Integrated analyses reveal CHD4 mediating establishment and maintenance of latent KSHV chromatin [Capture Hi-C]

lncRNAs regulate protein functions via formation of protein-RNA complexes. Previous studies have shown that expression of viral lncRNA, polyadenylated nuclear RNA (PAN RNA) is essential for inducible viral genomic looping and distal gene activation during Kaposi's sarcoma-associated herpesvirus (KSHV) reactivation. Here we show an underlying molecular mechanism and regulation of KSHV latency by a viral lncRNA-CHD4 (chromodomain helicase DNA binding protein 4) interaction. Knock-out of the viral RNA binding protein, ORF57 protein, leads to decreased inducible and static viral genomic loops in latent chromatin and a failure to form RNA polymerase II aggregates in the nucleus during reactivation. We identified that CHD4's enzymatic activity silences viral gene expression by preventing nuclear aggregate formation. Furthermore, integrated genomic and proteomic studies together show that KSHV episomes frequently tether near the host cell centromeres and colocalize with a CHD4 protein complex, ChAHP. KSHV episomes detached from these sites when reactivation is triggered, and PAN RNA binds and inhibits CHD4 DNA binding in vitro. Our studies suggest that CHD4 exhibits strong repressor function by preventing inducible enhancer-promoter looping, and is therefore important for the ability of KSHV to establish and maintain latency in a “poised” state at specific host genomic loci. Overall design: The overall goal of these studies was to define the long-range chromatin interactions occurring within the KSHV genome as well as between the KSHV and human host cell genomes both during latency and viral reactivation. These were defined in an unbiased manner using Hi-C analysis, which is chromosome conformation capture (3C) combined with next-generation sequencing (NGS). A modified protocol, referred to as Capture Hi-C, was utilized to selectively enrich interactions involving KSHV by performing target enrichment with a custom-designed KSHV genomic capture probe library. Experiments were performed in three different KSHV naturally infected human primary effusion lymphoma (PEL) cell lines (TREx-F3H3-K-Rta BCBL-1, BC1 and BC3) and wild type or ORF57-Stop recombinant KSHV infected iSLK cells.