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
Kaposi's Sarcoma-associated herpesvirus (KSHV) is maintained as a stable episome in latently infected pleural effusion lymphoma (PEL) cells. Episome maintenance is conferred by the binding of the KSHV-encoded LANA protein to the viral terminal repeats (TR). Here, we show that DNA replication in the KSHV TR is coupled with DNA recombination and mediated in part through the cellular replication fork protection factors Timeless (Tim) and Tipin. We show by two-dimensional (2D) agarose gel electrophoresis that replication forks naturally stall and form recombination-like structures at the TR during an unperturbed cell cycle. Chromatin immunoprecipitation (ChIP) assays revealed that Tim and Tipin are selectively enriched at the KSHV TR during S phase and in a LANA-dependent manner. Tim depletion inhibited LANA-dependent TR DNA replication and caused the loss of KSHV episomes from latently infected PEL cells. Tim depletion resulted in the aberrant accumulation of recombination structures and arrested MCM helicase at TR. Tim depletion did not induce the KSHV lytic cycle or apoptotic cell death. We propose that KSHV episome maintenance requires Tim-assisted replication fork protection at the viral terminal repeats and that Tim-dependent recombination-like structures form at TR to promote DNA repeat stability and viral genome maintenance.
LANA is the KSHV-encoded terminal repeat binding protein essential for viral replication and episome maintenance during latency. We have determined the X-ray crystal structure of LANA C-terminal DNA binding domain (LANADBD) to reveal its capacity to form a decameric ring with an exterior DNA binding surface. The dimeric core is structurally similar to EBV EBNA1 with an N-terminal arm that regulates DNA binding and is required for replication function. The oligomeric interface between LANA dimers is dispensable for single site DNA binding, but is required for cooperative DNA binding, replication function, and episome maintenance. We also identify a basic patch opposite of the DNA binding surface that is responsible for the interaction with BRD proteins and contributes to episome maintenance function. The structural features of LANADBD suggest a novel mechanism of episome maintenance through DNA-binding induced oligomeric assembly.
Kaposi's sarcoma-associated herpesvirus (KSHV) establishes latent infections that are associated with several cancers including Kaposi's sarcoma, pleural effusion lymphoma, and multicentric Caslteman's disease. One of the major viral proteins required for establishment and maintenance of the latent state is the latency-associated nuclear antigen (LANA). LANA binds to DNA sequences within the terminal repeats (TR) of the viral genome and stimulates both DNA replication and episome maintenance during latency. Here we present the X-ray crystal structure of the DNA binding domain of LANA (LANADBD) and show that it has the capacity to form oligomeric complexes upon DNA binding. We characterize structural features of LANADBD that are required for oligomerization, DNA binding, and interaction with host cell BET proteins, BRD2 and BRD4, which are important for mediating multiple functions of LANA, including episome maintenance.
Self-reinforcing negative feedback loops are commonly observed in biological systems. RNA-mediated negative feedback loops have been described in the formation of heterochromatin at centromeres in fission yeast and the inactive X chromosome in mammalian cells. The telomere repeat-containing RNA (TERRA) has also been implicated in the formation of telomeric heterochromatin through a self-reinforcing negative feedback loop. In cells derived from human ICF syndrome, TERRA levels are abnormally elevated and telomeres are abnormally shortened. We now show that telomere heterochromatin is also abnormal in ICF cells. We propose that ICF cells fail to reinforce the TERRA-dependent negative feedback loop as a result of the inability to establish heterochromatin at subtelomeres. This failure is likely due to the lack of DNMT3b and DNA methylation, which is a genetic lesion associated with ICF syndrome. Failure of this feedback mechanism leads to catastrophic telomere dysfunction and chromosome instability.
