The RNA Binding Proteins YTHDC1 and FMRP Regulate the Nuclear Export of N6-Methyladenosine-Modified Hepatitis B Virus Transcripts and Affect the Viral Life Cycle.

YTHDC1 and fragile X mental retardation protein (FMRP) bind N6-methyladenosine (m6A)-modified RNAs and facilitate their transport to the cytoplasm. Here, we investigated the role of these proteins in hepatitis B virus (HBV) gene expression and life cycle. We have previously reported that HBV transcripts are m6A methylated, and this modification regulates the viral life cycle. HBV is particularly interesting, as its DNA genome upon transcription gives rise to a pregenomic RNA (pgRNA), which serves as a template for reverse transcription to produce the relaxed circular DNA that transforms into a covalently closed circular DNA (cccDNA). While m6A modification negatively affects RNA stability and translation of viral transcripts, our current results revealed the possibility that it positively affects pgRNA encapsidation in the cytoplasm. Thus, it plays a differential dual role in the virus life cycle. YTHDC1 as well as FMRP recognize m6A-methylated HBV transcripts and facilitate their transport to the cytoplasm. In cells depleted with YTHDC1 or FMRP, viral transcripts accumulate in the nucleus to affect the viral life cycle. Most importantly, the core-associated DNA and subsequent cccDNA syntheses are dramatically affected in FMRP- or YTHDC1-silenced cells. This study highlights the functional relevance of YTHDC1 and FMRP in the HBV life cycle with the potential to arrest liver disease pathogenesis. IMPORTANCE YTHDC1 and FMRP have been recently implicated in the nuclear export of m6A modified mRNAs. Here, we show that FMRP and YTHDC1 proteins bind with m6A-modified HBV transcripts and facilitate their nuclear export. In the absence of FMRP and YTHDC1, HBV transcripts accumulate in the nucleus to reduce reverse transcription in HBV core particles and subsequently the cccDNA synthesis. Our study shows how m6A binding proteins can regulate the HBV life cycle by facilitating the nuclear export of m6A-modified HBV RNA.