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方面发挥着不同的作用。RNA修饰N6-甲基腺苷 (M6A)通过对黄病毒科正链RNA病毒感染的影响，调节其感染。 病毒和细胞RNA。M6A调节RNA生物学的关键方面，包括RNA的稳定性、翻译和 定位，以及与m6A结合蛋白的相互作用。而增加或结合M6A的碱性蛋白 MRNA是已知的，但对m6A-甲基转移酶复合体或m6A结合蛋白是如何变化的知之甚少 在感染期间针对病毒和细胞RNA的特定调控或靶向。我们之前的工作揭示了 黄病毒科病毒的RNA基因组在特定的位置被m6A修饰，而m6A在一个位点上被修饰 丙型肝炎病毒基因组中的位置通过抑制感染颗粒的产生而负向调节感染颗粒的产生 丙型肝炎病毒RNA基因组与病毒包装蛋白的相互作用。我们还发现黄病毒科诱导的 细胞信号通路改变了宿主对感染至关重要的mRNAs中m6A的变化。最后，我们的初选 数据鉴定新的m6A-甲基转移酶相互作用蛋白和具有所述作用的新的m6A-读取器 抗病毒干扰素(干扰素)反应。这项提议的目的是阐明 靶向病毒和细胞RNA的m6A如何调节黄病毒科特定病毒的感染。 根据我们的初步数据，这一提议的中心假设是特定的M6A调控途径 由病毒感染引起的增选和诱导可能是病毒复制的决定因素。在我们初步数据的指引下， 这一假设将通过追求以下三个具体目标来检验：1)定义M6A- 甲基转移酶复合体以丙型肝炎病毒RNA为靶标调节感染；2)确定黄病毒感染如何改变 M6A对特定宿主mRNAs的调节；3)干扰素诱导的限制黄病毒科的M6A阅读器的特征 感染。在目标1中，我们将定义细胞核m6A-甲基转移酶是如何被招募到胞质定位的 丙型肝炎病毒的RNA基因组。在目标2中，我们将研究m6A-甲基转移酶复合体是如何功能化的。 在病毒感染期间，以不同的靶向细胞mRNAs甲基化。在目标3中，我们将定义分子 干扰素诱导的m6A阅读器如何靶向病毒和宿主m6A修饰的RNA以抑制黄病毒的决定因素 感染。拟议的工作将具有重大意义和创新性，因为它将定义细胞路径和 在黄病毒感染过程中改变m6A-甲基转移酶组成和功能的机制以及 定义新的干扰素诱导的m6A调节因子，从而发现被病毒利用或利用的新宿主途径 由宿主抑制感染。重要的是，这项工作还将揭示基因表达方式的新方面 受m6A调控，包括特定的细胞信号通路改变m6A在特定细胞上的分布 MRNAs。因此，这项工作对理解由以下原因引起的多种人类疾病具有广泛的意义 改变基因表达，并通过RNA修饰进行调节。

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.