NSUN2-mediated m5C modification of HBV RNA positively regulates HBV replication

Chronic hepatitis B virus (HBV) infection is a major cause of liver cirrhosis and liver cancer, despite strong prevention and treatment efforts. The study of the epigenetic modification of HBV has become a research hotspot, including the N 6-methyladenosine (m 6 A) modification of HBV RNA, which plays complex roles in the HBV life cycle. In addition to m 6 A modification, 5-methylcytosine (m 5 C) is another major modification of eukaryotic mRNA. In this study, we explored the roles of m 5 C methyltransferase and demethyltransferase in the HBV life cycle. The results showed that m 5 C methyltransferase NSUN2 deficiency could negatively regulate the expression of HBV while m 5 C demethyltransferase TET2 deficiency positively regulates the expression of HBV. Subsequently, we combined both in vitro bisulfite sequencing and high-throughput bisulfite sequencing methods to determine the distribution and stoichiometry of m 5 C modification in HBV RNA. Two sites: C2017 and C131 with the highest-ranking methylation rates were identified, and mutations at these two sites could lead to the decreased expression and replication of HBV, while the mutation of the “fake” m 5 C site had no effect. Mechanistically, NSUN2-mediated m 5 C modification promotes the stability of HBV RNA. In addition, compared with wild-type HepG2-NTCP cells and primary human hepatocytes, the replication level of HBV after NSUN2 knockdown decreased, and the ability of the mutant virus to infect and replicate in wild-type HepG2-NTCP cells and PHHs was substantially impaired. Similar results were found in the experiments using C57BL/6JGpt- Nsun2 +/- mice. Interestingly, we also found that HBV expression and core protein promoted the endogenous expression of NSUN2, which implied a positive feedback loop. In summary, our study provides an accurate and high-resolution m 5 C profile of HBV RNA and reveals that NSUN2-mediated m 5 C modification of HBV RNA positively regulates HBV replication by maintaining RNA stability.