ebv-sisRNA-3 contributes to the formation of G4-associated R-loop upstream of EBV lytic replication origin in latently infected cells.

BACKGROUND: In EBV-associated epithelial cancers, only a limited number of viral proteins are translated, while multiple EBV-encoded non-coding RNAs are expressed to minimize activation of the host's immune response. These non-coding RNAs have been shown to play regulatory roles in maintaining latency and promoting cancer progression while many aspects of them remain to be elucidated. RESULTS: Here we revealed abundant expression of ebv-sisRNA-3, a novel EBV transcript in nasopharyngeal carcinoma and EBV-associated gastric cancer. This 5-7 kb non-polyA transcript is derived from RPMS1 intron and is partially complementary to LF3. We observed high expression level of ebv-sisRNA-3 in multiple EBV-positive cancer cells and clinical specimens, with accumulation in the cell nucleus. Notably, ebv-sisRNA-3 invades the double-strand DNA in trans upstream of lytic replication origin in EBV genome and leads to the formation of R-loop and G-quadruplex simultaneously in the latently EBV-infected epithelial cells. Additionally, we revealed the locations of R-loops within the EBV genome and identified endogenous G-quadruplexes near the EBER1 and EBNA1 promoters. CONCLUSIONS: In this study, we revealed and characterized a novel EBV transcript ebv-sisRNA-3 widely expressed in latently infected cells. The unique ebv-sisRNA-3-binding R-loop and G-quadruplex structures near lytic replication origin may play a significant role in EBV lytic replication.