NSUN2-mediated m5C modification of BHLF1 RNA facilitates the lytic replication of Epstein-Barr virus

The 5-methylcytosine (m5C) modification of mRNA mediates diverse cellular and viral functions. Epstein-Barr virus (EBV) is etiologically linked to multiple malignancies, including nasopharyngeal carcinoma (NPC), in which both viral latency and lytic replication play critical pathogenic roles. Here, we show that the transcripts derived from the EBV gene BHLF1 exhibit abundant m5C modification in patient-derived xenograft tissue as well as in NPC cells during the lytic stage of EBV infection. Mechanistically, NSUN2 mediates the m5C modification of EBV BHLF1 RNA, which not only enhances transcript stability but also promotes DNA methylation at the EBV origin of lytic replication (oriLyt) region near the m5C site through recruitment of DNA methyltransferase 1 (DNMT1) to facilitate BZLF1 binding, thereby promoting efficient EBV lytic replication. Taken together, our findings provide mechanistic insights into the roles of m5C modification in the EBV life cycle and highlight the importance of crosstalk between RNA m5C and DNA methylation in regulating viral replication.