A comprehensive single cell data analysis of lymphoblastoid cells reveals the role of super‐enhancers in maintaining EBV latency

We probed the lifecycle of Epstein‐Barr virus (EBV) on a cell‐by‐cell basis using single cell RNA sequencing (scRNA‐seq) data from nine publicly available lymphoblastoid cell lines (LCLs). While the majority of LCLs comprised cells containing EBV in the latent phase, two other clusters of cells were clearly evident and were distinguished by distinct expression of host and viral genes. Notably, both were high expressors of EBV LMP1/BNLF2 and BZLF1 compared to another cluster that expressed neither gene. The two novel clusters differed from each other in their expression of EBV lytic genes, including glycoprotein gene GP350. The first cluster, comprising GP350 (–) LMP1 ( hi ) cells, expressed high levels of HIF1A and was transcriptionally regulated by HIF1‐α. Treatment of LCLs with Pevonedistat, a drug that enhances HIF1‐α signaling, markedly induced this cluster. The second cluster, containing GP350 (+) LMP1 ( hi ) cells, expressed EBV lytic genes. Host genes that are controlled by super‐enhancers (SEs), such as transcription factors MYC and IRF4, had the lowest expression in this cluster. Functionally, the expression of genes regulated by MYC and IRF4 in GP350 (+) LMP1 ( hi ) cells were lower compared to other cells. Indeed, induction of EBV lytic reactivation in EBV(+) AKATA reduced the expression of these SE‐regulated genes. Furthermore, CRISPR‐mediated perturbation of the MYC or IRF4 SEs in LCLs induced the lytic EBV gene expression, suggesting that host SEs and/or SE target genes are required for maintenance of EBV latency. Collectively, our study revealed EBV‐associated heterogeneity among LCLs that may have functional consequence on host and viral biology.