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

TL;DR

A new EBV non-coding RNA, ebv-sisRNA-3, is found to form DNA structures that may influence viral replication in cancer cells.

Contribution

Discovery and characterization of ebv-sisRNA-3, a novel EBV transcript involved in forming R-loops and G-quadruplexes.

Findings

ebv-sisRNA-3 is abundantly expressed in EBV-positive cancer cells and clinical specimens.

ebv-sisRNA-3 forms R-loops and G-quadruplexes upstream of the EBV lytic replication origin.

Endogenous G-quadruplexes are identified near EBER1 and EBNA1 promoters in the EBV genome.

Abstract

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.

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.

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.

The online version contains supplementary material available at 10.1186/s13578-025-01437-3.

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