N6-Methyladenosine modification of hepatitis B and C viral RNAs attenuates host innate immunity via RIG-I signaling
2020; Elsevier BV; Volume: 295; Issue: 37 Linguagem: Inglês
10.1074/jbc.ra120.014260
ISSN1083-351X
AutoresGeon‐Woo Kim, Hasan Imam, Mohsin Khan, Aleem Siddiqui,
Tópico(s)interferon and immune responses
ResumoN6-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. 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. 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 (1Seeger C. Mason W.S. Molecular biology of hepatitis B virus infection.Virology. 2015; 479-480 (25759099): 672-68610.1016/j.virol.2015.02.031Crossref PubMed Scopus (539) Google Scholar, 2Suzuki T. Aizaki H. Murakami K. Shoji I. Wakita T. 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Wakita T. Rice C.M. et al.The RNA sensor RIG-I dually functions as an innate sensor and direct antiviral factor for hepatitis B virus.Immunity. 2015; 42 (25557055): 123-13210.1016/j.immuni.2014.12.016Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar, 6Saito T. Owen D.M. Jiang F.G. Marcotrigiano J. Gale M. Innate immunity induced by composition-dependent RIG-I recognition of hepatitis C virus RNA.Nature. 2008; 454 (18548002): 523-52710.1038/nature07106Crossref PubMed Scopus (577) Google Scholar). HCV nonstructural proteins (NS3/4 protease) cleave mitochondrial antiviral-signaling protein (MAVS) to block the innate immune signals (7Li X.D. Sun L.J. Seth R.B. Pineda G. Chen Z.J.J. Hepatitis C virus protease NS3/4A cleaves mitochondrial antiviral signaling protein off the mitochondria to evade innate immunity.Proc. Natl. Acad. Sci. U.S.A. 2005; 102 (16301520): 17717-1772210.1073/pnas.0508531102Crossref PubMed Scopus (657) Google Scholar). 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Chemical methods for the modification of RNA.Methods. 2019; 161 (30905751): 64-8210.1016/j.ymeth.2019.03.018Crossref PubMed Scopus (43) Google Scholar). Among the diverse internal RNA chemical modifications, m6A RNA methylation is the most prevalent mRNA modification. (21Shi H.L. Wei J.B. He C. Where, when, and how: context-dependent functions of RNA methylation writers, readers, and erasers.Mol. Cell. 2019; 74 (31100245): 640-65010.1016/j.molcel.2019.04.025Abstract Full Text Full Text PDF PubMed Scopus (689) Google Scholar). Over 25% of mammalian transcripts are m6A-modified. m6A methylation has been linked to various biological processes, which include; innate immune response, sex determination, stem cell differentiation, circadian clock, meiosis, stress response, and cancer (21Shi H.L. Wei J.B. He C. Where, when, and how: context-dependent functions of RNA methylation writers, readers, and erasers.Mol. Cell. 2019; 74 (31100245): 640-65010.1016/j.molcel.2019.04.025Abstract Full Text Full Text PDF PubMed Scopus (689) Google Scholar). A methyltransferase complex composed of METT3 (methyltransferase-like 3), METTL14, and WTAP place m6A modification on mRNA co-transcriptionally (21Shi H.L. Wei J.B. He C. Where, when, and how: context-dependent functions of RNA methylation writers, readers, and erasers.Mol. Cell. 2019; 74 (31100245): 640-65010.1016/j.molcel.2019.04.025Abstract Full Text Full Text PDF PubMed Scopus (689) Google Scholar). This modification is typically enriched in 3′-UTR and near the stop codons of cellular mRNA. The m6A-modified mRNA is especially recognized by YTH-domain family proteins (YTHDF1, YTHDF2, and YTHDF3) to regulate mRNA stability, translation, and localization (22Du H. Zhao Y. He J.Q. Zhang Y. Xi H.R. Liu M.F. Ma J.B. Wu L.G. YTHDF2 destabilizes m6A-containing RNA through direct recruitment of the CCR4-NOT deadenylase complex.Nat. 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Although differences in the role of m6A among viruses can lead to different consequences in their life cycle, these reports reinforce the notion that these RNA modifications might play an important role in various aspects of the viral life cycle and most importantly in disease pathogenesis. In this study, we define the role of m6A-modified HBV transcripts and the HCV RNA genome in regulating the host innate immune systems. We demonstrate that cellular m6A machinery regulates the RIG-I signaling pathway activated by virus infection. Importantly, m6A modification in the RIG-I–sensing region of viral RNAs reduces innate immune response by inhibition of RIG-I recognition of viral RNAs. Furthermore, we found that YTHDF2 binds m6A-modified viral RNAs preventing their recognition by RIG-I. Taken together, 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 RIG-I. 