomes. However, there is no direct evidence thus far for EBP2 being associated with EBNA-1 during mitosis. By combining video microscopy and Förster resonance energy transfer (FRET) microscopy, we demonstrate here for the first time that EBNA-1 and EBP2 interact in the nucleoplasm, as well as in the nucleoli during interphase. However, in strong contrast to the current proposed model, we were unable to observe any interaction between EBNA-1 and EBP2 on mitotic chromosomes. We also performed a yeast double-hybrid screening, followed by a FRET analysis, that led us to identify HMGB2 (high-mobility group box 2), a well-known chromatin component, as a new partner for EBNA-1 on chromatin during interphase and mitosis. Although the depletion of HMGB2 partly altered EBNA-1 association with chromatin in HeLa cells during interphase and mitosis, it did not significantly impact the maintenance of EBV episomes in Raji cells.
Epstein–Barr virus (EBV), a ubiquitous human herpesvirus, can latently infect the human population. EBV is associated with several types of malignancies originating from lymphoid and epithelial cell types. EBV latent antigen 3C (EBNA3C) is essential for EBV-induced immortalization of B-cells. The Moloney murine leukemia provirus integration site (PIM-1), which encodes an oncogenic serine/threonine kinase, is linked to several cellular functions involving cell survival, proliferation, differentiation, and apoptosis. Notably, enhanced expression of Pim-1 kinase is associated with numerous hematological and non-hematological malignancies. A higher expression level of Pim-1 kinase is associated with EBV infection, suggesting a crucial role for Pim-1 in EBV-induced tumorigenesis. We now demonstrate a molecular mechanism which reveals a direct role for EBNA3C in enhancing Pim-1 expression in EBV-infected primary B-cells. We also showed that EBNA3C is physically associated with Pim-1 through its amino-terminal domain, and also forms a molecular complex in B-cells. EBNA3C can stabilize Pim-1 through abrogation of the proteasome/Ubiquitin pathway. Our results demonstrate that EBNA3C enhances Pim-1 mediated phosphorylation of p21 at the Thr145 residue. EBNA3C also facilitated the nuclear localization of Pim-1, and promoted EBV transformed cell proliferation by altering Pim-1 mediated regulation of the activity of the cell-cycle inhibitor p21/WAF1. Our study demonstrated that EBNA3C significantly induces Pim-1 mediated proteosomal degradation of p21. A significant reduction in cell proliferation of EBV-transformed LCLs was observed upon stable knockdown of Pim-1. This study describes a critical role for the oncoprotein Pim-1 in EBV-mediated oncogenesis, as well as provides novel insights into oncogenic kinase-targeted therapeutic intervention of EBV-associated cancers.
The oncogenic serine/threonine kinase Pim-1 is upregulated in a number of human cancers including lymphomas, gastric, colorectal and prostate carcinomas. EBV nuclear antigen 3C (EBNA3C) is essential for EBV-induced transformation of human primary B-lymphocytes. Our current study revealed that EBNA3C significantly enhances Pim-1 kinase expression at both the transcript and protein levels. EBNA3C also interacts with Pim-1 and can form a complex in EBV-transformed cells. Moreover, EBNA3C increases nuclear localization of Pim-1 and stabilizes Pim-1 protein levels by inhibiting its poly-ubiquitination. Additionally, EBNA3C augments Pim-1 mediated phosphorylation of p21 and its proteosomal degradation. Stable knockdown of Pim-1 using si-RNA showed a significant decrease in proliferation of EBV transformed lymphoblastoid cell lines and subsequent induction of apoptosis by triggering the intrinsic apoptotic pathway. Therefore, our study demonstrated a new mechanism by which the oncogenic Pim-1 kinase targeted by EBV latent antigen 3C can inhibit p21 function, and is therefore a potential therapeutic target for the treatment of EBV-associated malignancies.
