Epigenetic addition of mC to HBV transcripts promotes viral replication and evasion of innate antiviral responses.

Eukaryotic five-methylcytosine (mC) is an important regulator of viral RNA splicing, stability, and translation. However, its role in HBV replication remains largely unknown. In this study, functional mC sites are identified in hepatitis B virus (HBV) mRNA. The mC 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 mC 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 mC modification of type I IFN mRNA in host cells, thus, positively regulating IFN expression. Hence, the delicate regulation of NSUN2 expression induces mC modification of HBV mRNA while decreasing the levels of mC 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.