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 5′ stem-loop (5′-MT), or the 3′ stem-loop (3′-MT) (Fig. 1A). The p-IRF-3 expression level was up-regulated in HBV 1.3-mer 5′-MT transfected cells compared with WT transfection, whereas mutation of m6A site in the 3′ stem-loop did not affect phosphorylation of IRF-3 (199 ± 25.7% increase of p-IRF-3 levels in 5′-MT transfection; Fig. 1B). Poly(I:C) serves as a positive control. Because the mutation of m6A site in ε structure leads to a base-pair mismatch in lower stem-loop, the secondary structure of lower stem-loop could be distorted by A1907C mutation (Fig. 1C). Because the induced p-IRF-3 by pHBV 1.3-mer 5′-MT transfection could be due to this structure alteration, we analyzed p-IRF-3 levels in cells transfected with compensatory mutant (CM) plasmid, in which U is mutated to G to restore base pairing (Fig. 1, C and D). Importantly, we found that p-IRF-3 levels were similar to the original mutant (5′-MT), suggesting that helical structural change did not cause this effect. Next, we determined whether cellular m6A machinery affects innate immune response activated by the presence of HBV RNA, and we depleted METTL3 and 14 by siRNA in HepG2 hepatoma cells and transfected these cells with HBV 1.3-mer plasmid. The silencing of m6A methyltransferases increased phosphorylation of IRF-3 induced by HBV transfection relative to its level in cells treated with control siRNA (178 ± 20.21% increase of p-IRF-3 level in METTL3 + 14 depletion) (Fig. 1E), whereas METTL3 and 14 overexpression reduced phosphorylation of IRF-3 (49 ± 5.69% decrease of p-IRF-3 in METTL3 and 14 overexpression) (Fig. 1F). Interestingly, HBV transfection in HepG2 cells induces IRF-3 activation but does not induce IFN synthesis (5Sato S. Li K. Kameyama T. Hayashi T. Ishida Y. Murakami S. Watanabe T. Iijima S. Sakurai Y. Watashi K. Tsutsumi S. Sato Y. Akita H. Wakita T. Rice C.M. et al.The RNA sensor RIG-I dually functions as an innate sensor and direct antiviral factor for hepatitis B virus.Immunity. 2015; 42 (25557055): 123-13210.1016/j.immuni.2014.12.016Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar). Thus, we could analyze only p-IRF-3 levels in HBV transfected cells. Previously, we reported that m6A modification of 5′ ε structure induces reverse-transcription activity, and m6A modification of 3′ ε structure reduces viral RNA stabilities. Thus, our observed effects of the silencing of m6A methyltransferases on IRF-3 activation could be due to the alterations of HBV replication and translation. To exclude this possibility, we depleted m6A machinery in pHBV 1.3-mer 5′-MT or 3′-MT transfected cells and analyzed phosphorylation of IRF-3 levels (Fig. S1, A and B). The knockdown of METTL3 and 14 did not affect p-IRF-3 levels in pHBV 1.3-mer 5′-MT transfected cells, whereas depletion of m6A methyltransferases induced phosphorylation of IRF-3 in pHBV 1.3-mer 3′-MT transfected cells. Collectively, these results suggest that m6A modification of HBV 5′ ε RNA critically determines IRF-3 activation during HBV replication. Because it is known that the 5′ ε structure is recognized by RIG-I to induce the innate immune response (5Sato S. Li K. Kameyama T. Hayashi T. Ishida Y. Murakami S. Watanabe T. Iijima S. Sakurai Y. Watashi K. Tsutsumi S. Sato Y. Akita H. Wakita T. Rice C.M. et al.The RNA sensor RIG-I dually functions as an innate sensor and direct antiviral factor for hepatitis B virus.Immunity. 2015; 42 (25557055): 123-13210.1016/j.immuni.2014.12.016Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar), our results imply that m6A modification of 5′ ε structure may affect RIG-I signal transduction. We also carried out similar experiments during HCV RNA transfection. We depleted METTL3 and 14 by siRNAs transfection of Huh7 cells and transfected these cells with in vitro transcribed HCV GND RNA from JFH-1 GND plasmid (Fig. 2, A–C). JFH-1 GND contains a point mutation in the viral polymerase region; hence it does not replicate but RIG-I sensing is normal (33Wakita T. Pietschmann T. Kato T. Date T. Miyamoto M. Zhao Z.J. Murthy K. Habermann A. Kräusslich H.G. Mizokami M. Bartenschlager R. Liang T.J. Production of infectious hepatitis C virus in tissue culture from a cloned viral genome.Nat. Med. 2005; 11 (15951748): 791-79610.1038/nm1268Crossref PubMed Scopus (2415) Google Scholar). Because HCV NS3/4 protein expression inhibits the immune response by cleaving the MAVS protein (7Li X.D. Sun L.J. Seth R.B. Pineda G. Chen Z.J.J. Hepatitis C virus protease NS3/4A cleaves mitochondrial antiviral