Biochemical Dissection of the RIG-I Antiviral Pathway

RIG-I 抗病毒途径的生化剖析

• 批准号: 8087900
• 负责人: Zhijian J Chen
• 金额: $ 39.63万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-02-15 至 2016-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8087900
• 关键词: Antiviral Agents; Autoimmune Diseases; Binding; Biochemical; Biological Assay; Cell Nucleus; Cell-Free System; Cells; Cytosol; Detection; Dissection; Foundations; Fractionation; Genes; Genetic; Homologous Gene; Human; IRF3 gene; Immune; Immune response; Immunology; Interferon Type I; Investigation; Length; Link; Lysine; Mediating; Mitochondria; Molecular; N-terminal; Natural Immunity; Organism; Outer Mitochondrial Membrane; Pathway interactions; Play; Polyubiquitin; Polyubiquitination; Production; Protein Binding; Protein Kinase; Proteins; RNA; Regulation; Research; Role; Signal Pathway; Signal Transduction; Signaling Protein; Staging; System; TBK1 gene; Ubiquitin; Virus; Virus Diseases; base; combat; in vitro Assay; induced pluripotent stem cell; innovation; insight; member; mitochondrial membrane; novel strategies; receptor; receptor binding; reconstitution; research study; transcription factor; viral RNA; virology

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.

描述(申请人提供)：RIG-I样受体(RLRs)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-I是RLRs中研究最深入的成员，它与细胞质中的病毒RNA结合，触发信号级联，导致I型干扰素和其他抗病毒分子的产生。我们之前已经发现了一种线粒体抗病毒信号蛋白MAVS(也称为IPS-1、VISA或Medicf)，它在RIG-I信号级联中发挥关键作用。MAVS位于线粒体外膜上，激活胞浆蛋白激酶，包括IKK和TBK1，分别使转录因子NF-βB和IRF3磷酸化。核转录因子？B、IRF3和其他转录因子在细胞核内共同作用，启动抗病毒基因的表达。为了了解RIG-I途径中的信号转导机制，我们最近建立了一个无细胞系统，它忠实地概括了病毒RNA激活IRF3的过程。对这个系统的初步剖析导致发现RIG-I的激活不仅需要病毒RNA，而且还需要一种独特的涉及泛素赖氨酸-63(K63)的多泛素化。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末端卡域结合，导致RIG-I的激活。这些研究为进一步剖析RIG-I信号通路奠定了坚实的基础。在本应用中，我们建议阐明RNA和K63泛素链(目标1)对RIG-I的调节机制。我们还将研究RIG-I如何激活线粒体膜上的MAV(目标2)。最后，我们将描绘MAV激活胞浆中的TBK1和IKK的途径(目标3)。总而言之，这些研究路线应该会填补我们目前对RIG-I信号级联的一些主要了解空白，并为抗病毒天然免疫提供机械性的见解。 与公共卫生相关：RIG-I途径在很大程度上负责人类和许多其他生物体对病毒感染的第一道免疫防御。在这一应用中，我们建议使用一种创新的生化方法来阐明RIG-I途径关键步骤中的信号转导机制。如果成功，我们的研究将极大地推动免疫学和病毒学领域的发展，并提供新的方法来利用宿主的先天性免疫来对抗病毒和自身免疫性疾病。