Carcinoma-risk variant of EBNA1 deregulates Epstein-Barr Virus episomal latency.
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ABSTRACT:
SUBMITTER: Dheekollu J
PROVIDER: S-EPMC5352318 | biostudies-literature | 2017 Jan
REPOSITORIES: biostudies-literature
Publications
Carcinoma-risk variant of EBNA1 deregulates Epstein-Barr Virus episomal latency.
Dheekollu Jayaraju J Malecka Kimberly K Wiedmer Andreas A Delecluse Henri-Jacques HJ Chiang Alan K S AK Altieri Dario C DC Messick Troy E TE Lieberman Paul M PM
Oncotarget 20170101 5
Epstein-Barr Virus (EBV) latent infection is a causative co-factor for endemic Nasopharyngeal Carcinoma (NPC). NPC-associated variants have been identified in EBV-encoded nuclear antigen EBNA1. Here, we solve the X-ray crystal structure of an NPC-derived EBNA1 DNA binding domain (DBD) and show that variant amino acids are found on the surface away from the DNA binding interface. We show that NPC-derived EBNA1 is compromised for DNA replication and episome maintenance functions. Recombinant virus ...[more]
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Project description:Latent Epstein-Barr virus (EBV) infection is strongly associated with several malignancies, including B-cell lymphomas and epithelial tumors. EBNA1 is a key antigen expressed in all EBV-associated tumors during latency that is required for maintenance of the EBV episome DNA and the regulation of viral gene transcription. However, the mechanism utilized by EBV to maintain latent infection at the levels of posttranslational regulation remains largely unclear. Here, we report that EBNA1 contains two SUMO-interacting motifs (SIM2 and SIM3), and mutation of SIM2, but not SIM3, dramatically disrupts the EBNA1 dimerization, while SIM3 contributes to the polySUMO2 modification of EBNA1 at lysine 477 in vitro. Proteomic and immunoprecipitation analyses further reveal that the SIM3 motif is required for the EBNA1-mediated inhibitory effects on SUMO2-modified STUB1, SUMO2-mediated degradation of USP7, and SUMO1-modified KAP1. Deletion of the EBNASIM motif leads to functional loss of both EBNA1-mediated viral episome maintenance and lytic gene silencing. Importantly, hypoxic stress induces the SUMO2 modification of EBNA1, and in turn the dissociation of EBNA1 with STUB1, KAP1 and USP7 to increase the SUMO1 modification of both STUB1 and KAP1 for reactivation of lytic replication. Therefore, the EBNA1SIM motif plays an essential role in EBV latency and is a potential therapeutic target against EBV-associated cancers.
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Project description:Expression of mRNAs in EBV-positive B-cell strains in the presence or absence of EBNA1 or a mutant of EBNA1 (ΔUR1-EBNA) that is unable to activate transcription.
2021-11-12 | GSE75198 | GEO
Project description:The EBNA1 protein of Epstein-Barr virus (EBV) plays multiple roles in EBV latent infection, including altering cellular pathways relevant for cancer. Here we used microRNA (miRNA) cloning coupled with high-throughput sequencing to identify the effects of EBNA1 on cellular miRNAs in two nasopharyngeal carcinoma cell lines. EBNA1 affected a small percentage of cellular miRNAs in both cell lines, in particular, upregulating multiple let-7 family miRNAs, including let-7a. The effects of EBNA1 on let-7a were verified by demonstrating that EBNA1 silencing in multiple EBV-positive carcinomas downregulated let-7a. Accordingly, the let-7a target, Dicer, was found to be partially downregulated by EBNA1 expression (at the mRNA and protein levels) and upregulated by EBNA1 silencing in EBV-positive cells. Reporter assays based on the Dicer 3' untranslated region with and without let-7a target sites indicated that the effects of EBNA1 on Dicer were mediated by let-7a. EBNA1 was also found to induce the expression of let-7a primary RNAs in a manner dependent on the EBNA1 transcriptional activation region, suggesting that EBNA1 induces let-7a by transactivating the expression of its primary transcripts. Consistent with previous reports that Dicer promotes EBV reactivation, we found that a let-7a mimic inhibited EBV reactivation to the lytic cycle, while a let-7 sponge increased reactivation. The results provide a mechanism by which EBNA1 could promote EBV latency by inducing let-7 miRNAs.The EBNA1 protein of Epstein-Barr virus (EBV) contributes in multiple ways to the latent mode of EBV infection that leads to lifelong infection. In this study, we identify a mechanism by which EBNA1 helps to maintain EBV infection in a latent state. This involves induction of a family of microRNAs (let-7 miRNAs) that in turn decreases the level of the cellular protein Dicer. We demonstrate that let-7 miRNAs inhibit the reactivation of latent EBV, providing an explanation for our previous observation that EBNA1 promotes latency. In addition, since decreased levels of Dicer have been associated with metastatic potential, EBNA1 may increase metastases by downregulating Dicer.
