Identification of Host-Chromosome Binding Sites and Candidate Gene Targets for Kaposi's Sarcoma-Associated Herpesvirus LANA

Abstract

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.

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

“…We compared our results with data from two independent ChIP-Seq analysis experiments where binding of LANA with KSHV episome was examined (45). Seven of the nine regions having a LANA binding peak score of Ͼ2 in ChIP-Seq analysis displayed a Ն2-fold binding with ␥H2AX in our experiments ( Table 1).…”

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

“…ChIP-Seq peaks for CTCF and SMC1 were identified using a Poisson distribution model (modified MACS program) and then normalized to an IgG control (Fig. 3A) (43). As reported previously, most cohesin (e.g., SMC1) peaks overlapped extensively with CTCF peaks (56,62,68,78).…”

“…ChIP with high-throughput sequencing (ChIP-Seq) was performed as described previously (43). Briefly, 1 ϫ 10 7 BCBL1 cells were used per assay, with either rabbit anti-cellular SMC1 (Bethyl) or anti-CTCF (Millipore) antibody or control rabbit IgG (Santa Cruz Biotechnology).…”

“…DNA fragments of ϳ150 to 300 bp were isolated by agarose gel purification, ligated to primers, and subjected to Solexa-based sequencing using the manufacturer's recommendations (Illumina, Inc.). Peak-calling analysis of the KSHV genome was performed as described previously (43).…”

Kaposi sarcoma–associated herpesvirus: immunobiology, oncogenesis, and therapy

“…Although it is primarily a DNA-binding protein and transcription factor, KSHV Rta/ORF50 can also induce the degradation of innate immune sensors such as IRF7, TLR3, and myeloid differentiation factor 88 (MyD88) (58)(59)(60)(61). The latencyassociated nuclear antigen (LANA) is another DNA-binding protein that inhibits IFN-β induction (62) and the transcription of IFN-γ-inducible genes (63). Recently, it was shown that cytoplasmic variants of LANA can inhibit the cGAS-STING DNA-sensing pathway by directly binding to cGAS (47).…”