TERRA; TRF2; CpG methylation; heterochromatin; ICF syndrome
Chromatin-organizing factors such as CTCF and cohesins have been implicated in the control of complex viral regulatory programs. We investigated the role of CTCF and cohesins in the control of the switch from latency to the lytic cycle for Kaposi's sarcoma-associated herpesvirus (KSHV). We found that cohesin subunits but not CTCF are required for the repression of KSHV immediate early gene transcription. Depletion of the cohesin subunits Rad21, SMC1, and SMC3 resulted in lytic cycle gene transcription and viral DNA replication. In contrast, depletion of CTCF failed to induce lytic transcription or DNA replication. Chromatin immunoprecipitation with high-throughput sequencing (ChIP-Seq) revealed that cohesins and CTCF bound to several sites within the immediate early control region for ORF50 and to more distal 5′ sites that also regulate the divergently transcribed ORF45-ORF46-ORF47 gene cluster. Rad21 depletion led to a robust increase in ORF45, ORF46, ORF47, and ORF50 transcripts, with similar kinetics to that observed with chemical induction by sodium butyrate. During latency, the chromatin between the ORF45 and ORF50 transcription start sites was enriched in histone H3K4me3, with elevated H3K9ac at the ORF45 promoter and elevated H3K27me3 at the ORF50 promoter. A paused form of RNA polymerase II (Pol II) was loosely associated with the ORF45 promoter region during latency but was converted to an active elongating form upon reactivation induced by Rad21 depletion. Butyrate treatment caused a rapid dissociation of cohesins and loss of CTCF binding at the immediate early gene locus, suggesting that cohesins may be a direct target of butyrate-mediated lytic induction. Our findings implicate cohesins as a major repressor of KSHV lytic gene activation and show that they function coordinately with CTCF to regulate the switch between latent and lytic gene activity.
Epstein-Barr Virus (EBV), which is associated with multiple human tumors, persists as a minichromosome in the nucleus of B-lymphocytes and induces malignancies through incompletely understood mechanisms. Here, we present a large-scale functional genomic analysis of EBV. Our experimentally generated nucleosome positioning maps and viral protein binding data were integrated with over 700 publicly available high-throughput sequencing data sets for human lymphoblastoid cell lines mapped to the EBV genome. We found that viral lytic genes are coexpressed with cellular cancer-associated pathways, suggesting that the lytic cycle may play an unexpected role in virus-mediated oncogenesis. Host regulators of viral oncogene expression and chromosome structure were identified and validated, revealing a role for the B-cell-specific protein Pax5 in viral gene regulation and the cohesin complex in regulating higher order chromatin structure. Our findings provide a deeper understanding of latent viral persistence in oncogenesis and establish a valuable viral genomics resource for future exploration.
CCCTC-binding factor (CTCF) has been implicated in various aspects of viral and host chromatin organization and transcriptional control. We showed previously that CTCF binds to a cluster of three sites in the first intron of the Kaposi's sarcoma-associated herpesvirus (KSHV) multicistronic latency-associated transcript that encodes latency-associated nuclear antigen (LANA), viral cyclin (vCyclin), vFLIP, viral microRNAs, and kaposin. We show here that these CTCF binding sites regulate mRNA production, RNA polymerase II (RNAPII) programming, and nucleosome organization of the KSHV latency transcript control region. We also show that KSHV bacmids lacking these CTCF binding sites have elevated and altered ratios of spliced latency transcripts. CTCF binding site mutations altered RNAPII and RNAPII-accessory factor interactions with the latency control region. CTCF binding sites were required for the in vitro recruitment of RNAPII to the latency control region, suggesting that direct interactions between CTCF and RNAPII contribute to transcription regulation. Histone modifications in the latency control region were also altered by mutations in the CTCF binding sites. Finally, we show that CTCF binding alters the regular phasing of nucleosomes in the latency gene transcript and intron, suggesting that nucleosome positioning can be an underlying biochemical mechanism of CTCF function. We propose that RNAPII interactions and nucleosome displacement serve as a biochemical basis for programming RNAPII in the KSHV transcriptional control region.
