Hepatitis B virus X (HBx) Protein Expression is Tightly Regulated by N6-methyladenosine Modification of its mRNA.

Hepatitis B virus (HBV) encodes a regulatory protein termed HBx, that has been intensely studied in the past and shown to play a key role(s) in viral transcription and replication. In addition, a huge body of work exists in the literature related to signal transduction and possible mechanism(s) leading to hepatocarcinogenesis associated with infection. We have previously reported that HBV transcripts are modified by N6-methyladenosine (m6A) at the single consensus DRACH motif at 1905-1909 nucleotide (nt) in the epsilon structural element and this m6A modification affects the HBV life cycle. In this study, we present evidence that additional variants of m6A (DRACH) motifs are located within 1606 to 1809 nt correspond on the coding region of HBx mRNA and 3' untranslated region (UTR) of other viral mRNAs. Using the mutants of additional m6A site in 1606 to 1809 nt and a depletion strategy of m6A methyltransferases (METTL3/14) and reader proteins (YTHDFs), we show that m6A modification at 1616 nt, located in HBx coding region, regulates HBx protein expression. The HBx RNA and protein expressions were notably increased by the silencing of m6A reader YTHDF2 and methyltransferases as well as the mutation of m6A sites in the HBx coding region. However, other viral protein expressions were not affected by the m6A modification at 1616 nt. Thus, m6A modifications in the HBx open reading frame (ORF), downregulate HBx protein expression, commonly seen during HBV transfections, transgenic mice, and natural infections of human hepatocytes. These studies identify the functional role of m6A modification in the subtle regulation of HBx protein expression consistent with its possible role in establishing chronic hepatitis. Importance N6-methyladenosien (m6A) modifications have been recently implicated in the HBV life cycle. Previously, we observed that m6A modification occurs in the adenosine at 1907 nt of HBV genome and this modification regulates the viral life cycle. Here, we identified an additional m6A site located in 1616 nt of the HBV genome. This modification negatively affects HBx RNA and protein expression. In the absence of m6A methyltransferases (METTL3/14) and reader protein (YTHDF2), the HBx RNA and protein expression were increased. Using HBV mutants that lack m6A in the HBx coding region, we present the unique positional effects of m6A in the regulation of HBx protein expression.