Epstein-Barr virus encodes competing long non-coding RNAs from the oriLyt locus that control the viral lytic switch through opposing regulation of CTCF recruitment and DNA looping: a pro-lytic lncRNA containing an RNA G-quadruplex structure recruits CTCF to drive cohesin-mediated looping between oriLyt enhancers and the BZLF1 immediate early promoter to trigger reactivation, while a pro-latency lncRNA forms duplexes with the pro-lytic RNA to disrupt its G-quadruplex structure and prevent CTCF association, thereby establishing a threshold for reactivation that is controlled by MYC and the LSD1/CoREST/ZNF217 repressor complex.

Epstein-Barr virus (EBV) uses a biphasic lifecycle, switching between latent and lytic phases to persistently infect most adults. Latency is observed in most tumor cells of the 200,000 EBV-associated cancers/year. EBV reactivation is increasingly implicated in autoimmune diseases, including multiple sclerosis. However, mechanisms that regulate EBV reactivation have remained incompletely understood. Here, we leveraged multi-omic approaches to reveal the existence of pro-latency and a pro-lytic viral long noncoding RNAs (lncRNAs) that counter-regulate the lytic switch. Known reactivation triggers rapidly induced expression of the pro-lytic lncRNA, which encodes an RNA G-quadruplex that mediated its interaction with CTCF. The pro-lytic lncRNA occupies viral origin of lytic replication enhancers and promotes their looping to the immediate early lytic promoter to trigger reactivation. The pro-latency lncRNA duplexes with the pro-lytic RNA to impede its interactions with CTCF. These studies lay a foundation for therapeutic approaches to manipulate the EBV lytic switch.