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（由适用提供）：RIG-I喜欢受体（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扩散。 RIG-I是RLR的最佳研究成员，它与细胞质中的病毒RNA结合，并触发一个信号级联，该级联导致产生I型干扰素和其他抗病毒分子。我们先前已经鉴定出称为MAV的线粒体抗病毒信号传导蛋白（也称为IPS-1，Visa或cardif），该蛋白在RIG-I信号级联中起关键作用。 MAVS位于线粒体外膜上，它分别激活包括IKK和TBK1在内的胞质蛋白激酶，分别磷酸化了转录因子NF-κB和IRF3。 NF-κB，IRF3和其他转录因子在细胞核中共同发挥抗病毒基因的表达。为了了解RIG-I途径中信号转导的机理，我们最近建立了一个无细胞的系统，该系统完美地概括了病毒RNA的IRF3激活。该系统的初始解剖导致发现，RIG-I激活不仅需要病毒RNA，而且还需要一种独特的多泛素化形式，涉及泛素的赖氨酸63（K63）。进一步的研究表明，未与任何与任何细胞蛋白结合的未经锚定的K63多泛素链结合了RIG-I的N端卡结构域，从而导致RIG-I激活。这些研究为进一步解剖RIG-I信号通路提供了坚实的基础。在此应用中，我们建议通过RNA和K63泛素链阐明RIG-I调节的机制（AIM 1）。我们还将研究RIG-1如何激活线粒体膜上的MAV（AIM 2）。最后，我们将描述MAVS在细胞质中激活TBK1和IKK的途径（AIM 3）。总之，这些投资应填补我们当前对RIG-I信号级联的一些主要空白，并提供对抗病毒先天免疫力的机械见解。