Son KN, Liang ZG, Lipton HL. of the type I interferon signaling pathway, including by virion RNA. Collectively, our results suggest that several families in NNS RNA viruses acquire m6A in viral RNA as a common strategy to evade host innate immunity. IMPORTANCE The nonsegmented negative-sense (NNS) RNA viruses share many common replication and gene expression strategies. There are no vaccines or antiviral drugs for many of these viruses. We found that representative members of the families among the NNS RNA viruses acquire m6A methylation in their genome and antigenome as a means to escape recognition by host innate immunity via a RIG-I-dependent signaling pathway. Viral RNA lacking m6A methylation induces a significantly higher type I interferon response than m6A-sufficient viral RNA. In addition to uncovering m6A methylation as a common mechanism for many NNS RNA viruses to evade host innate immunity, this study discovered a novel strategy to enhance type I interferon responses, which may have important applications in vaccine development, as strong innate immunity will likely promote the subsequent adaptive immunity. include a wide range of human, animal, and herb pathogens. The NNS RNA viruses are classified into five families: mRNA, leading to elevated levels of type I IFN (28, 29). Viruses are obligate intracellular parasites; they must utilize host machinery to synthesize their own genomes and proteins. The presence of m6A in their genome RNA and/or mRNA has been reported for many RNA viruses Zafirlukast and DNA viruses (30, 31). Unexpectedly, viral m6A RNA can play either antiviral or proviral functions in the computer virus life cycle via poorly comprehended mechanisms (30, 31). Similar to host mRNA, m6A in viral mRNA (such as some positive-sense RNA viruses and DNA viruses) has been shown to be important for RNA stability and translation (31, 32). The genome and antigenome (replicative intermediate) of NNS RNA viruses are triphosphorylated and cannot be directly translated into viral proteins. However, why the genomes and SF3a60 antigenomes of NNS RNA viruses are m6A methylated is usually mystical. Recently, we provided the first evidence that m6A methylation in the hMPV genome and antigenome prevents innate immune detection in a RIG-I-dependent manner (33). Here, we demonstrate that this novel function of viral m6A in innate immunity is usually universally conserved in the representative members of the (hMPV), (MeV and SeV), and (VSV) in the order among NNS RNA computer virus families. RESULTS RNA purified from virions produced in METTL3 knockout cells is usually defective in m6A methylation. We previously showed that this genome, antigenome, and mRNAs of RSV and hMPV, two members of the family and 0.05) (Fig. 3b to ?toe).e). For VSV RNA extracted from virus-infected cells, a methylated RNA immunoprecipitation (MeRIP) assay was used for quantification of m6A levels. Both VSV genome and antigenome from METTL3 KO U2OS cells were defective in m6A methylation compared to those from WT U2OS cells (Fig. 3f). However, in all cases, m6A content in virion RNAs from METTL3 KO U2OS cells was not completely defective in m6A, consistent with the previous observation that METTL3 is the major but not the only host RNA m6A methyltransferase. Unlike m6A-deficient virion RNA generated by mutagenesis to Zafirlukast disrupt m6A addition sites, these virion RNAs were naturally defective in m6A methylation due to METTL3 KO, but their nucleotide sequences had not been altered. Open in a separate windows FIG 3 The viral RNA of computer virus produced in METTL3 knockout U2OS cells is defective in m6A methylation. (a) Western blot showing METTL3 expression in METTL3-knockout U2OS cells and wild-type U2OS cells. (b to d) Quantification of m6A level in virion RNA. Shown are the relative m6A levels in virion RNAs from SeV (b), MeV Zafirlukast (c), hMPV (d), and VSV (e) produced on METTL3 KO/WT U2OS cells. Each computer virus was purified through 30 to 50% linear sucrose gradient ultracentrifugation. Virion RNA was extracted, and the total m6A level of each virion RNA was quantified by m6A RNA methylation assay. (f) Total viral RNA from VSV-infected METTL3 KO U2OS cells is defective in binding to m6A antibody by MeRIP assay. An MeRIP assay was carried out to determine the binding of RNA.