Epstein-Barr virus-associated chromatin activation and enhancer engagement in T/NK-cell lymphoproliferative diseases.

Epstein-Barr virus (EBV) infects many people and causes T-cell and natural killer (NK)-cell disorders, such as chronic active EBV disease and extranodal NK/T-cell lymphoma, which are aggressive and lack a common genetic driver. This suggests that virus-associated, non-genetic mechanisms contribute to disease. We studied primary T cells, NK cells, Epstein-Barr virus-positive cell lines derived from these disorders, virus-negative controls, and clinical chronic active EBV disease samples. Using genome-wide and single-cell multiome approaches, we examined three-dimensional chromatin organization, histone modifications, viral-host chromatin proximity, regulatory element activity, and transcription. EBV-positive lines shared a chromatin compartment pattern distinct from primary and virus-negative cancer cells. Regions that changed from inactive to active chromatin states showed increased activating histone marks and higher expression of nearby genes. EBV-interacting regions were enriched in inactive chromatin and inactive-to-active shifting regions, and contained activated promoters and enhancers associated with increased expression of neighboring genes in both T-cell and NK-cell backgrounds. Locus-level analyses identified CACNA2D1 and RGS1 as representative target genes with chromatin activation, and knockdown of each gene impaired cell growth in EBV-positive lines. Single-cell multiome profiling of clinical chronic active EBV disease samples identified EBV read-positive T-cell or NK-cell populations consistent with independently defined infected lineages and validated epigenomic and transcriptional rewiring at EBV-interacting regions. These findings support a model in which EBV contributes to oncogenic transcriptional programs in these diseases by coupling three-dimensional chromatin reorganization and regulatory element activation to physical interactions with the host genome.