Biochemical Dissection of the RIG-I Antiviral Pathway

RIG-I 抗病毒途径的生化剖析

基本信息

批准号：
8810636
负责人：
Zhijian J Chen
金额：
$ 39.75万
依托单位：
UT SOUTHWESTERN MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-02-15 至 2017-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8810636
关键词：
Antiviral Agents Autoimmune Diseases Binding Biochemical Biological Assay Cell Nucleus Cell-Free System Cells Cytosol Detection Dissection Foundations Fractionation Genes Genetic study Health 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

项目摘要

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-κB and IRF3, respectively. NF-κB, 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.

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-I 是 RLR 中研究最深入的成员，它与细胞质中的病毒 RNA 结合并触发。导致 I 型干扰素和其他抗病毒分子产生的信号级联反应我们之前已经鉴定了一种称为 MAVS（也称为 IPS-1、VISA）的线粒体抗病毒信号蛋白。或 CARDIF），它在 RIG-I 信号级联中发挥关键作用，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，分别磷酸化转录因子 NF-κB 和 IRF3。 κB、IRF3和其他转录因子在细胞核中共同发挥作用，开启抗病毒基因的表达，以了解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，该系统忠实地重现了病毒 RNA 对 IRF3 的激活，对该系统的初步剖析发现，RIG-I 激活不仅需要病毒 RNA，还需要一种涉及赖氨酸的独特形式的多泛素化。进一步研究表明，未锚定的 K63 多聚泛素链未与任何细胞蛋白缀合，可与 N 端 CARD 结合。这些研究为进一步剖析 RIG-I 信号通路奠定了坚实的基础。在本申请中，我们建议阐明 RNA 和 K63 泛素调节 RIG-I 的机制。我们还将研究 RIG-I 如何激活线粒体膜上的 MAVS（目标 2）。最后，我们将描述 MAVS 激活 TBK1 和 IKK 的途径。总之，这些研究方向应该填补我们目前对 RIG-I 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。