Defining novel Riplet-activated antiviral innate immune signaling pathways

 描述（由申请人提供）：干扰素抗病毒防御程序在感知RNA病毒感染时被宿主先天免疫系统激活。然而，许多RNA病毒已经进化出破坏这种防御程序的机制。决定病毒感染结果的潜在宿主-病毒相互作用，包括调节抗病毒先天免疫信号传导以驱动对病毒感染的有效免疫应答的分子机制，尚未完全理解。理解这些过程是必要的，以确定驱动宿主防御RNA病毒感染的第一道防线的关键组件。肝炎 丙型肝炎病毒（HCV）NS 3/4A蛋白酶切割宿主抗病毒信号蛋白Riplet和MAVS以阻断这种抗病毒信号传导。初步研究已经鉴定了NS 3/4A中抑制Riplet切割同时维持MAVS切割的突变。我们使用这种突变型HCV的研究发现，E3泛素连接酶Riplet调节的信号通路独立于已知的模式识别受体和抗病毒蛋白RIG-I，这是先天免疫HCV所必需的。因此，本提案的目标是定义这种Riplet依赖的、RIG-I独立的抗病毒信号传导途径。基于我们的初步数据，该提议的中心假设是Riplet泛素化一种蛋白，该蛋白驱动在HCV感染期间受调节的RIG-I独立的抗病毒信号传导途径，以控制抗病毒应答和感染的结果。在我们的初步数据的指导下，这一假设将通过追求以下两个具体目标来检验：1）确定由Riplet调节的RIG-I独立的先天免疫信号传导途径; 2）确定HCV感染期间Riplet泛素化的靶点。 在目标1中，将定义由NS 3/4A调节的这种RIG-I独立信号传导途径有助于先天免疫信号传导和抗病毒应答的分子机制。在目标2中，将鉴定和验证Riplet的特异性泛素化靶标。 总之，本申请中提出的工作将是重要的，因为它将定义一种新的抗病毒先天免疫信号传导途径，其将对RNA病毒感染和干扰素介导的自身免疫性疾病的治疗和预防产生影响。这项工作具有创新性，因为它是第一个将HCV NS 3/4A蛋白中不同的先天免疫调节机制解偶联的工作，该机制将用于充分定义Riplet和MAVS在抗病毒反应中的作用，并揭示一种新的Riplet调节的抗病毒信号通路。最终，对先天免疫途径调节的更多理解将提高我们对干扰素产生失调的机制原因的认识，干扰素产生失调可导致自身免疫性疾病，并且它将确定RNA病毒感染的免疫保护机制，这将对限制RNA病毒感染的治疗和疫苗策略产生影响。 病毒感染。

 DESCRIPTION (provided by applicant): The interferon antiviral defense program is activated by the host innate immune system upon sensing RNA virus infection. However, many RNA viruses have evolved mechanisms to subvert this defense program. The underlying host-viral interactions that dictate the outcome of viral infection, including the molecular mechanisms that regulate antiviral innate immune signaling to drive an effective immune response to virus infection, are not fully understood. An understanding of these processes is required to define the critical components that drive the first line of host defense to RNA virus infection. The hepatitis C virus (HCV) NS3/4A protease cleaves the host antiviral signaling proteins Riplet and MAVS to block this antiviral signaling. Preliminary studies have identified a mutation in NS3/4A that inhibits Riplet cleavage while maintaining MAVS cleavage. Our studies using this mutant HCV have found that the E3 ubiquitin ligase Riplet regulates a signaling pathway independent of the known pattern recognition receptor and antiviral protein RIG-I that is essential for innate immunity to HCV. Therefore, the goal of this proposal is to define this Riplet-dependent, RIG-I independent antiviral signaling pathway. Based on our preliminary data, the central hypothesis of this proposal is that Riplet ubiquitinate a protein that drives a RIG-I independent antiviral signaling pathway that is regulated during HCV infection to control the antiviral response and outcome of infection. Guided by our preliminary data, this hypothesis will be tested by pursuing the following two specific aims: 1) Define the RIG-I independent innate immune signaling pathway regulated by Riplet; 2) Identify the targets of Riplet ubiquitination during HCV infection. In Aim 1, the molecular mechanisms by which this RIG-I independent signaling pathway regulated by NS3/4A contributes to innate immune signaling and the antiviral response will be defined. In Aim 2, the specific ubiquitinated target(s) of Riplet will be identified and validated. Taken together, the work proposed in this application will be significant because it will define a new antiviral innate immune signaling pathway that will have implications for the treatment and prevention of RNA virus infection and interferon-mediated autoimmune disease. The proposed work is innovative because it is the first to uncouple distinct innate immune regulatory mechanisms within the HCV NS3/4A protein that will be used to fully define the roles of Riplet and MAVS in the antiviral response and to uncover a new Riplet-regulated antiviral signaling pathway. Ultimately, an increased understanding of the regulation of innate immune pathways will improve our knowledge of the mechanistic causes of dysregulated interferon production that can lead to autoimmune disease, and it will define the mechanisms of immune protection for RNA virus infection that will have implications for therapeutic and vaccine strategies to limit RNA virus infection.