signaling protein off the mitochondria to evade innate immunity.Proc. Natl. Acad. Sci. U.S.A. 2005; 102 (16301520): 17717-1772210.1073/pnas.0508531102Crossref PubMed Scopus (657) Google Scholar), we surmised that transfection with the replication-defective mutant HCV genome instead of replication efficient HCV RNA was suitable for immune response studies. We determined IFN-β mRNA and IRF-3 phosphorylation levels in cells harvested at 16 h post-transfected with HCV GND RNA. We observed that METTL3 and 14 depletion significantly increased IFN-β synthesis and IRF-3 activation relative to its level in cells treated with control siRNA (1.6-fold increase of IFN-β mRNA in METTL3 + 14 depleted cells; 177 ± 15.5% increase of p-IRF-3 level in METTL3 + 14 depleted cells; Fig. 2, A and C). Conversely, overexpression of m6A methyltransferases complexes decreased IFN-β mRNA levels and phosphorylation of IRF-3 (2.1-fold decrease of IFN-β mRNA in METTL3 + 14 overexpression; 58 ± 17.1% decrease of p-IRF-3 level in METTL3 + 14 overexpression) (Fig. 2, D and F). These results suggest that host m6A machinery regulates the IRF-3–mediated IFN signaling pathway induced by the HCV RNAs in the early step of HCV infection. After determining that m6A machinery affects IFN signaling pathway activated by the HCV RNA, we next tested whether m6A modification within pathogen-associated molecular patterns (PAMPs) recognized by RIG-I affects IFN-β mRNA level and phosphorylation of IRF-3. Saito et al. (6Saito T. Owen D.M. Jiang F.G. Marcotrigiano J. Gale M. Innate immunity induced by composition-dependent RIG-I recognition of hepatitis C virus RNA.Nature. 2008; 454 (18548002): 523-52710.1038/nature07106Crossref PubMed Scopus (577) Google Scholar) identified RIG-I–sensing nucleotides 8872–9616 in the 3′ end of the HCV genome, which acts as a PAMP. There are approximately 19 regions that are m6A-modified within the HCV RNA genome (27Gokhale N.S. McIntyre A.B.R. McFadden M.J. Roder A.E. Kennedy E.M. Gandara J.A. Hopcraft S.E. Quicke K.M. Vazquez C. Willer J. Ilkayeva O.R. Law B.A. Holley C.L. Garcia-Blanco M.A. Evans M.J. et al.N6-Methyladenosine in flaviviridae viral RNA genomes regulates infection.Cell Host Microbe. 2016; 20 (27773535): 654-66510.1016/j.chom.2016.09.015Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). We analyzed whether the HCV PAMP RNA contains m6A-methylated sites. Unexpectedly, the HCV PAMP RNA did not contain any m6A modification (DRACH motif), but an m6A modification site was identified at nucleotide 8766 of HCV RNA ∼100 bp upstream of the PAMP RNA (Fig. 2G) (27Gokhale N.S. McIntyre A.B.R. McFadden M.J. Roder A.E. Kennedy E.M. Gandara J.A. Hopcraft S.E. Quicke K.M. Vazquez C. Willer J. Ilkayeva O.R. Law B.A. Holley C.L. Garcia-Blanco M.A. Evans M.J. et al.N6-Methyladenosine in flaviviridae viral RNA genomes regulates infection.Cell Host Microbe. 2016; 20 (27773535): 654-66510.1016/j.chom.2016.09.015Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). Because m6A methyltransferases regulated IFN signaling pathway activated by HCV RNA transfection, we assessed the possibility that m6A modification of HCV 8766 nucleotide affects the IRF-3–mediated IFN signaling pathway (Fig. 2, H and J). We addressed this question by mutating m6A sites at nucleotides 8766 (A8766C) and 331 (A331C) of HCV GND RNA, respectively. HCV A331C mutant genome was used as a negative control because it lacks an m6A DRACH consensus and is not a RIG-I–sensitive site. Initially, we analyzed IFN-β mRNA levels in A8766C-mutated HCV genome-transfected cells and found that A8766C mutation, but not A331C mutation, substantially increased IFN-β mRNA levels compared with the HCV GND WT infected cells. The A8766C mutated HCV RNA transfection also increased phosphorylation of IRF-3 (2.6-fold increase of IFN-β mRNA level and 253 ± 41.8% increase of p-IRF-3 level in HCV GND A8766C RNA transfected cells; Fig. 2, H and J). We next tested whether the depletion of m6A machinery affects IFN signals activated by HCV GND A8766C because other m6A sites of the HCV genome might affect IFN-β mRNA and IRF-3 activation. Interestingly, the silencing of METTL3 and 14 did not increase IFN-β mRNA and phosphorylation of IRF-3 in HCV GND A8766C transfected cells but induced IRF-3–mediated IFN-β expression in HCV GND A331C transfected cells (Fig. S2). These results illustrate that m6A modification of HCV 8766 nucleotide affects the function of HCV PAMP RNA. In particular, the deficiency of m6A modification in other nucleo
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