Viral N6-methyladenosine upregulates replication and pathogenesis of human respiratory syncytial virus: m6A-seq of vesicular stomatitis virus. Viral N6-methyladenosine upregulates replication and pathogenesis of human respiratory syncytial virus: m6A-seq of vesicular stomatitis virus

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 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 sequesters m6A-sufficient virion RNA which suppresses type I interferon signaling pathway. Collectively, our results suggest that NNS RNA viruses acquire m6A in viral RNA as a common strategy to evade host innate immunity. Overall design: A few other (NNS) RNA viruses including RSV and hMPV has been sequenced and published before by us. Here, we performed m6A-seq of vesicular stomatitis virus (VSV). VSV virions were purified from supernatants of VSV-infected Hela cell lines. Virus derived mRNA as well as host mRNA were purified from total RNA, which were extracted from VSV infected cells.

N6-甲基腺嘌呤（N6-methyladenosine, m6A）是宿主RNA甲基转移酶催化的最丰富的内部RNA修饰。作为专性细胞内寄生生物，众多病毒的RNA会发生m6A甲基化修饰。然而，病毒m6A甲基化的生物学功能仍鲜为人知。本研究发现，病毒m6A甲基化可作为宿主先天免疫区分自身与非自身RNA的分子标记，且病毒m6A甲基化的这一新型生物学功能在不分节段负链（non-segmented negative-sense, NNS）RNA病毒中普遍保守。我们利用m6A甲基转移酶（METTL3）敲除细胞，从肺病毒科（Pneumoviridae）、副黏病毒科（Paramyxoviridae）和弹状病毒科（Rhabdoviridae）的代表性毒株中制备了m6A缺失的病毒粒子RNA，并发现相较于m6A充足的病毒RNA，这些m6A缺失的病毒RNA以视黄酸诱导基因-I（RIG-I）依赖的方式，显著诱导更高水平的I型干扰素表达。对RIG-I信号通路的重构实验表明，m6A缺失的病毒粒子RNA可诱导更高水平的RIG-I表达，更高效地结合RIG-I，增强RIG-I的泛素化修饰，并促进RIG-I的构象重排与寡聚化。此外，m6A结合蛋白YTHDF2会螯合m6A充足的病毒粒子RNA，从而抑制I型干扰素信号通路。综上，本研究结果表明，NNS RNA病毒通过在病毒RNA中获取m6A修饰，作为逃逸宿主先天免疫的通用策略。总体实验设计：此前我们已对包括呼吸道合胞病毒（RSV）和人偏肺病毒（hMPV）在内的数种其他NNS RNA病毒完成测序并发表相关研究成果。本研究中，我们对水疱性口炎病毒（vesicular stomatitis virus, VSV）开展了m6A测序（m6A-seq）。我们从VSV感染的HeLa细胞系上清液中纯化VSV病毒粒子，从感染细胞提取的总RNA中分别纯化病毒来源mRNA与宿主mRNA。