Interferon (IFN) stimulates a whole repertoire of cellular genes, collectively referred to as ISGs (Interferon-stimulated genes). ISG20, a 3´-5´ exonuclease enzyme, has been previously shown to bind and degrade hepatitis B Virus (HBV) transcripts. Here, we show that the N6-methyladenosine (m6A)-modified HBV transcripts are selectively recognized and processed for degradation by ISG20. Moreover, this effect of ISG20 is critically regulated by m6A reader protein, YTHDF2 (YTH-domain family 2). Previously, we identified a unique m6A site within HBV transcripts and confirmed that methylation at nucleotide A1907 regulates HBV lifecycle. In this report, we now show that the methylation at A1907 is a critical regulator of IFN-α mediated decay of HBV RNA. We observed that the HBV RNAs become less sensitive to ISG20 mediated degradation when methyltransferase enzymes or m6A reader protein YTHDF2 are silenced in HBV expressing cells. By using an enzymatically inactive form ISG20D94G, we further demonstrated that ISG20 forms a complex with m6A modified HBV RNA and YTHDF2 protein. Due to terminal redundancy, HBV genomic nucleotide A1907 position is acquired twice by pregenomic RNA (pgRNA) during transcription and therefore the sites of methylation are encoded within 5´ and 3´ epsilon stem loops. We generated HBV mutants that lack m6A site at either one (5´ or 3´) or both the termini (5´& 3´). Using these mutants, we demonstrated that m6A modified HBV RNAs are subjected to ISG20-mediated decay and propose sequence of events, in which ISG20 binds with YTHDF2 and recognizes m6A-modified HBV transcripts to carry out the ribonuclease activity. This is the first study, which identifies a hitherto unknown role of m6A modification of RNA in IFN-α induced viral RNA degradation and proposes a new role of YTHDF2 protein as a cofactor required for IFN-α mediated viral RNA degradation.

Background and aims

Epitranscriptomic modification of RNA has emerged as the most prevalent form of regulation of gene expression that affects development, differentiation, metabolism, viral infections, and most notably cancer. We have previously shown that hepatitis B virus (HBV) transcripts are modified by N6 methyladenosine (m⁶ A) addition. HBV also affects m⁶ A modification of several host RNAs, including phosphatase and tensin homolog (PTEN), a well-known tumor suppressor. PTEN plays a critical role in antiviral innate immunity and the development of hepatocellular carcinoma (HCC). Reports have shown that PTEN controlled interferon regulatory factor 3 (IRF-3) nuclear localization by negative phosphorylation of IRF-3 at Ser97, and PTEN reduced carcinogenesis by inhibiting the phosphatidylinositol-3-kinase (PI3K)/AKT pathway.

Approach and results

Here, we show that HBV significantly increases the m⁶ A modification of PTEN RNA, which contributes to its instability with a corresponding decrease in PTEN protein levels. This is reversed in cells in which the expression of m⁶ A methyltransferases is silenced. PTEN expression directly increases activated IRF-3 nuclear import and subsequent interferon synthesis. In the absence of PTEN, IRF-3 dephosphorylation at the Ser97 site is decreased and interferon synthesis is crippled. In chronic HBV patient biopsy samples, m⁶ A-modified PTEN mRNA levels were uniformly up-regulated with a concomitant decrease of PTEN mRNA levels. HBV gene expression also activated the PI3K/AKT pathway by regulating PTEN mRNA stability in HCC cell lines.

Conclusions

The m⁶ A epitranscriptomic regulation of PTEN by HBV affects innate immunity by inhibiting IRF-3 nuclear import and the development of HCC by activating the PI3K/AKT pathway. Our studies collectively provide new insights into the mechanisms of HBV-directed immune evasion and HBV-associated hepatocarcinogenesis through m⁶ A modification of the host PTEN mRNAs.

N6-methyladenosine (m⁶A) RNA methylation is the most abundant epitranscriptomic modification of eukaryotic messenger RNAs (mRNAs). Previous reports have found m⁶A on both cellular and viral transcripts and defined its role in regulating numerous biological processes, including viral infection. Here, we show that m⁶A 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 m⁶A to RNA resulted in increased HBV protein expression, but overall reduced reverse transcription of the pgRNA. We mapped the m⁶A site in the HBV RNA and found that a conserved m⁶A consensus motif situated within the epsilon stem loop structure, is the site for m⁶A 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 m⁶A site in the 5' epsilon stem loop of pgRNA revealed that m⁶A at this site is required for efficient reverse transcription of pgRNA, while m⁶A methylation of the 3' epsilon stem loop results in destabilization of all HBV transcripts, suggesting that m⁶A has dual regulatory function for HBV RNA. Overall, this study reveals molecular insights into how m⁶A regulates HBV gene expression and reverse transcription, leading to an increased level of understanding of the HBV life cycle.