Interferon stimulated genes that regulate viral infection by binding to modified RNAs

摘要 抗病毒的先天免疫反应是抵御病毒感染的第一道防线。抗病毒免疫是 由细胞蛋白启动，可感知受感染细胞内病毒的存在。因此，这一信号 级联最终产生I型干扰素(干扰素)和数百个干扰素刺激基因(ISGs)。 干扰素反应性RNA结合蛋白，抑制RNA病毒复制或具有m6A选择性识别 细胞RNA的选择性结合先前已经描述过；然而，如果N6-甲基腺苷(M6A)的选择性结合是 这些蛋白质在抗病毒反应中所需的功能尚不清楚。我们的初步研究 我发现了一种ISG，它优先与含有m6A的RNA结合，并对RNA病毒进行负面调控 感染。PAR-CLIP(与下一代耦合的光活化交联剂和免疫沉淀 测序)分析表明，它与已知含有m6A的区域的细胞RNA结合。然而， M6A选择性对该ISG的抗病毒天然免疫效应机制的影响尚不清楚。因此， 这项提议的目标是定义在干扰素过程中起作用的一个人ISG的生化相互作用 对病毒RNA上m6A修饰核苷酸的识别反应。根据我们的初步数据，中央 该提议的假设是，该ISG以m6A依赖的方式与RNA结合，以调节宿主 对病毒感染的反应。在我们初步数据的指导下，这一假设将通过以下两个方面进行检验 具体目标：1)确定干扰素刺激的RNA识别和m6A识别的机制 M6A-Reader；2)确定干扰素刺激的M6A-Reader在限制RNA病毒中的RNA识别作用 感染。在目标1中，将确定该ISG识别含有M6A的RNA的分子基础。 在目标2中，这种蛋白质的RNA结合和m6A识别如何定义病毒感染的结果 在感染了全球公共卫生问题的RNA病毒的人类细胞中进行了探索。总而言之，这项工作 本申请中提出的建议将具有重大意义和创新性，因为它将定义ISG在 识别m6A修饰的RNA调节抗病毒先天免疫系统和病毒感染。这项工作将 为RNA病毒感染和自身免疫性疾病的治疗和治疗策略提供信息。此外，一个 进一步了解病毒感染是如何由先天免疫效应器控制的将具有重要意义 用于治疗和疫苗反应，以限制RNA病毒感染的负担。

ABSTRACT The antiviral innate immune response is the first line of defense against viral infection. Antiviral immunity is initiated by cellular proteins that sense the presence of virus inside an infected cell. As a result, this signaling cascade culminates in the production of type I interferons (IFN) and hundreds of IFN-stimulated genes (ISGs). IFN-responsive RNA binding proteins that either inhibit RNA virus replication or have m6A-selective recognition of cellular RNAs have been described previously; however, if selective binding of N6-methyladenosine (m6A) is required for the function of these proteins during an antiviral response is not clear. Our preliminary studies have identified an ISG that preferentially binds to m6A-containing RNAs and negatively regulates RNA virus infection. PAR-CLIP (photoactivatable crosslinking and immunoprecipitation coupled to next generation sequencing) analysis reveals that it binds to cellular RNAs in regions known to contain m6A. However, the impact of m6A-selectivity on the antiviral innate immune effector mechanisms of this ISG is unclear. Therefore, the goal of this proposal is to define the biochemical interactions of one human ISG that acts during the IFN response to recognize m6A-modified nucleotides on viral RNA. Based on our preliminary data, the central hypothesis of this proposal is that this ISG binds to RNAs in an m6A-dependent manner to regulate the host response to viral infection. Guided by our preliminary data, this hypothesis will be tested by the following two specific aims: 1) Define the mechanisms of RNA recognition and m6A discrimination of this IFN-stimulated m6A-reader; 2) Determine the role RNA recognition by this IFN-stimulated m6A-reader in restricting RNA virus infection. In Aim 1, the molecular basis for recognition of m6A-containing RNAs by this ISG will be determined. In Aim 2, how RNA-binding and m6A-recognition of this protein define the outcome of viral infection will be explored in human cells infected with RNA viruses of global public health concern. Taken together, the work proposed in this application will be significant and innovative because it will define a new role for ISGs in recognizing m6A-modified RNA to regulate the antiviral innate immune system and viral infection. This work will inform therapeutics and treatment strategies for RNA virus infection and autoimmune diseases. Further, an increased understanding of how viral infection is controlled by innate immune effectors will have implications for therapeutics and vaccine responses to limit the burden of RNA virus infection.