Epigenetic addition of m 5 C to HBV transcripts promotes viral replication and evasion of innate antiviral responses

Eukaryotic five-methylcytosine (m 5 C) is an important regulator of viral RNA splicing, stability, and translation. However, its role in HBV replication remains largely unknown. In this study, functional m 5 C sites are identified in hepatitis B virus (HBV) mRNA. The m 5 C modification at nt 1291 is not only indispensable for Aly/REF export factor (ALYREF) recognition to promote viral mRNA export and HBx translation but also for the inhibition of RIG-I binding to suppress interferon-β (IFN-β) production. Moreover, NOP2/Sun RNA methyltransferase 2 (NSUN2) catalyzes the addition of m 5 C to HBV mRNA and is transcriptionally downregulated by the viral protein HBx, which suppresses the binding of EGR1 to the NSUN2 promoter. Additionally, NSUN2 expression correlates with m 5 C modification of type I IFN mRNA in host cells, thus, positively regulating IFN expression. Hence, the delicate regulation of NSUN2 expression induces m 5 C modification of HBV mRNA while decreasing the levels of m 5 C in host IFN mRNA, making it a vital component of the HBV life cycle. These findings provide new molecular insights into the mechanism of HBV-mediated IFN inhibition and may inform the development of new IFN-α based therapies.