The Single RBP-Jκ Site within the LANA Promoter Is Crucial for Establishing Kaposi's Sarcoma-Associated Herpesvirus Latency during Primary Infection

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

“…A previously described protocol was used with minor changes (28). Approximately 500 million exponentially growing HEK-293-BAC36 -KSHV cells were induced with 20 ng/ml of tetradecanoyl phorbol acetate (TPA) and 1.5 mM sodium butyrate (Sigma-Aldrich Corp., St. Louis, MO) for 4 to 5 days at 37°C in 5% CO 2 .…”

Section: Methodsmentioning

confidence: 99%

H2AX Phosphorylation Is Important for LANA-Mediated Kaposi's Sarcoma-Associated Herpesvirus Episome Persistence

Jha

Upadhyay

et al. 2013

Self Cite

The DNA damage response (DDR) of host cells is utilized by a number of viruses to establish and propagate their genomes in the infected cells. We examined the expression of the DDR genes during Kaposi's sarcoma-associated herpesvirus (KSHV) infection of human peripheral blood mononuclear cells (PBMCs). The genes were mostly downregulated, except H2AX, which was upregulated during infection. H2AX is important for gammaherpesvirus infectivity, and its phosphorylation at serine 139 is crucial for maintenance of latency during mouse gamma-herpesvirus 68 (MHV-68) infection. We now also observed phosphorylation of H2AX at serine 139 during KSHV infection. H2AX is a histone H2A isoform shown to interact with the latency-associated nuclear antigen (LANA) encoded by KSHV. Here, we show that LANA directly interacted with H2AX through domains at both its N and C termini. The phosphorylated form of H2AX (␥H2AX) was shown to colocalize with LANA. Chromatin immunoprecipitation (ChIP) assays showed that a reduction in H2AX levels resulted in reduced binding of LANA with KSHV terminal repeats (TRs). Binding preferences of H2AX and ␥H2AX along the KSHV episome were examined by whole-episome ChIP analysis. We showed that ␥H2AX had a higher relative binding activity along the TR regions than that of the long unique region (LUR), which highlighted the importance

“…The primary infection was performed as described previously (61,62). Briefly, 3.0 ϫ 10 6 Bac36-293 cells were seeded on T175 flask 24 h prior to induction.…”

Section: Methodsmentioning

confidence: 99%

“…Immunofluorescence assays were performed as previously described (61,62). Briefly, BC3 and JSC-1 cells were fixed and incubated with primary antibodies with mouse anti-LANA and rabbit anti-AK-B or antisurvivin.…”

Section: Methodsmentioning

confidence: 99%

Kaposi's Sarcoma-Associated Herpesvirus-Encoded LANA Contributes to Viral Latent Replication by Activating Phosphorylation of Survivin

Jha

Verma

et al. 2014

Self Cite

Kaposi's sarcoma-associated herpesvirus (KSHV) is a human gammaherpesvirus casually linked to Kaposi's sarcoma (KS), multicentric Castleman's disease (MCD), and primary effusion lymphoma (PEL). Previously, we showed that LANA encoded by KSHV upregulates expression of survivin, a member of the inhibitor of apoptosis (IAP) family. This leads to an increase in the rate of cell proliferation of KSHV-infected B cells. LANA is required for tethering of the KSHV episome to the host chromosomes and efficiently segregates the viral genomes into dividing tumor cells. Here we show that LANA interacts with Aurora kinase B (AK-B) and induces phosphorylation of survivin at residue T34. Phosphorylation of survivin specifically on residue T34 enhances the activity of p300 and inhibits the activity of histone deacetylase 1 (HDAC-1), which then leads to an increase in acetylation of histone H3 on the viral genome. Phosphorylation of survivin specifically on residue T34 upregulates the activities of histone acetyltransferases and deacetylases, which then leads to an increase in viral copy number in KSHV-infected B cells. This results in a boost of KSHV replication in latently infected B-lymphoma cells. The studies showed that LANA can also function to regulate viral replication prior to mitosis of the latently infected cells, suggesting that LANA possesses a novel role in regulating KSHV replication in infected B cells. IMPORTANCE