LANA is essential for tethering the Kaposi's sarcoma-associated herpesvirus (KSHV) genome to metaphase chromosomes and for modulating host-cell gene expression, but the binding sites in the host-chromosome remain unknown. Here, we use LANA-specific chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq) to identify LANA binding sites in the viral and host-cell genomes of a latently infected pleural effusion lymphoma cell line BCBL1. LANA bound with high occupancy to the KSHV genome terminal repeats (TR) and to a few minor binding sites in the KSHV genome, including the LANA promoter region. We identified 256 putative LANA binding site peaks with P < 0.01 and overlap in two independent ChIP-Seq experiments. We validated several of the high-occupancy binding sites by conventional ChIP assays and quantitative PCR. Candidate cellular LANA binding motifs were identified and assayed for binding to purified recombinant LANA protein in vitro but bound with low affinity compared to the viral TR binding site. More than half of the LANA binding sites (170/256) could be mapped to within 2.5 kb of a cellular gene transcript. Pathways and Gene Ontogeny (GO) analysis revealed that LANA binds to genes within the p53 and tumor necrosis factor (TNF) regulatory network. Further analysis revealed partial overlap of LANA and STAT1 binding sites in several gamma interferon (IFN-γ)-regulated genes. We show that ectopic expression of LANA can downmodulate IFN-γ-mediated activation of a subset of genes, including the TAP1 peptide transporter and proteasome subunit beta type 9 (PSMB9), both of which are required for class I antigen presentation. Our data provide a potential mechanism through which LANA may regulate several host cell pathways by direct binding to gene regulatory elements.
A variety of telomere protection programs are utilized to preserve telomere structure. However, the complex nature of telomere maintenance remains elusive. The Timeless protein associates with the replication fork and is thought to support efficient progression of the replication fork through natural impediments, including replication fork block sites. However, the mechanism by which Timeless regulates such genomic regions is not understood. Here, we report the role of Timeless in telomere length maintenance. We demonstrate that Timeless depletion leads to telomere shortening in human cells. This length maintenance is independent of telomerase, and Timeless depletion causes increased levels of DNA damage, leading to telomere aberrations. We also show that Timeless is associated with Shelterin components TRF1 and TRF2. Timeless depletion slows telomere replication in vitro, and Timeless-depleted cells fail to maintain TRF1-mediated accumulation of replisome components at telomeric regions. Furthermore, telomere replication undergoes a dramatic delay in Timeless-depleted cells. These results suggest that Timeless functions together with TRF1 to prevent fork collapse at telomere repeat DNA and ensure stable maintenance of telomere length and integrity.
TRF1; replication efficiency; telomere; telomere aberration; the fork protection complex; timeless
The Epstein-Barr virus (EBV) double-stranded DNA genome is subject to extensive epigenetic regulation. Large consortiums and individual labs have generated a vast number of genome-wide data sets on human lymphoblastoid and other cell lines latently infected with EBV. Analysis of these data sets reveals important new information on the properties of the host and viral chromosome structure organization and epigenetic modifications. We discuss the mapping of these data sets and the subsequent insights into the chromatin structure and transcription factor binding patterns on latent EBV genomes. Colocalization of multiple histone modifications and transcription factors at regulatory loci are considered in the context of the biology and regulation of EBV.
Epstein-Barr virus; gammaherpesvirus; chromatin; histone modification; CTCF; OriP
Hydroxyurea (HU) is a chemotherapeutic agent commonly used for various malignancies and hematological disorders, including chronic myelogenous leukemia and sickle cell anemia. We show here that chronic, low-level treatment with HU induces a variety of defects in telomere replication and maintenance. HU treatment preferentially decreased the rate of telomere DNA synthesis and altered the cell cycle timing of telomere replication. HU reduced the expression levels of telomere repeat RNA (TERRA). In some cells, HU caused a rapid loss of telomere restriction fragment length. Chromatin immunoprecipitation (ChIP) assay indicated that telomere repeat binding factors TRF1 and TRF2 dissociate from telomere DNA after HU treatment. TRF2 protein purified from HU treated cells showed a modest reduction in DNA binding activity and a change in isoelectric point as measured by 2D gel electrophoresis. However, chronic low level HU treatment did not evoke a DNA replication checkpoint response, suggesting that the mechanism of action is distinct from the well-characterized S-phase checkpoint pathway. We conclude that therapeutic doses of HU preferentially effects telomere replication and maintenance, through a mechanism that may involve the direct modification of TRF2. These findings provide new insight into the potential mechanisms of action of HU at telomeres and in cancer chemotherapies.
hydroxyurea; telomere; TRF2; replication; TERRA
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 we describe the development of a biochemical high-throughput screening (HTS) method using a homogenous 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. We 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 three 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 high-throughput screening of compound libraries. Further screening in conjunction with medicinal chemistry optimization may provide a selective inhibitor of EBNA1 and EBV latent infection.
