The Epstein-Barr Virus BRRF1 Protein, Na, Induces Lytic Infection in a TRAF2- and p53-Dependent Manner

114

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.

Section: Discussionmentioning

confidence: 99%

Cohesins Repress Kaposi's Sarcoma-Associated Herpesvirus Immediate Early Gene Transcription during Latency

Chen

Wikramasinghe

Showe

et al. 2012

114

“…pSG5-Z, containing the genomic Z sequence transcribed from a simian virus 40 (SV40) promoter, and pSG5-R, containing the genomic sequence encoding R beginning from the second exon but lacking the Na-encoding first exon, were a gift from Diane Hayward (39). pSG5-FLAGNa contains a FLAG tag on the amino terminus of the Na open reading frame (38). pRK-BALF4 codes for the EBV glycoprotein 110 and was a gift from H.-J.…”

Section: Methodsmentioning

confidence: 99%

“…In addition, expression of the BRRF1-encoded early gene product, Na, is sufficient to reactivate the lytic form of EBV infection in some latently infected epithelial cell lines, due to the ability of Na to activate the Z promoter indirectly through cellular factors (38,42). Therefore, the cellular and viral proteins that regulate Z, R, and Na gene expression play a key role in determining if EBV infection is latent or lytic.…”

mentioning

confidence: 99%

“…Since we recently showed that the tumor suppressor protein p53 greatly facilitates lytic reactivation induced by overexpression of the EBV Na (BRRF1) and R proteins (38), and DNA damage potently activates p53 transcriptional function (84,86), DNAdamaging agents could potentially induce EBV reactivation (at least partially) via activation of p53. Recently, p53 was also shown to be required for HDACi-mediated lytic reactivation in both EBV-positive nasopharyngeal carcinoma cell lines and lymphoblastoid B cell lines (13,17,38), further confirming a critical role of p53 function in controlling EBV reactivation in a variety of different settings.…”

mentioning

confidence: 99%

See 1 more Smart Citation

The Cellular Ataxia Telangiectasia-Mutated Kinase Promotes Epstein-Barr Virus Lytic Reactivation in Response to Multiple Different Types of Lytic Reactivation-Inducing Stimuli

Hagemeier

Barlow

Qiao

et al. 2012

Self Cite

The Epstein-Barr virus (EBV) latent-to-lytic switch is mediated by the viral proteins BZLF1 (Z), BRLF1 (R), and BRRF1 (Na). Since we previously showed that DNA-damaging agents (including chemotherapy and irradiation) can induce EBV lytic reactivation and recently demonstrated that wild-type p53 contributes to lytic reactivation, we investigated the role of the ATM kinase during EBV reactivation. ATM phosphorylates and activates p53, as well as numerous other substrates involved in the cellular DNA damage response. Using an ATM inhibitor (KU55933), we found that ATM activity is required for efficient induction of EBV lytic gene expression by a variety of different stimuli, including a histone deacetylase (HDAC) inhibitor, the transforming growth factor ␤ (TGF-␤) cytokine, a demethylating agent (5-azacytidine), B cell receptor engagement with anti-IgG antibody, hydrogen peroxide, and the proteosome inhibitor bortezomib. In EBV-infected AGS (gastric) cells, knockdown of ATM, or p53, expression inhibits EBV reactivation. Conversely, treatment of these cells with nutlin-3 (which activates p53 and ATM) robustly induces lytic reactivation in a p53-and ATM-dependent manner. The ability of the EBV R and Na proteins to induce lytic reactivation in EBV-infected AGS cells is ATM dependent. However, overexpression of Z induces lytic gene expression in the presence or absence of ATM activity. Our results suggest that ATM enhances Z promoter activity in the context of the intact EBV genome and that p53 contributes to the ATM effect. Nevertheless, since we found that ATM inhibitors also reduce lytic reactivation in Burkitt lymphoma cells that have no p53, additional ATM substrates must also contribute to the ATM effect. E pstein-Barr virus (EBV) is a gammaherpesvirus that is the cause of infectious mononucleosis and is associated with a variety of epithelial and B cell cancers, including nasopharyngeal carcinoma (NPC), a subset of gastric carcinomas, Burkitt lymphoma (BL), and Hodgkin's disease (77,102). Like all herpesviruses, EBV can infect cells in either latent or lytic forms. Following infection of humans, EBV establishes long-term viral latency in the memory B cell compartment but can be reactivated to undergo the lytic form of infection following plasma cell differentiation (51). EBV infection of normal epithelial cells generally results in lytic infection (32,93,94), although EBV ϩ epithelial cell tumors such as NPCs and gastric carcinomas are primarily composed of cells with latent forms of infection (48,77). Both the latent and lytic forms of EBV infection are essential for the long-term success of the virus, and the latent-lytic switch is tightly controlled by both cellular and viral factors.The two EBV immediate-early (IE) proteins, BZLF1 (Z, also known as Zta, ZEBRA, or EB1) and BRLF1 (R), are transcription factors that activate expression of lytic viral promoters, and overexpression of either the Z or R IE protein is sufficient to reactivate the lytic form of EBV infection in many latently infected cell lines (2, ...

