Regulation of RIG-I signaling and viral immune evasion by ufmylation

摘要 I型和III型干扰素（IFN）限制RNA病毒感染。感染细胞通过激活信号传导产生IFN 通过模式识别受体如RIG-I。RIG-I和下游信号必须高度协调 有效的抗病毒反应。因此，这些过程受到几种翻译后调节。 修改（PTM）。这些PTM对于RIG-I的激活和最终终止都是必不可少的。 信号虽然RIG-I信号转导是通过泛素化和磷酸化来协调的，但RIG-I信号转导的机制是不确定的。 其它可能调节RIG-I信号传导的PTM，如泛素样修饰剂，在很大程度上是未知的。我们的初步 数据显示，称为ufmylation的泛素样修饰物调节参与RIG-I信号传导的多种蛋白质 用于响应病毒感染的最佳IFN诱导。此外，我们的数据表明， 病毒逃避宿主细胞内先天免疫反应。因此，本提案的目标是 确定ufmylation如何调节细胞内对病毒感染和病毒逃逸的先天免疫应答。 基于我们的初步数据，中心假设是，ufmylation调节宿主和病毒的功能， 调节抗病毒先天免疫和病毒逃避的蛋白质。根据我们的初步数据，这个假设 将通过追求以下三个具体目标进行测试：1）定义UFL 1，即磺酰化E3连接酶， 促进RIG-I的活化; 2）理解关键蛋白的磺酰化的分子机制， 在RIG-I信号通路中下调其功能和信号传导; 3）确定登革病毒如何选择 用于免疫逃避的磺酰化缀合系统。在目标1中，UFL 1 诱导RIG-I响应RNA病毒传感的活化。在目标2中，机制和 将确定在RIG-I途径中，磺酰基化如何调节信号蛋白功能的动力学。在 目的3，在人细胞中，DENV蛋白的ufmylation促进DENV免疫逃避的功能， 在原代细胞中，将被确定。综上所述，本申请中提出的工作将 因为它将赋予ufmylation新免疫调节功能，有助于 我们对其基本功能的理解，并揭示了一种新的控制（ufmylation）的抗病毒先天免疫。 此外，这项工作将提供对病毒用于免疫的宿主导向过程的理解。 逃避以促进病毒复制。总之，这项工作将定义一个新的PTM，协调RIG-I信令 这将提高我们对抗病毒免疫和先天免疫途径调节的认识 这将导致增加对IFN失调的机制原因的理解， 自身免疫性疾病它还将定义黄病毒免疫逃避的新机制， 对限制其感染的治疗和疫苗策略的影响。

ABSTRACT Type I and III interferons (IFN) restrict RNA virus infection. Infected cells produce IFN through signaling activated by pattern recognition receptors such as RIG-I. Both RIG-I and downstream signaling must be highly coordinated for efficient antiviral responses. As such, these processes are regulated by several post-translational modifications (PTMs). These PTMs are essential for both the activation and eventual termination of RIG-I- signaling. While RIG-I-signaling is coordinated by ubiquitination and phosphorylation, the mechanisms by which other PTMs, such as ubiquitin-like modifiers, may regulate RIG-I-signaling are largely unknown. Our preliminary data reveal that the ubiquitin-like modifier called ufmylation regulates multiple proteins involved in RIG-I signaling for optimal IFN induction in response to viral infection. Further, our data suggest that ufmylation is utilized by viruses to evade the host intracellular innate immune response. Therefore, the goal of this proposal is to determine how ufmylation regulates the intracellular innate immune response to virus infection and viral evasion. Based on our preliminary data, the central hypothesis is that ufmylation modulates the function of host and viral proteins to regulate antiviral innate immunity and viral evasion. Guided by our preliminary data, this hypothesis will be tested by pursuing the following three specific aims: 1) Define how UFL1, the ufmylation E3 ligase, promotes the activation of RIG-I; 2) Understand the molecular mechanism by which ufmylation of a key protein in the RIG-I signaling pathway downregulates its function and signaling; 3) Determine how dengue virus co-opts the ufmylation conjugation system for immune evasion. In Aim 1, the molecular mechanisms by which UFL1 induces the activation of RIG-I in response to RNA virus sensing will be defined. In Aim 2, the mechanism and dynamics of how ufmylation regulates the function of a signaling protein in RIG-I pathway will be determined. In Aim 3, the function by which ufmylation of a DENV protein promotes DENV immune evasion, both in human cell lines and in primary cells, will be determined. Taken together, the work proposed in this application will be significant and innovative because it will attribute a novel immune regulatory function to ufmylation, contribute to our understanding of its basic functions, and uncover a novel control (ufmylation) of antiviral innate immunity. Additionally, this work will provide understanding of a host-directed process that is utilized by viruses for immune evasion to facilitate viral replication. Overall, this work will define a new PTM that coordinates the RIG-I signaling pathway, which will improve our knowledge of both antiviral immunity and regulation of innate immune pathways that will lead to increased understanding of the mechanistic causes of dysregulated IFN that can ultimately result in autoimmune disease. It will also define a new mechanism of immune evasion by flaviviruses that will have implications for therapeutic and vaccine strategies to limit their infection.