| S-EPMC4178782 | biostudies-other
Project description:Epstein-Barr virus (EBV) is the causative agent of infectious mononucleosis and a risk factor for developing a variety of lymphomas and carcinomas. EBV nuclear antigen 1 (EBNA1) is the only viral protein found in all EBV-related malignancies. It plays a key role in establishing and maintaining the altered state of cells transformed with EBV. EBNA1 is required for a variety of functions, including gene regulation, replication and maintenance of the viral genome, but the regulation of EBNA1's functions is poorly understood. We demonstrate that phosphorylation affects the functions of EBNA1. By using electron-transfer dissociation tandem mass spectrometry, ten specific phosphorylated EBNA1 residues were identified. A mutant derivative preventing the phosphorylation of all ten phosphosites retained the unusually long half-life and the ability to translocate into the nucleus of wild-type EBNA1. This phosphorylation-deficient mutant, however, had a significantly reduced ability to activate transcription and to maintain EBV's plasmids in cells.
| S-EPMC2786306 | biostudies-literature
Project description:Epstein-Barr virus (EBV) escapes host immunity by the reversible and epigenetic silencing of immunogenic viral genes. We previously presented evidence that a dynamic chromatin domain, which we have referred to as the latency control region (LCR), contributes to the reversible repression of EBNA2 and LMP1 gene transcription. We now explore the protein-DNA interaction profiles for a few known regulatory factors and histone modifications that regulate LCR structure and activity. A chromatin immunoprecipitation assay combined with real-time PCR analysis was used to analyze protein-DNA interactions at approximately 500-bp intervals across the first 60,000 bp of the EBV genome. We compared the binding patterns of EBNA1 with those of the origin recognition complex protein ORC2, the chromatin boundary factor CTCF, the linker histone H1, and several histone modifications. We analyzed three EBV-positive cell lines (MutuI, Raji, and LCL3459) with distinct transcription patterns reflecting different latency types. Our findings suggest that histone modification patterns within the LCR are complex but reflect differences in each latency type. The most striking finding was the identification of CTCF sites immediately upstream of the Qp, Cp, and EBER transcription initiation regions in all three cell types. In transient assays, CTCF facilitated EBNA1-dependent transcription activation of Cp, suggesting that CTCF coordinates interactions between different chromatin domains. We also found that histone H3 methyl K4 clustered with CTCF and EBNA1 at sites of active transcription or DNA replication initiation. Our findings support a model where CTCF delineates multiple domains within the LCR and regulates interactions between these domains that correlate with changes in gene expression.
| S-EPMC1900095 | biostudies-literature
Project description:BackgroundThe Epstein-Barr virus (EBV)-encoded EBNA1 protein is expressed in all EBV-associated tumours, including undifferentiated nasopharyngeal carcinoma (NPC), where it is indispensable for viral replication, genome maintenance and viral gene expression. EBNA1's transcription factor-like functions also extend to influencing the expression of cellular genes involved in pathways commonly dysregulated during oncogenesis, including elevation of AP-1 activity in NPC cell lines resulting in enhancement of angiogenesis in vitro. In this study we sought to extend these observations by examining the role of EBNA1 upon another pathway commonly deregulated during carcinogenesis; namely NF-kappaB.ResultsIn this report we demonstrate that EBNA1 inhibits the canonical NF-kappaB pathway in carcinoma lines by inhibiting the phosphorylation of IKKalpha/beta. In agreement with this observation we find a reduction in the phosphorylation of IkappaBalpha and reduced phosphorylation and nuclear translocation of p65, resulting in a reduction in the amount of p65 in nuclear NF-kappaB complexes. Similar effects were also found in carcinoma lines infected with recombinant EBV and in the EBV-positive NPC-derived cell line C666-1. Inhibition of NF-kappaB was dependent upon regions of EBNA1 essential for gene transactivation whilst the interaction with the deubiquitinating enzyme, USP7, was entirely dispensable. Furthermore, in agreement with EBNA1 inhibiting p65 NF-kappaB we demonstrate that p65 was exclusively cytoplasmic in 11 out of 11 NPC tumours studied.ConclusionsInhibition of p65 NF-kappaB in murine and human epidermis results in tissue hyperplasia and the development of squamous cell carcinoma. In line with this, p65 knockout fibroblasts have a transformed phenotype. Inhibition of p65 NF-kappaB by EBNA1 may therefore contribute to the development of NPC by inducing tissue hyperplasia. Furthermore, inhibition of NF-kappaB is employed by viruses as an immune evasion strategy which is also closely linked to oncogenesis during persistent viral infection. Our findings therefore further implicate EBNA1 in playing an important role in the pathogenesis of NPC.
