Defining the role of the RNA modification N6-methyladenosine during Flaviviridae virus infection

摘要 RNA修饰在调节病毒感染中发挥着不同的作用。RNA修饰N6-甲基腺苷 (m6A)调节感染的正链RNA病毒在黄病毒科，通过其影响， 病毒和细胞RNA。m6 A调节RNA生物学的关键方面，包括RNA稳定性、翻译和转录。 定位，以及与m6 A结合蛋白的相互作用。而添加或结合m6 A的碱性蛋白质， mRNA是已知的，很少有人知道如何m6 A-甲基转移酶复合物或m6 A结合蛋白， 在感染期间特异性调节或靶向病毒和细胞RNA。我们之前的研究表明， 黄病毒科病毒的RNA基因组在特定位点被m6 A修饰，而在一个位点m6 A 丙型肝炎病毒（HCV）基因组中的一个位点通过抑制 HCV RNA基因组与病毒包装蛋白的相互作用。我们还发现黄病毒诱导的 细胞信号传导途径改变宿主mRNA中对感染重要的m6 A变化。最后，我们初步的 数据鉴定了新的m6 A-甲基转移酶相互作用蛋白和具有所述作用的新的m6 A-阅读器， 抗病毒干扰素（IFN）反应。该提案的目标是阐明 靶向病毒和细胞RNA的m6 A如何调节黄病毒科中特定病毒的感染。 根据我们的初步数据，该提议的中心假设是，特定的m6 A调节途径 被病毒感染所选择和诱导的基因可能是病毒复制的决定因素。根据我们的初步数据， 这一假设将通过追求以下三个具体目标来检验：1）定义m6 A- 甲基转移酶复合物靶向HCV RNA以调节感染; 2）确定黄病毒感染如何改变 特异性宿主mRNA的m6 A调节; 3）表征限制黄病毒科的IFN诱导的m6 A阅读器 感染在目标1中，我们将定义核m6 A-甲基转移酶是如何被募集到细胞质定位的 HCV的RNA基因组。在目标2中，我们将研究m6 A-甲基转移酶复合物是如何官能化的 在病毒感染过程中差异靶向细胞mRNA进行甲基化。在目标3中，我们将定义分子 IFN诱导的m6 A阅读器如何靶向病毒和宿主m6 A修饰的RNA以抑制黄病毒科的决定因素 感染拟议的工作将是重要的和创新的，因为它将定义细胞通路， 在黄病毒科感染期间改变m6 A-甲基转移酶组成和功能的机制，以及 定义新的IFN诱导的m6 A调节因子，从而揭示病毒利用或利用的新宿主途径 以抑制感染。重要的是，这项工作还将揭示基因表达是如何影响人类基因表达的新方面。 由m6 A调节，包括特定的细胞信号传导途径改变m6 A如何被放置在特定的 mRNA。因此，这项工作对于理解由以下原因引起的许多人类疾病具有广泛的意义： 改变基因表达并受RNA修饰调控。

ABSTRACT RNA modifications play diverse roles in regulating viral infection. The RNA modification N6-methyladenosine (m6A) regulates infection by the positive-strand RNA viruses in the Flaviviridae family, through its effect on both viral and cellular RNA. m6A regulates key aspects of RNA biology, including RNA stability, translation, and localization, as well as interactions with m6A-binding proteins. While the basic proteins that add or bind m6A on mRNA are known, little is known about how the m6A-methyltransferase complex or m6A-binding proteins are specifically regulated or targeted to viral and cellular RNA during infection. Our previous work has revealed that the RNA genomes of viruses in the Flaviviridae family are m6A-modified at specific sites, and that m6A at one site in the hepatitis C virus (HCV) genome negatively regulates infectious particle production by inhibiting interaction of the HCV RNA genome with the viral packaging protein. We also found that Flaviviridae-induced cellular signaling pathways alter m6A changes in host mRNAs important for infection. Finally, our preliminary data identify novel m6A-methyltransferase interacting proteins and a novel m6A-reader with a described role in the antiviral interferon (IFN) response. The goal of this proposal is to elucidate the molecular mechanisms of how m6A-targeting to viral and cellular RNA regulates infection by specific viruses in the Flaviviridae family. Based on our preliminary data, the central hypothesis of this proposal is that the specific m6A regulatory pathways co-opted and induced by viral infection can be determinants of viral replication. Guided by our preliminary data, this hypothesis will be tested by pursuing the following three specific aims: 1) Define how the m6A- methyltransferase complex targets HCV RNA to regulate infection; 2) Determine how Flaviviridae infection alters m6A regulation of specific host mRNAs; 3) Characterize an IFN-induced m6A-reader that restricts Flaviviridae infection. In Aim 1, we will define how the nuclear m6A-methyltransferase is recruited to the cytoplasmic-localized RNA genome of HCV. In Aim 2, we will investigate how the m6A-methyltransferase complex is functionalized during viral infection to differentially target cellular mRNAs for methylation. In Aim 3, we will define the molecular determinants of how an IFN-induced m6A reader targets viral and host m6A-modified RNA to inhibit Flaviviridae infection. The proposed work will be significant and innovative because it will define cellular pathways and mechanisms that alter m6A-methyltransferase composition and function during Flaviviridae infection, as well as define new IFN-induced regulators of m6A, thus uncovering new host pathways exploited by viruses or utilized by the host to inhibit infection. Importantly, this work will also reveal novel aspects of how gene expression is regulated by m6A, including the fact that specific cellular signaling pathways alter how m6A is placed on specific mRNAs. Thus, this work has broad implications for understanding a host of human diseases that are caused by altered gene expression and regulated by RNA modifications.