“…Another KSHV-encoded protein, the open reading frame 49 protein (ORF49 KSHV ), has been shown to upregulate the transcriptional activity of RTA (18). The protein homologs of ORF49 KSHV in Epstein-Barr virus (EBV) and murine herpesvirus 68 (MHV-68) display similar effects in the respective viruses (19)(20)(21)(22). ORF49 KSHV has been demonstrated to enhance lytic replication in KSHV, while the lack of the ORF49 MHV-68 protein has been shown to attenuate viral growth of MHV-68 (23).…”

mentioning

confidence: 99%

Structure of the Open Reading Frame 49 Protein Encoded by Kaposi's Sarcoma-Associated Herpesvirus

Hew

Veerappan

Sim

et al. 2017

Herpesviruses alternate between the latent and the lytic life cycle. Switching into the lytic life cycle is important for herpesviral replication and disease pathogenesis. Activation of a transcription factor replication and transcription activator (RTA) has been demonstrated to govern this switch in Kaposi's sarcomaassociated herpesvirus (KSHV). The protein encoded by open reading frame 49 from KSHV (ORF49 KSHV ) has been shown to upregulate lytic replication in KSHV by enhancing the activities of the RTA. We have solved the crystal structure of the ORF49 KSHV protein to a resolution of 2.4 Å. The ORF49 KSHV protein has a novel fold consisting of 12 alpha-helices bundled into two pseudodomains. Most notably are distinct charged patches on the protein surface, which are possible protein-protein interaction sites. Homologs of the ORF49 KSHV protein in the gammaherpesvirus subfamily have low sequence similarities. Conserved residues are mainly located in the hydrophobic regions, suggesting that they are more likely to play important structural roles than functional ones. Based on the identification and position of three sulfates binding to the positive areas, we performed some initial protein-DNA binding studies by analyzing the thermal stabilization of the protein in the presence of DNA. The ORF49 KSHV protein is stabilized in a dose-responsive manner by doublestranded oligonucleotides, suggesting actual DNA interaction and binding. Biolayer interferometry studies also demonstrated that the ORF49 KSHV protein binds these oligonucleotides. IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) is a tumorigenic gammaherpesvirus that causes multiple cancers and lymphoproliferative diseases. The virus exists mainly in the quiescent latent life cycle, but when it is reactivated into the lytic life cycle, new viruses are produced and disease symptoms usually manifest. Several KSHV proteins play important roles in this reactivation, but their exact roles are still largely unknown. In this study, we report the crystal structure of the open reading frame 49 protein encoded by KSHV (ORF49 KSHV ). Possible regions for protein interaction that could harbor functional importance were found on the surface of the ORF49 KSHV protein. This led to the discovery of novel DNA binding properties of the ORF49 KSHV protein. Evolutionary conserved structural elements with the functional homologs of ORF49 KSHV were also established with the structure.