| S-EPMC2818691 | biostudies-literature
Project description:Latent infection with Epstein-Barr virus (EBV) is a carcinogenic cofactor in several lymphoid and epithelial cell malignancies. At present, there are no small-molecule inhibitors that specifically target EBV latent infection or latency-associated oncoproteins. EBNA1 is an EBV-encoded sequence-specific DNA binding protein that is consistently expressed in EBV-associated tumors and required for stable maintenance of the viral genome in proliferating cells. EBNA1 is also thought to provide cell survival function in latently infected cells. In this work, the authors describe the development of a biochemical high-throughput screening (HTS) method using a homogeneous fluorescence polarization (FP) assay monitoring EBNA1 binding to its cognate DNA binding site. An FP-based counterscreen was developed using another EBV-encoded DNA binding protein, Zta, and its cognate DNA binding site. The authors demonstrate that EBNA1 binding to a fluorescent-labeled DNA probe provides a robust assay with a Z factor consistently greater than 0.6. A pilot screen of a small-molecule library of ~14,000 compounds identified 3 structurally related molecules that selectively inhibit EBNA1 but not Zta. All 3 compounds had activity in a cell-based assay specific for the disruption of EBNA1 transcription repression function. One of the compounds was effective in reducing EBV genome copy number in Raji Burkitt lymphoma cells. These experiments provide a proof of concept that small-molecule inhibitors of EBNA1 can be identified by biochemical HTS of compound libraries. Further screening in conjunction with medicinal chemistry optimization may provide a selective inhibitor of EBNA1 and EBV latent infection.
| S-EPMC3310380 | biostudies-literature
Project description:BackgroundAlternative splicing (AS) is an important mRNA maturation step that allows increased variability and diversity of proteins in eukaryotes. AS is dysregulated in numerous diseases, and its implication in the carcinogenic process is well known. However, progress in understanding how oncogenic viruses modulate splicing, and how this modulation is involved in viral oncogenicity has been limited. Epstein-Barr virus (EBV) is involved in various cancers, and its EBNA1 oncoprotein is the only viral protein expressed in all EBV malignancies.MethodsIn the present study, the ability of EBNA1 to modulate the AS of cellular genes was assessed using a high-throughput RT-PCR approach to examine AS in 1238 cancer-associated genes. RNA immunoprecipitation coupled to RNA sequencing (RIP-Seq) assays were also performed to identify cellular mRNAs bound by EBNA1.ResultsUpon EBNA1 expression, we detected modifications to the AS profiles of 89 genes involved in cancer. Moreover, we show that EBNA1 modulates the expression levels of various splicing factors such as hnRNPA1, FOX-2, and SF1. Finally, RNA immunoprecipitation coupled to RIP-Seq assays demonstrate that EBNA1 immunoprecipitates specific cellular mRNAs, but not the ones that are spliced differently in EBNA1-expressing cells.ConclusionThe EBNA1 protein can modulate the AS profiles of numerous cellular genes. Interestingly, this modulation protein does not require the RNA binding activity of EBNA1. Overall, these findings underline the novel role of EBNA1 as a cellular splicing modulator.
