Biochemical Dissection of the RIG-I Antiviral Pathway

RIG-I 抗病毒途径的生化剖析

• 批准号: 8602822
• 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
• 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/8602822
• 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; public health relevance; 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.

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 中研究最充分的成员，它与细胞质中的病毒 RNA 结合，触发信号级联反应，从而产生 I 型干扰素和其他抗病毒分子。我们之前发现了一种名为 MAVS（也称为 IPS-1、VISA 或 CARDIF）的线粒体抗病毒信号蛋白，它在 RIG-I 信号级联中发挥着关键作用。 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，它们分别磷酸化转录因子 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，该系统忠实地再现了病毒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，还需要涉及泛素赖氨酸 63 (K63) 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 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-I 信号通路奠定了坚实的基础。在此应用中，我们建议阐明 RNA 和 K63 泛素链调节 RIG-I 的机制（目标 1）。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 激活细胞质中的 TBK1 和 IKK 的途径（目标 3）。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。