Interferon-stimulated gene 20 (ISG20) selectively degrades N6-methyladenosine modified Hepatitis B Virus transcripts - PubMed (original) (raw)
Interferon-stimulated gene 20 (ISG20) selectively degrades N6-methyladenosine modified Hepatitis B Virus transcripts
Hasan Imam et al. PLoS Pathog. 2020.
Abstract
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.
Conflict of interest statement
The authors have declared that no competing interests exist.
Figures
Fig 1. IFN-α induced ISG20 and ectopically expressed ISG20 can degrade m6A modified HBV RNA.
(A) HBV-WT (B) HBV-M1 (C) HBV-M2 (D) HBV-M3 plasmids were transfected into HepG2 cells and incubated for 48h until harvest and IFN-α was added (2000 IU/ml) 24h before harvesting the cells. After RNA isolation relative expression of pg/pc RNA was analyzed through RT-qPCR. (E) HBV-WT (F) HBV-M1 (G) HBV-M2 (H) HBV-M3 plasmids were co-transfected with FLAG-ISG20 into HepG2 cells and incubated for 48h until harvest. After RNA isolation relative expression of pg/pc RNA was checked through RT-qPCR. (I) HBV-WT (J) HBV-M1 (K) HBV-M2 (L) HBV-M3 plasmids were co-transfected with FLAG-ISG20M (mutated FLAG-ISG20) into HepG2 cells and incubated for 48h before harvesting the cells. After RNA isolation relative expression of pg/pc RNA was checked through RT-qPCR. The data for this figure are from three independent experiments and the bars represent the mean ± SD. ND, not detected. *P ≤0.05 by unpaired Student’s t test.
Fig 2. HBV-independent interaction between YTHDF2 proteins with ISG20.
(A) FLAG-YTHDF2 and HA-ISG20D94G plasmids were co-transfected into HepG2 cells. After 48h of incubation cells were harvested and lysates were prepared. FLAG antibody was used for IP and then probed with FLAG and HA antibody after Western Blot. (B) FLAG-YTHDF2 and HA-ISG20D94G plasmids were co-transfected into HBV expressing HepG2 cells. After 48 hour of incubation cells were harvested and lysates were prepared. IP was done with FLAG antibody and then probed with FLAG and HA antibody after Western Blot. (C) FLAG-YTHDF2 and HA-ISG20D94G plasmids were co-transfected into HepG2 cells and then incubated for 48h until harvest and IFN-α was added (2000 IU/ml) 24h before harvesting the cells. After preparing the lysates IP was done with FLAG antibody and probed with FLAG and HA antibody after Western Blot. (D) Confocal microscopy of HepG2 cells transfected with HA-ISG20D94G and FLAG-YTHDF2, showing signals for DAPI stained nuclei (blue), ISG20D94G (green), YTHDF2 (red) and the merged images (yellow) (upper panel). Confocal microscopy of HBV expressing HepG2 cells transfected with HA-ISG20D94G and FLAG-YTHDF2, showing signals for DAPI stained nuclei (blue), ISG20D94G (green), YTHDF2 (red) and the merged images (yellow) (lower panel). The data for this figure are from two independent experiments and the bars represent the mean ± SD.
Fig 3. IFN-α induced ISG20 degrades m6A methylated HBV RNA via YTHDF2.
(A) HBV-WT, HBV-M1, HBV-M2 and HBV-M3 plasmids were separately co-transfected with FLAG-YTHDF2 into HepG2 cells and incubated for 48h until harvest and IFN-α was added (2000 IU/ml) 24h before harvesting the cells. After preparing the lysates FLAG-IP was done and probed with FLAG and ISG20 antibody after Western Blot. (B) RNA was isolated from the final eluted products from co-IP experiment and checked for pg/pc RNA expression by RT-qPCR. The data for this figure are from two independent experiments and the bars represent the mean ± SD.
Fig 4. YTHDF2 protein facilitates the degradation of m6A methylated HBV RNA by IFN-α induced ISG20.
(A) YTHDF2 was knocked down in HBV expressing HepG2 cells along with scrambled siRNA. IFN-α was added (2000 IU/ml) 24h before harvesting the cells. RNA was isolated from the cells and checked for pg/pc RNA expression using RT-qPCR. (B) METTL3/14 was knocked down in HBV expressing HepG2 cells along with scrambled siRNA. IFN-α was added (2000 IU/ml) 24h before harvesting the cells. RNA was isolated from the cells and checked for pg/pc RNA expression using RT-qPCR. (C) RT-qPCR analysis of pg/pc RNA relative to GAPDH in HBV-WT expressing HepG2 cells. The HBV-WT transfected HepG2 cells were depleted for YTHDF2 by specific siRNA, following Actinomycin D treatment at 24h post-siRNA transfection with and without IFN-α treatment. RNA was harvested at 0, 8, 16, and 24h post Actinomycin D treatment and relative levels of remaining HBV transcripts were analyzed. IFN-α was treated (2000 IU/ml) 24h before harvesting the cells. (D) Proposed model for ISG20 mediated degradation of m6A modified HBV RNA degradation. ISG20 exonuclease enzyme is released upon IFN-α treatment and is recruited by m6A reader protein YTHDF2 which facilitates m6A modified HBV RNA to degrade. The data for this figure are from two independent experiments and the bars represent the mean ± SD. *P ≤0.05 by unpaired Student’s t test.
Fig 5. Schematics indicating the location of ISG20 binding site in the lower ε stem loop of HBV RNA.
(A) m6A consensus motif (red) (B) ISG20 binding site (green) (C) m6A consensus and ISG20 binding site overlap (yellow) (D) ISG20 binding site in m6A methylated and unmethylated stem-loop of HBV RNA. (E) Proposed model for HBV long transcript for showing how cis-elements can juxtapose both the termini as described in discussion.