Epitranscriptomic cytidine methylation of the hepatitis B viral RNA is essential for viral reverse transcription and particle production.

Epitranscriptomic RNA modifications have emerged as important regulators of the fate and function of viral RNAs. One prominent modification, the cytidine methylation 5-methylcytidine (mC), is found on the RNA of HIV-1, where mC enhances the translation of HIV-1 RNA. However, whether mC functionally enhances the RNA of other pathogenic viruses remains elusive. Here, we surveyed a panel of commonly found RNA modifications on the RNA of hepatitis B virus (HBV) and found that HBV RNA is enriched with mC as well as ten other modifications, at stoichiometries much higher than host messenger RNA (mRNA). Intriguingly, mC is mostly found on the epsilon hairpin, an RNA element required for viral RNA encapsidation and reverse transcription, with these mC mainly deposited by the cellular methyltransferase NSUN2. Loss of mC from HBV RNA due to NSUN2 depletion resulted in a partial decrease in viral core protein (HBc) production, accompanied by a near-complete loss of the reverse transcribed viral DNA. Similarly, mutations introduced to remove the methylated cytidines resulted in a loss of HBc production and reverse transcription. Furthermore, pharmacological disruption of mC deposition led to a significant decrease in HBV replication. Thus, our data indicate mC methylations as a critical mediator of the epsilon elements' function in HBV virion production and reverse transcription, suggesting the therapeutic potential of targeting the mC methyltransfer process on HBV epsilon as an antiviral strategy.