Interferon-stimulated gene 20 (ISG20) selectively degrades N6-methyladenosine modified Hepatitis B Virus transcripts

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 (m(6)A)-modified HBV transcripts are selectively recognized and processed for degradation by ISG20. Moreover, this effect of ISG20 is critically regulated by m(6)A reader protein, YTHDF2 (YTH-domain family 2). Previously, we identified a unique m(6)A 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-alpha mediated decay of HBV RNA. We observed that the HBV RNAs become less sensitive to ISG20 mediated degradation when methyltransferase enzymes or m(6)A reader protein YTHDF2 are silenced in HBV expressing cells. By using an enzymatically inactive form ISG20(D94G), we further demonstrated that ISG20 forms a complex with m(6)A 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 m(6)A site at either one (5 ' or 3 ') or both the termini (5 '& 3 '). Using these mutants, we demonstrated that m(6)A modified HBV RNAs are subjected to ISG20-mediated decay and propose sequence of events, in which ISG20 binds with YTHDF2 and recognizes m(6)A-modified HBV transcripts to carry out the ribonuclease activity. This is the first study, which identifies a hitherto unknown role of m(6)A modification of RNA in IFN-alpha induced viral RNA degradation and proposes a new role of YTHDF2 protein as a cofactor required for IFN-alpha mediated viral RNA degradation. Author summary Hepatitis B Virus (HBV) is a DNA virus but replicates through a transitional pregenomic RNA (pgRNA). Interferon stimulated antiviral RNase, ISG20 selectively binds to the lower epsilon stem loop of HBV RNA and causes their degradation. Surprisingly this ISG20 binding site is chemically modified by N6-methyladenosine addition to A1907 residue, which resides in the lower region of the epsilon stem loop. This single m(6)A site occurs twice due to terminal redundancy of sequences in the pgRNA. We demonstrated herein that IFN-alpha-induced ISG20 can selectively degrade m(6)A modified HBV RNA. Using a combined strategy of silencing cellular methyltransferases, m(6)A binding protein YTHDF2 and the m(6)A sites mutants, we show that HBV transcripts are resistant to either IFN-alpha treatment or ectopically introduced ISG20 mediated degradation. YTHDF2 is an m(6)A binding protein which makes the HBV RNAs less stable. YTHDF2 protein forms a complex with IFN-alpha stimulated ISG20 and executes the nuclease digestion of the recruited m(6)A modified transcripts. Absence of cellular m(6)A machinery (methyltransferases or m(6)A reader proteins) makes the HBV RNA unresponsive to ISG20 mediated decay. This study provides molecular explanation of IFN-alpha mediated degradation of m(6)A modified HBV RNAs.