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N6-methyladenosine is a molecular signature for discrimination of self and non-self RNA by cytoplasmic RNA sensor RIG-I

ABSTRACT: Internal N6-methyladenosine (m6A) modification of RNA is one of the most common and abundant modifications in eukaryotic cells as well as in viruses. However, the biological role(s) of RNA m6A in virus-host interaction remains elusive. Using human metapneumovirus (hMPV), a medically important non-segmented negative-sense RNA virus as a model, we demonstrate that m6A serves as a molecular marker for innate immune discrimination self and nonself RNAs. We show that hMPV RNAs are m6A methylated and that viral m6A methylation promotes hMPV replication and gene expression. HMPV infection leads to differential expression of interferon-related genes involved in innate immune signaling pathways. Inactivating these m6A sites with synonymous mutations resulted in m6A deficient recombinant hMPVs that induced significantly higher expression of type I interferon that restricted viral replication. Notably, the induction of type I interferons by m6A-deficient rhMPVs and virion RNA was dependent on the cytoplasmic RNA sensor RIG-I, not MDA5. Mechanistically, m6A-deficient virion RNA induces higher expression of RIG-I, enhances its binding affinity to RIG-I, and facilitates the conformational change of RIG-I, leading to enhanced induction of type I IFN expression. The replication of m6A-deficient rhMPVs was attenuated in wild type A459 cells but was restored in cells knocked out for RIG-I and MAVS. Furthermore, m6A-deficient rhMPVs triggered higher type I interferon in vivo and were significantly attenuated in the lower respiratory tract yet retained high immunogenicity in cotton rats. Collectively, our results highlight that (i) virus acquires m6A in their RNAs as a means of mimicking cellular RNA to avoid the detection by innate immunity; and (ii) viral m6A RNA can serve as a novel target to attenuate hMPV for vaccine purposes.

ORGANISM(S): Homo sapiens

PROVIDER: GSE136139 | GEO | 2019/10/18

REPOSITORIES: GEO

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Project description:The RIG-I like receptor pathway is stimulated during RNA virus infection by interaction between cytosolic RIG-I and viral RNA structures that contain short hairpin dsRNA and 5M-bM-^@M-^Y triphosphate (5M-bM-^@M-^Yppp) terminal structure. In the present study, an RNA agonist of RIG-I was synthesized in vitro and shown to stimulate RIG-I-dependent antiviral responses at concentrations in the picomolar range. In human lung epithelial A549 cells, 5M-bM-^@M-^YpppRNA specifically stimulated multiple parameters of the innate antiviral response, including IRF3, IRF7 and STAT1 activation, andinduction of inflammatory and interferon stimulated genes - hallmarks of a fully functional antiviral response. Evaluation of the magnitude and duration of gene expression by transcriptional profiling identified a robust, sustained and diversified antiviral and inflammatory response characterized by enhanced pathogen recognition and interferon (IFN)signaling. Bioinformatics analysis further identified a transcriptional signature uniquely induced by 5M-bM-^@M-^YpppRNA, and not by IFNM-NM-1-2bthat included a constellation of IRF7 and NF-kB target genes capable of mobilizing multiple arms of the innate and adaptive immune response. Treatment of primary PBMCs or lung epithelial A549 cells with 5M-bM-^@M-^YpppRNA provided significant protection against a spectrum of RNA and DNA viruses. In C57Bl/6 mice, intravenous administration of 5M-bM-^@M-^YpppRNA protected animals from a lethal challenge with H1N1 Influenza, reduced virus titers in mouse lungs and protected animals from virus-induced pneumonia. Strikingly, the RIG-I-specific transcriptional response afforded partial protection from influenza challenge, even in the absence of type I interferon signaling. This systems approach providestranscriptional, biochemical, and in vivo analysis of the antiviral efficacy of 5M-bM-^@M-^YpppRNA and highlights the therapeutic potential associated with the use of RIG-I agonists as broad spectrum antiviral agents. Kinetic analysis of A549 cells treated with 5'pppRNA and analyzed at 1h, 2h, 4h, 6h, 8h, 12h, 24h or 48h.

Project description:The RIG-I like receptor pathway is stimulated during RNA virus infection by interaction between cytosolic RIG-I and viral RNA structures that contain short hairpin dsRNA and 5M-bM-^@M-^Y triphosphate (5M-bM-^@M-^Yppp) terminal structure. In the present study, an RNA agonist of RIG-I was synthesized in vitro and shown to stimulate RIG-I-dependent antiviral responses at concentrations in the picomolar range. In human lung epithelial A549 cells, 5M-bM-^@M-^YpppRNA specifically stimulated multiple parameters of the innate antiviral response, including IRF3, IRF7 and STAT1 activation, andinduction of inflammatory and interferon stimulated genes - hallmarks of a fully functional antiviral response. Evaluation of the magnitude and duration of gene expression by transcriptional profiling identified a robust, sustained and diversified antiviral and inflammatory response characterized by enhanced pathogen recognition and interferon (IFN)signaling. Bioinformatics analysis further identified a transcriptional signature uniquely induced by 5M-bM-^@M-^YpppRNA, and not by IFNM-NM-1-2bthat included a constellation of IRF7 and NF-kB target genes capable of mobilizing multiple arms of the innate and adaptive immune response. Treatment of primary PBMCs or lung epithelial A549 cells with 5M-bM-^@M-^YpppRNA provided significant protection against a spectrum of RNA and DNA viruses. In C57Bl/6 mice, intravenous administration of 5M-bM-^@M-^YpppRNA protected animals from a lethal challenge with H1N1 Influenza, reduced virus titers in mouse lungs and protected animals from virus-induced pneumonia. Strikingly, the RIG-I-specific transcriptional response afforded partial protection from influenza challenge, even in the absence of type I interferon signaling. This systems approach providestranscriptional, biochemical, and in vivo analysis of the antiviral efficacy of 5M-bM-^@M-^YpppRNA and highlights the therapeutic potential associated with the use of RIG-I agonists as broad spectrum antiviral agents. A549 cells were either non-treated, treated with RNAiMax only, transfected with 5'pppRNA, or treated with IFNa-2b and analysed at 6h or 24h.

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N6-methyladenosine is a molecular signature for discrimination of self and non-self RNA by cytoplasmic RNA sensor RIG-I

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