Biochemical Dissection of the RIG-I Antiviral Pathway

描述（由申请方提供）：RIG-I样受体（RLR）检测病毒感染并触发适当的免疫应答以限制病毒感染和传播。RIG-I是RLR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。我们先前已经鉴定了一种称为MAVS（也称为IPS-1、VISA或CARDIF）的线粒体抗病毒信号蛋白，其在RIG-I信号级联中起关键作用。MAVS位于线粒体外膜上，它激活细胞溶质蛋白激酶，包括IKK和TBK 1，磷酸化转录因子NF-？分别为B和IRF 3。NF-？B、IRF 3和其他转录因子在细胞核中共同发挥作用，以开启抗病毒基因的表达。为了了解RIG-I通路中的信号转导机制，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IRF 3的激活。该系统的初步解剖导致发现RIG-I激活不仅需要病毒RNA，而且还需要一种独特形式的多聚泛素化，涉及泛素的赖氨酸-63（K63）。进一步的研究表明，未锚定的K63多聚泛素链，其不与任何细胞蛋白缀合，结合到RIG-I的N-末端CARD结构域，导致RIG-I活化。这些研究为进一步研究RIG-I信号通路提供了坚实的基础。在本申请中，我们建议阐明RIG-I通过RNA和K63泛素链调控的机制（目的1）。我们还将研究RIG-I如何激活线粒体膜上的MAVS（目的2）。最后，我们将描述MAVS激活细胞溶质中TBK 1和IKK的途径（目的3）。总之，这些研究应该填补了我们目前对RIG-I信号级联的理解中的一些主要空白，并提供了对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.