Genome maintenance mechanisms actively suppress genetic instability associated with cancer and aging. Some viruses provoke genetic instability by subverting the host’s control of genome maintenance. Viruses have their own specialized strategies for genome maintenance, which can mimic and modify host cell processes. Here, we review some of the common features of genome maintenance utilized by viruses and host chromosomes, with a particular focus on terminal repeat (TR) elements. The TRs of cellular chromosomes, better known as telomeres, have well-established roles in cellular chromosome stability. Cellular telomeres are themselves maintained by viral-like mechanisms, including self-propagation by reverse transcription, recombination, and retrotransposition. Viral TR elements, like cellular telomeres, are essential for viral genome stability and propagation. We review the structure and function of viral repeat elements and discuss how they may share telomere-like structures and genome protection functions. We consider how viral infections modulate telomere regulatory factors for viral repurposing and can alter normal host telomere structure and chromosome stability. Understanding the common strategies of viral and cellular genome maintenance may provide new insights into viral–host interactions and the mechanisms driving genetic instability in cancer.
virus; telomere; replication; EBV; KSHV; HHV6; MDV
The gammaherpesviruses are a subclass of the herpesvirus family that establish stable latent infections in proliferating lymphoid and epithelial cells. The latent genomes are maintained as multicopy chromatinized episomes that replicate in synchrony with the cellular genome. Importantly, most of the episomes do not integrate into the host chromosome. Therefore, it is essential that the viral “minichromosome” establish a chromatin structure that is suitable for gene expression, DNA replication, and chromosome segregation. Evidence suggests that chromatin organization is important for each of these functions and plays a regulatory role in the establishment and maintenance of latent infection. Here, we review recent studies on the chromatin organization of the human gammaherpesviruses, Epstein-Barr Virus (EBV) and Kaposi’s Sarcoma-Associated Herpesvirus (KSHV). We discuss the potential role of viral origins of DNA replication and viral encoded origin-binding proteins like EBNA1 and LANA in establishment of viral chromosome organization during latent infection. We also discuss the roles of host cell factors, like CTCF and Cohesins, that contribute to higher order chromosome structures that may be important for stable gene expression programs during latent infection in proliferating cells.
Glycyrrhizic acid (GA), a derivative of licorice, selectively inhibits the growth of lymphocytes latently infected with Kaposi's sarcoma-associated herpesvirus. The mechanism involves the deregulation of the multicistronic latency transcript, including the failure to generate the mature forms of viral mRNA encoding LANA. We show here that GA disrupts an RNA polymerase II (RNAPII) complex that accumulates at the CTCF-cohesin binding site within the first intron of the latency transcript. GA altered the enrichment of the RNAPII pausing complex, along with pausing factors SPT5 and NELF-A, at the intragenic CTCF-cohesin binding sites. GA blocked the interaction of cohesin subunit SMC3 with another cohesin subunit, RAD21, and reduced SPT5 interaction with RNAPII. Covalent coupling of GA to a solid support revealed that GA interacts with several cellular proteins, including SMC3 and SPT5, but not their respective interaction partners RAD21 and RNAPII. GA treatment also inhibited the transcription of some cellular genes, like c-myc, which contain a similar CTCF-cohesin binding site within the first intron. We also found that GA leads to a more general loss of sister chromatid cohesion for cellular chromosomes. These findings suggest that RNAPII pauses at intragenic CTCF-cohesin binding sites and that abrogation of this pausing by GA leads to loss of proper mRNA production and defects in sister chromatid cohesion, a process important for both viral and cellular chromosome stability.
Herpesviruses are a complex family of dsDNA viruses that are a major cause of human disease. All family members share highly related viral replication proteins, such as DNA polymerase, ssDNA-binding proteins and processivity factors. Consequently, it is generally thought that lytic replication occurs through a common and conserved mechanism. However, considerable evidence indicates that proteins controlling initiation of DNA replication vary greatly among the herepesvirus subfamilies. In this article, we focus on some of the known mechanisms that regulate Epstein-Barr virus lytic-cycle replication, and compare this to other herpesvirus family members. Our reading of the literature leads us to conclude that diverse viral mechanisms generate a common nucleoprotein prereplication structure that can be recognized by a highly conserved family of viral replication enzymes.