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Project description:Deciphering the molecular pathogenesis of virally induced cancers is challenging due, in part, to the heterogeneity of both viral gene expression and host gene expression. Epstein-Barr virus (EBV) is a ubiquitous herpesvirus prevalent in B-cell lymphomas of immune-suppressed individuals. EBV infection of primary human B cells leads to their immortalization into lymphoblastoid cell lines (LCLs), serving as a model of these lymphomas. In previous studies, reports from our laboratory have described a temporal model for immortalization with an initial phase characterized by expression of Epstein-Barr nuclear antigens (EBNAs), high levels of c-Myc activity, and hyperproliferation in the absence of the latent membrane proteins (LMPs), called latency IIb. This is followed by the long-term outgrowth of LCLs expressing the EBNAs along with the LMPs, particularly NFκB-activating LMP1, defining latency III. However, LCLs express a broad distribution of LMP1 such that a subset of these cells express LMP1 at levels similar to those seen in latency IIb, making it difficult to distinguish these two latency states. In this study, we performed mRNA sequencing (mRNA-Seq) on early EBV-infected latency IIb cells and latency III LCLs sorted by NFκB activity. We found that latency IIb transcriptomes clustered independently from latency III independently of NFκB. We identified and validated mRNAs defining these latency states. Indeed, we were able to distinguish latency IIb cells from LCLs expressing low levels of LMP1 using multiplex RNA-fluorescence in situ hybridization (RNA-FISH) targeting EBV EBNA2 or LMP1 and human CCR7 or MGST1 This report defines latency IIb as a bona fide latency state independent from latency III and identifies biomarkers for understanding EBV-associated tumor heterogeneity.IMPORTANCE EBV is a ubiquitous pathogen, with >95% of adults harboring a life-long latent infection in memory B cells. In immunocompromised individuals, latent EBV infection can result in lymphoma. The established expression profile of these lymphomas is latency III, which includes expression of all latency genes. However, single-cell analysis of EBV latent gene expression in these lymphomas suggests heterogeneity where most cells express the transcription factor, EBNA2, and only a fraction of the cells express membrane protein LMP1. Our work describes an early phase after infection where the EBNAs are expressed without LMP1, called latency IIb. However, LMP1 levels within latency III vary widely, making these states hard to discriminate. This may have important implications for therapeutic responses. It is crucial to distinguish these states to understand the molecular pathogenesis of these lymphomas. Ultimately, better tools to understand the heterogeneity of these cancers will support more-efficacious therapies in the future.
| S-EPMC6606803 | biostudies-literature
Project description:Epstein-Barr virus (EBV) is a human herpesvirus that causes infectious mononucleosis and contributes to both B-cell and epithelial-cell malignancies. EBV-infected epithelial cell tumors, including nasopharyngeal carcinoma (NPC), are largely composed of latently infected cells, but the mechanism(s) maintaining viral latency are poorly understood. Expression of the EBV BZLF1 (Z) and BRLF1 (R) encoded immediate-early (IE) proteins induces lytic infection, and these IE proteins activate each other's promoters. ΔNp63α (a p53 family member) is required for proliferation and survival of basal epithelial cells and is over-expressed in NPC tumors. Here we show that ΔNp63α promotes EBV latency by inhibiting activation of the BZLF1 IE promoter (Zp). Furthermore, we find that another p63 gene splice variant, TAp63α, which is expressed in some Burkitt and diffuse large B cell lymphomas, also represses EBV lytic reactivation. We demonstrate that ΔNp63α inhibits the Z promoter indirectly by preventing the ability of other transcription factors, including the viral IE R protein and the cellular KLF4 protein, to activate Zp. Mechanistically, we show that ΔNp63α promotes viral latency in undifferentiated epithelial cells both by enhancing expression of a known Zp repressor protein, c-myc, and by decreasing cellular p38 kinase activity. Furthermore, we find that the ability of cis-platinum chemotherapy to degrade ΔNp63α contributes to the lytic-inducing effect of this agent in EBV-infected epithelial cells. Together these findings demonstrate that the loss of ΔNp63α expression, in conjunction with enhanced expression of differentiation-dependent transcription factors such as BLIMP1 and KLF4, induces lytic EBV reactivation during normal epithelial cell differentiation. Conversely, expression of ΔNp63α in undifferentiated nasopharyngeal carcinoma cells and TAp63α in Burkitt lymphoma promotes EBV latency in these malignancies.
| S-EPMC8601603 | biostudies-literature