N6-methyladenosine modification of the 5′ epsilon structure of the HBV pregenome RNA regulates its encapsidation by the viral core protein

Hepatitis B virus (HBV) contains a partially double-stranded DNA genome. During infection, its replication is mediated by reverse transcription (RT) of an RNA intermediate termed pregenomic RNA (pgRNA) within core particles in the cytoplasm. An epsilon structural element located in the 5′ end of the pgRNA primes the RT activity. We have previously identified the N6-methyladenosine (m(6)A)–modified DRACH motif at 1905 to 1909 nucleotides in the epsilon structure that affects myriad functions of the viral life cycle. In this study, we investigated the functional role of m(6)A modification of the 5′ ε (epsilon) structural element of the HBV pgRNA in the nucleocapsid assembly. Using the m(6)A site mutant in the HBV 5′ epsilon, we present evidence that m(6)A methylation of 5′ epsilon is necessary for its encapsidation. The m(6)A modification of 5′ epsilon increased the efficiency of viral RNA packaging, whereas the m(6)A of 3′ epsilon is dispensable for encapsidation. Similarly, depletion of methyltransferases (METTL3/14) decreased pgRNA and viral DNA levels within the core particles. Furthermore, the m(6)A modification at 5′ epsilon of HBV pgRNA promoted the interaction with core proteins, whereas the 5′ epsilon m(6)A site–mutated pgRNA failed to interact. HBV polymerase interaction with 5′ epsilon was independent of m(6)A modification of 5′ epsilon. This study highlights yet another pivotal role of m(6)A modification in dictating the key events of the HBV life cycle and provides avenues for investigating RNA–protein interactions in various biological processes, including viral RNA genome encapsidation in the context of m(6)A modification.