Abstract
N6-Methyladenosine (m6A), the methylation of the adenosine base at the nitrogen 6 position, is the most common epitranscriptomic modification of mRNA that affects a wide variety of biological functions. We have previously reported that hepatitis B viral RNAs are m6A-modified, displaying a dual functional role in the viral life cycle. Here, we show that cellular m6A machinery regulates host innate immunity against hepatitis B and C viral infections by inducing m6A modification of viral transcripts. The depletion of the m6A writer enzymes (METTL3 and METTL14) leads to an increase in viral RNA recognition by retinoic acid-inducible gene I (RIG-I), thereby stimulating type I interferon production. This is reversed in cells in which m6A METTL3 and METTL14 are overexpressed. The m6A modification of viral RNAs renders RIG-I signaling less effective, whereas single nucleotide mutation of m6A consensus motif of viral RNAs enhances RIG-I sensing activity. Importantly, m6A reader proteins (YTHDF2 and YTHDF3) inhibit RIG-I-transduced signaling activated by viral RNAs by occupying m6A-modified RNAs and inhibiting RIG-I recognition. Collectively, our results provide new insights into the mechanism of immune evasion via m6A modification of viral RNAs.
Highlights
Hepatitis B virus (HBV) and hepatitis C virus (HCV) are diverse viruses that belong to the Hepadnaviridae and Flaviviridae families, respectively, but share common pathologies [1, 2]
Our results reveal a novel mechanism of immune evasion via m6A modification of viral RNA, where YTHDF2 binds m6A-modified motifs of viral RNAs, preventing recognition by retinoic acid–inducible gene I (RIG-I)
We found that mutation on the m6A site of the 59 e structure of pregenomic RNA (pgRNA) decreased binding affinity with YTHDF2 and YTHDF3 compared with WT, but did not affect interaction with YTHDF1 (Fig. S6A)
Summary
To determine whether m6A modification of viral RNA affects host immune response, we used the previously developed mutants of m6A modification sites (A1907C) of HBV 1.3-mer; in the pgRNA 59 stem-loop (59-MT), or the 39 stem-loop (39MT) (Fig. 1A). We investigated the possibility that YTHDF proteins affect RIG-I signal transduction induced by HBV and HCV via interacting with m6Amodified viral RNAs. We transfected each HBV 1.3-mer or 59MT–expressing cells with plasmids encoding FLAG-YTHDF1, YTHDF2, or YTHDF3 and analyzed p-IRF-3 expression levels (Fig. 4, A and B, and Fig. S5, A and B). YTHDF2 and YTHDF3 did not affect p-IRF-3 and IFN-b mRNA levels in cells transfected with HCV GND RNA mutated within the m6A site (A8766C) (Fig. 4, F and H, and Fig. S5, F and H) These results reveal that YTHDF2 and YTHDF3 play a critical role in the RIG-I signal pathway activated by the presence of viral m6A containing RNAs. In addition, we investigated whether YTHDF2 and YTHDF3 interact with the m6A site-mutated HBV pgRNA. YTHDF2 inhibits RIG-I recognition of viral RNAs via interacting with m6A-modified viral RNAs, leading to disrupt RIG-I– mediated immune response (Fig. 6)
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