BZLF1; EBV; Epstein–Barr; OriLyt; recombination; repair; replication; Zta
Many viruses introduce DNA into the host-cell nucleus, where they must either embrace or confront chromatin-factors as a support or obstacle to completion of its life cycle. Compared to the eukaryotic cell, viruses have compact and rapidly evolving genomes. Despite their smaller size, viruses have complex life-cycles that involve dynamic changes in DNA structure. Nuclear entry, transcription, replication, genome stabilization, and virion packaging involve complex changes in chromosome organization and structure. Chromatin dynamics and epigenetic modifications play major roles in viral and host chromosome biology. In some cases, viruses may use novel or viral-specific epigenetic modifying activities, which may reflect variant pathways that distinguish their behavior from the bulk of the cellular chromosome. This review examines several recent discoveries that highlight the role of chromatin dynamics in the life cycle of DNA viruses.
chromatin; nucleosome; virus; latency; reactivation; epigenetic
The Epstein-Barr virus (EBV) genome is maintained as an extrachromosomal episome during latent infection of B lymphocytes. Episomal maintenance is conferred by the interaction of the EBV-encoded nuclear antigen 1 (EBNA1) with a tandem array of high-affinity binding sites, referred to as the family of repeats (FR), located within the viral origin of plasmid replication (OriP). How this nucleoprotein array confers episomal maintenance is not completely understood. Previous studies have shown that DNA replication forks pause and terminate with high frequency at OriP. We now show that cellular DNA replication fork pausing and protection factors Timeless (Tim) and Tipin (Timeless-interacting protein) accumulate at OriP during S phase of the cell cycle. Depletion of Tim inhibits OriP-dependent DNA replication and causes a complete loss of the closed-circular form of EBV episomes in latently infected B lymphocytes. Tim depletion also led to the accumulation of double-strand breaks at the OriP region. These findings demonstrate that Tim is essential for sustaining the episomal forms of EBV DNA in latently infected cells and suggest that DNA replication fork protection is integrally linked to the mechanism of plasmid maintenance.
Productive infection by herpesviruses involve the disabling of host-cell intrinsic defenses by viral encoded tegument proteins. Epstein-Barr Virus (EBV) typically establishes a non-productive, latent infection and it remains unclear how it confronts the host-cell intrinsic defenses that restrict viral gene expression. Here, we show that the EBV major tegument protein BNRF1 targets host-cell intrinsic defense proteins and promotes viral early gene activation. Specifically, we demonstrate that BNRF1 interacts with the host nuclear protein Daxx at PML nuclear bodies (PML-NBs) and disrupts the formation of the Daxx-ATRX chromatin remodeling complex. We mapped the Daxx interaction domain on BNRF1, and show that this domain is important for supporting EBV primary infection. Through reverse transcription PCR and infection assays, we show that BNRF1 supports viral gene expression upon early infection, and that this function is dependent on the Daxx-interaction domain. Lastly, we show that knockdown of Daxx and ATRX induces reactivation of EBV from latently infected lymphoblastoid cell lines (LCLs), suggesting that Daxx and ATRX play a role in the regulation of viral chromatin. Taken together, our data demonstrate an important role of BNRF1 in supporting EBV early infection by interacting with Daxx and ATRX; and suggest that tegument disruption of PML-NB-associated antiviral resistances is a universal requirement for herpesvirus infection in the nucleus.
Persistent infection by Epstein-Barr virus (EBV) is associated with a variety of diseases, including lymphoid and epithelial tumors. Despite a wealth of information on the mechanism of viral persistence, relatively little is known about the early steps of EBV infection and viral gene activation. Host cells actively mount resistances against viral infection, which viruses need to overcome to invade the cell. We have found that among the proteins packaged in the EBV viral particle, BNRF1 plays an important role of counteracting cellular defenses. We show that EBV protein BNRF1 binds to the cellular protein Daxx and disassembles the Daxx-ATRX complex, where both Daxx and ATRX are cellular proteins known to inhibit viral gene expression. We also confirm that BNRF1 can promote expression of early viral genes, and that Daxx-binding by BNRF1 is required for this function. Finally, we demonstrate that Daxx and ATRX repress viral gene expression during latency. We conclude that BNRF1 disassembles cellular antiviral defense machinery to promote expression of viral genes in the host cell.
