Native epitranscriptome sequencing reveals Hepatitis B virus RNA stability protected by heavy m ⁶ A modification

Although the RNA of Hepatitis B virus (HBV) is known to be regulated by the epitranscriptomic modification N ⁶ -methyladenosine (m ⁶ A), it remains controversial whether if m ⁶ A is beneficial or detrimental for viral replication. Here, we employ Nanopore direct RNA sequencing (DRS) to analyze the HBV m ⁶ A epitranscriptome on a single molecule level and elucidate how m ⁶ A regulates the HBV replication cycle. We found more than 60% of all HBV RNA in cells are m ⁶ A methylated, ∼3x more methylated than host mRNAs. Previously unreported m ⁶ A sites were found in pol gene, which when mutated reduced viral production. A sizable proportion of HBV RNAs carry poly(A) tails at least twice as long (>120nt) as cellular mRNAs, with the shorter-tailed population more pronounced among m ⁶ A-free transcripts. In contrast, encapsidated RNAs have shorter poly(A) tails and less m ⁶ A. Concordantly, pharmacological reduction of viral m ⁶ A using the m ⁶ A methyltransferase inhibitor STM2457, shortens viral poly(A) tails and reduces viral RNA stability. Our findings thus suggest that the abundance of m ⁶ A on viral RNAs protects the surprisingly long viral poly(A) tails and thus enhances RNA stability. Overall, m ⁶ A strongly enhances HBV viral replication, in support of RNA modification machinery as a promising class of antiviral targets.