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

N6-methyladenosine (m(6)A) RNA methylation is the most abundant epitranscriptomic modification of eukaryotic messenger RNAs (mRNAs). Previous reports have found m(6)A on both cellular and viral transcripts and defined its role in regulating numerous biological processes, including viral infection. Here, we show that m(6)A 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 m(6)A to RNA resulted in increased HBV protein expression, but overall reduced reverse transcription of the pgRNA. We mapped the m(6)A site in the HBV RNA and found that a conserved m(6)A consensus motif situated within the epsilon stem loop structure, is the site for m(6)A 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 m(6)A site in the 5' epsilon stem loop of pgRNA revealed that m(6)A at this site is required for efficient reverse transcription of pgRNA, while m(6)A methylation of the 3' epsilon stem loop results in destabilization of all HBV transcripts, suggesting that m(6)A has dual regulatory function for HBV RNA. Overall, this study reveals molecular insights into how m(6)A regulates HBV gene expression and reverse transcription, leading to an increased level of understanding of the HBV life cycle.