Internal N 6 -methyladenosine (m 6 A) modification is one of the most common and abundant modifications of RNA. However, the biological role(s) of viral RNA m 6 A remains elusive. Using human metapneumovirus (hMPV) as a model, we demonstrate that m 6 A serves as a molecular marker for innate immune discrimination of self from nonself RNAs. We show that hMPV RNAs are m 6 A methylated and that viral m 6 A methylation promotes hMPV replication and gene expression. Inactivating m 6 A addition sites with synonymous mutations or demethylase resulted in m 6 A deficient recombinant hMPVs and virion RNAs that induced significantly higher expression of type I interferon (IFN) which was dependent on the cytoplasmic RNA sensor RIG-I, not MDA5. Mechanistically, m 6 A-deficient virion RNA induces higher expression of RIG-I, binds more efficiently to RIG-I, and facilitates the conformational change of RIG-I, leading to enhanced IFN expression. Furthermore, m 6 A-deficient rhMPVs triggered higher IFN in vivo and were significantly attenuated in cotton rats yet retained high immunogenicity. Collectively, our results highlight that (i) virus acquires m 6 A in their RNAs as a means of mimicking cellular RNA to avoid detection by innate immunity; and (ii) viral RNA m 6 A can serve as a target to attenuate hMPV for vaccine purposes.

N6-methyladenosine (m6A) is the most prevalent internal modification of mRNAs in most eukaryotes. Here we show that RNAs of human respiratory syncytial virus (RSV) are modified by m6A within discreet regions and that these modifications enhance viral replication and pathogenesis. Knockdown of m6A methyltransferases decreases RSV replication and gene expression whereas knockdown of m6A demethylases has the opposite effect. The G gene transcript contains the most m6A modifications. Recombinant RSV variants expressing G transcripts that lack particular clusters of m6A display reduced replication in A549 cells, primary well differentiated human airway epithelial cultures, and respiratory tracts of cotton rats. One of the m6A-deficient variants is highly attenuated yet retains high immunogenicity in cotton rats. Collectively, our results demonstrate that viral m6A methylation upregulates RSV replication and pathogenesis and identify viral m6A methylation as a target for rational design of live attenuated vaccine candidates for RSV and perhaps other pneumoviruses.

Current efforts to develop Zika virus (ZIKV) subunit vaccines have been focused on pre-membrane (prM) and envelope (E) proteins, but the role of NS1 in ZIKV-specific immune response and protection is poorly understood. Here, we develop an attenuated recombinant vesicular stomatitis virus (rVSV)-based vaccine expressing ZIKV prM-E-NS1 as a polyprotein. This vectored vaccine candidate is attenuated in mice, where a single immunization induces ZIKV-specific antibody and T cell immune responses that provide protection against ZIKV challenge. Co-expression of prM, E, and NS1 induces significantly higher levels of Th2 and Th17 cytokine responses than prM-E. In addition, NS1 alone is capable of conferring partial protection against ZIKV infection in mice even though it does not induce neutralizing antibodies. These results demonstrate that attenuated rVSV co-expressing prM, E, and NS1 is a promising vaccine candidate for protection against ZIKV infection and highlights an important role for NS1 in ZIKV-specific cellular immune responses.

N6-methyladenosine (m6A) is the most abundant internal RNA modification catalyzed by host RNA methyltransferases. As obligate intracellular parasites, many viruses acquire m6A methylation in their RNAs. However, the biological functions of viral m6A methylation are poorly understood. Here, we found that viral m6A methylation serves as a molecular marker for host innate immunity to discriminate self from nonself RNA and that this novel biological function of viral m6A methylation is universally conserved in several families in non-segmented negative-sense (NNS) RNA viruses. Using m6A methyltransferase (METTL3)-knockout cells, we produced m6A-deficient virion RNA from the representative members of the families Pneumoviridae, Paramyxoviridae, and Rhabdoviridae and found that these m6A-deficient viral RNAs triggered significantly higher levels of type I interferon compared to the m6A-sufficient viral RNAs, in a RIG-I dependent manner. Reconstitution of the RIG-I pathway revealed that m6A-deficient virion RNA induced higher expression of RIG-I, bound to RIG-I more efficiently, enhanced RIG-I ubiquitination, and facilitated RIG-I conformational rearrangement and oligomerization. Furthermore, the m6A binding protein YTHDF2 is essential for suppression of type I interferon signaling pathway included 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 non-segmented negative-sense (NNS) RNA viruses share many common replication and gene expression strategies. There is no vaccine or antiviral drugs for many of these viruses. We found that representative members in the families of Pneumoviridae, Paramyxoviridae, and Rhabdoviridae in NNS RNA viruses acquire m6A methylation in their genome and antigenome as a means to escape the recognition by host innate immunity via a RIG-I dependent signaling pathway. Viral RNA lacking m6A methylation induces a significantly higher type I interferon compared to 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 a robust innate immunity will likely promote the subsequent adaptive immunity.

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