Abstract
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.
Highlights
Kaposis’s Sarcoma-Associated Herpesvirus (KSHV) is the human gammaherpesvirus identified as the causative agent of Kaposi’s Sarcoma (KS), pleural effusion lymphoma (PEL), and Castleman’s Disease
Mutations in the CTCF binding sites of the Kaposi’s Sarcoma-Associated Herpesvirus (KSHV) latency control region caused a loss of cohesin binding, and derepression of latent transcripts
Loss of CTCF binding caused a loss of KSHV DNA copy number, and a failure to express lytic genes, including the immediate early gene Rta
Summary
Kaposis’s Sarcoma-Associated Herpesvirus (KSHV) is the human gammaherpesvirus identified as the causative agent of Kaposi’s Sarcoma (KS), pleural effusion lymphoma (PEL), and Castleman’s Disease ([1,2,3] (reviewed in [4,5,6]). KSHV pathogenesis depends on the long-term persistence of the viral genome and a restricted pattern of viral gene expression characteristic of latent infection. Most forms of latent infection and tissue biopsies express a complex multicistronic transcript encoding the viral genes for LANA (ORF73), vCyclin (ORF72), vFLIP (ORF71), v-miRNAs, and Kaposin (K12) [7,8]. These genes are required for viral episome maintenance, host-cell survival, and suppression of lytic gene activation [4,5,9]. The extent to which lytic gene expression is required for maintaining a stable copy number of viral genomes in a population of cells, and the mechanisms that regulate spontaneous lytic gene expression in latently infected cell pools remains enigmatic, yet central to our understanding KSHV persistence and pathogenesis
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