Epstein-Barr virus (EBV) immortalizes resting B-cells and is a key etiologic agent in the development of numerous cancers. The essential EBV-encoded protein EBNA 2 activates the viral C promoter (Cp) producing a message of ∼120 kb that is differentially spliced to encode all EBNAs required for immortalization. We have previously shown that EBNA 2-activated transcription is dependent on the activity of the RNA polymerase II (pol II) C-terminal domain (CTD) kinase pTEFb (CDK9/cyclin T1). We now demonstrate that Cp, in contrast to two shorter EBNA 2-activated viral genes (LMP 1 and 2A), displays high levels of promoter-proximally stalled pol II despite being constitutively active. Consistent with pol II stalling, we detect considerable pausing complex (NELF/DSIF) association with Cp. Significantly, we observe substantial Cp-specific pTEFb recruitment that stimulates high-level pol II CTD serine 2 phosphorylation at distal regions (up to +75 kb), promoting elongation. We reveal that Cp-specific pol II accumulation is directed by DNA sequences unfavourable for nucleosome assembly that increase TBP access and pol II recruitment. Stalled pol II then maintains Cp nucleosome depletion. Our data indicate that pTEFb is recruited to Cp by the bromodomain protein Brd4, with polymerase stalling facilitating stable association of pTEFb. The Brd4 inhibitor JQ1 and the pTEFb inhibitors DRB and Flavopiridol significantly reduce Cp, but not LMP1 transcript production indicating that Brd4 and pTEFb are required for Cp transcription. Taken together our data indicate that pol II stalling at Cp promotes transcription of essential immortalizing genes during EBV infection by (i) preventing promoter-proximal nucleosome assembly and ii) necessitating the recruitment of pTEFb thereby maintaining serine 2 CTD phosphorylation at distal regions.
Epstein-Barr virus (EBV) is associated with the development of a number of human cancers including Burkitt's lymphoma, Hodgkin's lymphoma, nasopharyngeal carcinoma and post-transplant lymphoproliferative disease. The virus infects B cells rendering them immortal through the production of a small number of viral proteins in the latently infected cell. Many of the viral proteins required for B-cell immortalization are produced from a very long protein-coding RNA message that initiates at the main viral latency promoter C, and our results provide important new information on how this message is produced. Specifically we show that the production of this long RNA is driven by the recruitment of the elongation factor (pTEFb) to paused transcription complexes at the C promoter. We show that pTEFb is recruited by the chromatin-associated protein, Brd4. Treatment of cells with a recently developed Brd4 inhibitor and inhibitors of the pTEFb elongation factor inhibits production of transcripts derived from the long EBV message thereby highlighting Brd4 and pTEFb inhibitors as potential anti-EBV agents.
The genetic elements of herpesvirus origins of lytic replication have been characterized in detail; however, much remains to be elucidated concerning their functional role in replication initiation. In the case of the Epstein-Barr virus (EBV), we have found that in addition to the two well-defined critical elements required for lytic replication (the upstream and downstream essential elements, UEE and DEE), the origin of lytic replication (OriLyt) also requires the presence of a GC-rich RNA in cis. The BHLF1 transcript is similar to the essential K5 transcript identified at the Kaposi's sarcoma-associated herpesvirus OriLyt. We have found that truncation of the BHLF1 transcript or deletion of the TATA box, but not the putative ATG initiation codon, reduce OriLyt function to background levels. By using an antibody specific for RNA-DNA hybrid molecules, we found the BHLF1 RNA stably annealed to its DNA template during the early steps of lytic reactivation. Furthermore, expression of human RNase H1, which degrades RNA in RNA-DNA hybrids, drastically reduces OriLyt-dependent DNA replication as well as recruitment of the viral single-stranded DNA binding protein BALF2 to OriLyt. These studies suggest that a GC-rich OriLyt transcript is an important component of gammaherpesvirus lytic origins and is required for initial strand separation and loading of core replication proteins.