The Epstein-Barr virus (EBV) latent origin of DNA replication (oriP) is composed of two elements that contain binding sites for the sole viral gene product required for latent cycle replication, EBNA-1. One of these elements, region I, functions as an EBNA-1-dependent enhancer for RNA polymerase II-transcribed genes, may play a role in plasmid segregation, and is required for origin function in B cells latently infected with EBV. The second element, region II, contains or is very near the site of initiation of DNA replication. A genetic approach was taken to determine the contribution of the EBNA-1 binding sites in oriP to origin function. Although region I is required for the transient replication of plasmids bearing region II in EBV-infected B cells, a plasmid lacking region I but containing region II, was observed to replicate transiently in both D98/Raji and HeLa cells expressing EBNA-1. Thus, binding of EBNA-1 to region I is not absolutely required for the molecular events that lead to initiation of DNA replication at region II. Site-directed mutagenesis of the four EBNA-1-binding sites in region II, individually and in various combinations, demonstrated that only two EBNA-1-binding sites are required for region II function. The results obtained with these mutants, together with the analysis of the replicative ability of plasmids containing insertions between EBNA-1-binding sites, suggested that the spatial relationship of the two sites is critical. Mutants that contain only two EBNA-1-binding sites separated by 26 to 31 bp in region II were not maintained as plasmids over many cell generations and were greatly reduced in their ability to replicate transiently in D98/Raji cells. The EBNA-1-induced bending or untwisting of the DNA in EBNA-1-binding sites 1 and 4 in region II did not, however, demonstrate this spatial constraint. It may be concluded from these results that specific protein-protein interactions between EBNA-1 and/or between EBNA-1 and a cellular protein(s) are required for origin function.
The Epstein-Barr virus (EBV)-encoded latency product EBNA-1 is functionally pleiotropic, being required for replication of the episomal form of the EBV genome and having a role in the regulation of latency transcription. EBNA-1 is a direct DNA-binding protein, and both replication and transactivation are dependent on the interaction of EBNA-1 with its cognate DNA recognition sequences. To better understand EBNA-1 function, we have further characterized the DNA-binding domain of EBNA-1 and have examined the contributions of other domains of the protein to EBNA-1 transactivation activity. A Bal31 deletional analysis of the carboxy-terminal region of EBNA-1 identified a core DNA-binding domain located between amino acids 493 and 584. Column chromatographic, sedimentation, and cross-linking studies indicated that EBNA-1 exists in solution as a dimer. Mobility retardation assays using in vitro-translated variants of EBNA-1 showed that the active DNA-binding form of EBNA-1 is also a dimer. In short-term cotransfections, a pFRTK-CAT target containing EBNA-1-binding sites from the EBV origin of plasmid replication, ori-P, was transactivated by a carboxy-terminal EBNA-1 construction (amino acids 450 to 641) that also carried a c-myc nuclear localization signal. These reconstruction experiments demonstrated that a transactivation domain exists within the carboxy-terminal region of EBNA-1, that transactivation is more efficient when a nuclear localization signal is present, and that the natural karyophilic signal lies outside of the carboxy-terminal 191 amino acids. To identify the EBNA-1 nuclear localization signal, small oligonucleotides representing EBNA-1 sequences that encode clusters of basic peptides were transferred into two different vectors expressing cytoplasmic proteins (pyruvate kinase and herpes simplex virus delta IE175 protein) and the cellular locations of the fusion constructions were determined by immunofluorescence staining of transfected cells. In this way we identified a functional nuclear localization signal, Leu-Lys-Arg-Pro-Arg-Ser-Pro-Ser-Ser, encompassing amino acids 379 to 386 of the EBNA-1 protein.
Epstein-Barr virus (EBV) nuclear proteins EBNA-LP and EBNA-2 are the first two proteins expressed in latent infection of primary B lymphocytes. EBNA-2 is essential for lymphocyte transformation, and EBNA-LP is at least critical. While EBNA-2 activates specific viral and cellular promoters, EBNA-LP's role has been obscure. We now show that EBNA-LP stimulates EBNA-2 activation of the LMP1 promoter and of the LMP1/LMP2B bidirectional transcriptional regulatory element. EBNA-LP alone has only a negative effect. EBNA-LP also stimulates EBNA-2 activation of a multimerized regulatory element from the BamC EBNA promoter. Since both viral regulatory elements can bind the EBNA-2-associated cell protein RBPJ kappa, consensus RBPJ kappa binding sites were positioned upstream of the herpes simplex virus type 1 thymidine kinase promoter and were found to be sufficient for EBNA-LP and EBNA-2 coactivation. EBNA-LP strongly stimulated activation of an adenovirus E1b promoter with upstream Gal4 binding sites by a Gal4 DNA binding domain/ EBNA-2 acidic domain fusion protein, indicating that EBNA-LP coactivation requires only the EBNA-2 acidic domain to be localized near a promoter. The EBNA-LP stimulatory activity resides in the amino-terminal 66-amino-acid repeat domain. The carboxyl-terminal unique 45 amino acids appear to regulate EBNA-LP's effects. The first 11 amino acids of the 45 have a strong negative effect, while the last 10 are critical for the ability of the last 34 to relieve the negative effect. These results indicate that EBNA-LP's critical role in EBV-mediated cell growth transformation is in stimulating (and probably regulating) EBNA-2-mediated transcriptional activation.