Biochemical Dissection of the RIG-I Antiviral Pathway

描述（由申请人提供）：RIG-I样受体（rlr）检测病毒感染并触发适当的免疫反应以限制病毒感染和传播。rig - 1是RLRs中研究得最好的成员，它与细胞质中的病毒RNA结合，并触发信号级联反应，导致i型干扰素和其他抗病毒分子的产生。我们之前已经确定了一种称为MAVS的线粒体抗病毒信号蛋白（也称为IPS-1， VISA或CARDIF），它在rig -1信号级联中起关键作用。MAVS位于线粒体外膜上，它激活包括IKK和TBK1在内的胞质蛋白激酶，使转录因子NF-？分别为B和IRF3。NF - ?B、IRF3等转录因子在细胞核内共同作用，开启抗病毒基因的表达。为了了解rig - 1通路的信号转导机制，我们最近建立了一个无细胞系统，忠实地再现了IRF3被病毒RNA激活的过程。对该系统的初步解剖发现，rig - 1的激活不仅需要病毒RNA，还需要一种独特的多泛素化形式，包括泛素的赖氨酸-63 （K63）。进一步的研究表明，未锚定的K63多泛素链（不与任何细胞蛋白结合）与RIG-I的n端CARD结构域结合，导致RIG-I激活。这些研究为进一步剖析rig - 1信号通路提供了坚实的基础。在本应用中，我们建议阐明RNA和K63泛素链调控rig - 1的机制（Aim 1）。我们还将研究RIG-I如何激活线粒体膜上的MAVS （Aim 2）。最后，我们将描述MAVS激活细胞质中TBK1和IKK的途径（目的3）。总之，这些研究路线应该填补我们目前对rig - 1信号级联的理解中的一些主要空白，并提供抗病毒先天免疫的机制见解。

DESCRIPTION (provided by applicant): RIG-I like receptors (RLRs) detect viral infections and triggers appropriate immune responses to limit viral infection and spread. RIG-I, the best studied member of RLRs, binds to viral RNA in the cytosol and triggers a signaling cascade that leads to the production of type-I interferons and other antiviral molecules. We have previously identified a mitochondrial antiviral signaling protein termed MAVS (also known as IPS-1, VISA or CARDIF), which plays a pivotal role in the RIG-I signaling cascade. MAVS resides on the mitochondrial outer membrane and it activates cytosolic protein kinases including IKK and TBK1, which phosphorylate the transcription factors NF-?B and IRF3, respectively. NF-?B, IRF3 and other transcription factors function together in the nucleus to turn on the expression of antiviral genes. In order to understand the mechanism of signal transduction in the RIG-I pathway, we have recently established a cell-free system that faithfully recapitulates the activation of IRF3 by viral RNA. Initial dissection of this system leads to the discovery that RIG-I activation requires not only viral RNA, but also a unique form of polyubiquitination involving lysine-63 (K63) of ubiquitin. Further investigation shows that unanchored K63 polyubiquitin chains, which are not conjugated to any cellular protein, binds to the N-terminal CARD domains of RIG-I, resulting in RIG-I activation. These studies provide a strong foundation for further dissection of the RIG-I signaling pathway. In this application, we propose to elucidate the mechanism of RIG-I regulation by RNA and K63 ubiquitin chains (Aim 1). We will also investigate how RIG-I activates MAVS on the mitochondrial membrane (Aim 2). Finally, we will delineate the pathways through which MAVS activate TBK1 and IKK in the cytosol (Aim 3). Together, these lines of investigation should fill some of the major gaps in our current understanding of the RIG-I signaling cascade, and provide mechanistic insights into antiviral innate immunity. PUBLIC HEALTH RELEVANCE: The RIG-I pathway is largely responsible for the first line of immune defense against viral infection in human and many other organisms. In this application, we propose to elucidate the mechanism of signal transduction in key steps of the RIG-I pathway using an innovative biochemical approach. If successful, our research should significantly advance the fields of immunology and virology, and provide new approaches to harness the host innate immunity to combat viral and autoimmune diseases.