Herpesvirus persistence requires a dynamic balance between latent and lytic cycle gene expression, but how this balance is maintained remains enigmatic. We have previously shown that the Kaposi's Sarcoma-Associated Herpesvirus (KSHV) major latency transcripts encoding LANA, vCyclin, vFLIP, v-miRNAs, and Kaposin are regulated, in part, by a chromatin organizing element that binds CTCF and cohesins. Using viral genome-wide chromatin conformation capture (3C) methods, we now show that KSHV latency control region is physically linked to the promoter regulatory region for ORF50, which encodes the KSHV immediate early protein RTA. Other linkages were also observed, including an interaction between the 5′ and 3′ end of the latency transcription cluster. Mutation of the CTCF-cohesin binding site reduced or eliminated the chromatin conformation linkages, and deregulated viral transcription and genome copy number control. siRNA depletion of CTCF or cohesin subunits also disrupted chromosomal linkages and deregulated viral latent and lytic gene transcription. Furthermore, the linkage between the latent and lytic control region was subject to cell cycle fluctuation and disrupted during lytic cycle reactivation, suggesting that these interactions are dynamic and regulatory. Our findings indicate that KSHV genomes are organized into chromatin loops mediated by CTCF and cohesin interactions, and that these inter-chromosomal linkages coordinate latent and lytic gene control.
Multiple mechanisms have been implicated in the control of herpesvirus latent and lytic gene regulation, but few mechanisms account for coordinate regulation of these two life cycles. Here, we show that the transcription control elements for KSHV latent and lytic genes are in close physical proximity. Mutations in the CTCF binding sites of the KSHV latency control region caused a loss of cohesin binding, and derepression of latent transcripts. Loss of CTCF binding also caused a loss of KSHV DNA copy number, and a failure to express lytic genes, including the immediate early gene Rta. Chromatin conformation capture (3C) methods indicated that the CTCF binding sites in the latency control region are linked to the promoter region of Rta. Additional chromatin linkages were detected between the 5′ and 3′ ends of the major latency transcripts, suggesting that chromatin loops organize both latent and lytic gene clusters. The interaction between latent and lytic control regions was subject to cell cycle regulation, consistent with earlier studies implicating cell cycle control of cohesin binding and viral transcription patterns. KSHV chromosome conformation was also disrupted by lytic cycle reactivation. We propose that CTCF-cohesin form dynamic linkages between viral regulatory domains to both insulate and coordinate latent and lytic gene expression.
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.
Epstein-Barr Virus (EBV) can establish latent infections with distinct gene expression patterns referred to as latency types. These different latency types are epigenetically stable and correspond to different promoter utilization. Here we explore the three-dimensional conformations of the EBV genome in different latency types. We employed Chromosome Conformation Capture (3C) assay to investigate chromatin loop formation between the OriP enhancer and the promoters that determine type I (Qp) or type III (Cp) gene expression. We show that OriP is in close physical proximity to Qp in type I latency, and to Cp in type III latency. The cellular chromatin insulator and boundary factor CTCF was implicated in EBV chromatin loop formation. Combining 3C and ChIP assays we found that CTCF is physically associated with OriP-Qp loop formation in type I and OriP-Cp loop formation in type III latency. Mutations in the CTCF binding site located at Qp disrupt loop formation between Qp and OriP, and lead to the activation of Cp transcription. Mutation of the CTCF binding site at Cp, as well as siRNA depletion of CTCF eliminates both OriP-associated loops, indicating that CTCF plays an integral role in loop formation. These data indicate that epigenetically stable EBV latency types adopt distinct chromatin architectures that depend on CTCF and mediate alternative promoter targeting by the OriP enhancer.
Epstein-Barr Virus (EBV) latent infection is associated with several human malignancies. The viral genes expressed during latent infection can vary depending on host cell or tumor type. The different gene expression programs, referred to as latency types, are determined by alternative viral promoter usage. In this work, we investigate how differential DNA loop formation regulates viral promoter selection in different latency types. We use chromatin conformation capture methods to demonstrate that the transcriptional enhancer at OriP forms a stable loop with one of two different promoters, depending on the latency type. In type I latency, OriP forms a loop with the active Q promoter (Qp). In type III latency, OriP forms a loop with the active C promoter (Cp). Loop formation was mediated, in part, by CTCF binding sites located within the loops. Mutation in the CTCF binding site located at Qp caused a loss of OriP-Qp loop formation, a loss of Qp transcription, and a reactivation of Cp transcription from an alternative loop formed with OriP-Cp. These findings indicate that OriP loop formation is an integral component of promoter selection, and that chromatin conformation may determine EBV latency type.