N6-methyladenosine modification of hepatitis B virus RNA differentially regulates the viral life cycle

N6-methyladenosine (m⁶A) RNA methylation is the most abundant epitranscriptomic modification of eukaryotic messenger RNAs (mRNAs). Previous reports have found m⁶A on both cellular and viral transcripts and defined its role in regulating numerous biological processes, including viral infection. Here, we show that m⁶A and its associated machinery regulate the life cycle of hepatitis B virus (HBV). HBV is a DNA virus that completes its life cycle via an RNA intermediate, termed pregenomic RNA (pgRNA). Silencing of enzymes that catalyze the addition of m⁶A to RNA resulted in increased HBV protein expression, but overall reduced reverse transcription of the pgRNA. We mapped the m⁶A site in the HBV RNA and found that a conserved m⁶A consensus motif situated within the epsilon stem loop structure, is the site for m⁶A modification. The epsilon stem loop is located in the 3' terminus of all HBV mRNAs and at both the 5' and 3' termini of the pgRNA. Mutational analysis of the identified m⁶A site in the 5' epsilon stem loop of pgRNA revealed that m⁶A at this site is required for efficient reverse transcription of pgRNA, while m⁶A methylation of the 3' epsilon stem loop results in destabilization of all HBV transcripts, suggesting that m⁶A has dual regulatory function for HBV RNA. Overall, this study reveals molecular insights into how m⁶A regulates HBV gene expression and reverse transcription, leading to an increased level of understanding of the HBV life cycle.