Molecular Mechanisms for Antiviral Signal Activation by MDA5 and RIG-I

描述（由申请人提供）：先天免疫系统中的模式识别受体是抵抗病原体感染的第一道防线。它们识别通常与病原体相关的保守分子特征，并迅速引发抗微生物免疫应答。这种受体的一个重要家族是病毒RNA受体RIG-I和MDA 5，它们与它们的共同接头MAVS合作以激活I型干扰素应答。RIG-I/MDA 5和MAVS之间的相互作用代表了启动抗病毒免疫应答的关键步骤，并且通常受到宿主和入侵病毒的多层调节。尽管重要性，RIG-I和MDA 5与MAVS相互作用并将上游病毒检测事件与下游信号事件联系起来的分子机制尚不清楚。这部分是由于分析信号激活期间发生的蛋白质聚集或寡聚化的挑战。在这里，我们建议调查的信号激活过程的RIG-I，MDA 5和MAVS使用创新的“混合”的方法，系统地整合结构和生化分析与细胞功能验证。特别地，我们将集中于两个关键步骤：（i）RIG-I和MDA 5的te信号传导结构域（串联半胱天冬酶激活募集结构域，2CARD）的同源寡聚化，其在它们的病毒RNA识别后发生，和（ii）MAVS CARD的细丝形成，其在与RIG-I/MDA 5 2CARD寡聚体相互作用后发生。我们将从由分离的信号结构域（即2CARD和CARD）组成的模型系统开始，以了解RIG-I和MDA 5 2CARD如何寡聚化（Aim 1）以及2CARD寡聚体如何触发MAVS CARD细丝形成（Aim 2）的详细分子和结构机制。然后，我们将研究在病毒RNA识别过程中，在全长RIG-I和MDA 5的背景下，信号结构域之间的寡聚化和相互作用是如何调节的（目的3）。 该提议基于我们的新发现，包括MDA 5和RIG-I的细丝形成（Peisley等人，PNAS，2010和2011; Mol Cell，2013）、MDA 5：dsRNA复合物的第一晶体结构（Wu等人，Cell，2013）以及RIG-I 2CARD四聚体（在Aim 1A中）和MAVS CARD细丝（在Aim 2A中）的最近未公开的结构。这些发现为解决RIG-I和MDA 5信号激活过程中的关键未决问题提供了前所未有的机会，这些问题既包括该领域长期争论的问题，也包括我们的发现所产生的新问题。我们希望，拟议的研究将揭示新的分子原理的“组装介导”的信号传导机制，一个新兴的模式，在先天免疫和细胞死亡的信号转导。此外，我们的机制理解可以提供新的治疗策略，利用RIG-I/MDA 5/MAVS途径治疗免疫疾病和开发抗病毒或抗癌疫苗疗法。

DESCRIPTION (provided by applicant): Pattern Recognition Receptors in the innate immune system serve as the first line of defense against pathogen infection. They recognize conserved molecular features commonly associated with pathogens and rapidly elicit anti-microbial immune response. One important family of such receptors are viral RNA receptors, RIG-I and MDA5, which cooperate with their common adaptor, MAVS, to activate the type I interferon response. The interaction between RIG-I/MDA5 and MAVS represents a committed step in initiation of the antiviral immune response and is often subject to multiple layers of regulation from both the host and invading viruses. Despite the importance, the molecular mechanism by which RIG-I and MDA5 interact with MAVS and link the upstream viral-detection events to the downstream signaling event is yet unclear. This is partly due to challenges of analyzing protein aggregation or oligomerization, which occurs during signal activation. We here propose to investigate the signal activation process of RIG-I, MDA5 and MAVS using an innovative "hybrid" approach that systematically integrates structural and biochemical analysis with cellular functional validation. In particular, we will focus on two key steps: (i) homo-oligomerization of te signaling domains (tandem caspase activation recruitment domain, 2CARD) of RIG-I and MDA5, which occurs upon their viral RNA recognition, and (ii) filament formation of MAVS CARD, which occurs upon its interaction with RIG-I/MDA5 2CARD oligomers. We will start with a model system consisting of the isolated signaling domains (i.e. 2CARD and CARD) to understand the detailed molecular and structural mechanisms for how RIG-I and MDA5 2CARDs oligomerize (Aim 1) and how the 2CARD oligomers trigger MAVS CARD filament formation (Aim 2). We will then investigate how the oligomerization and interactions among the signaling domains are regulated in the context of full-length RIG-I and MDA5 during viral RNA recognition (Aim 3). This proposal builds upon our novel findings, including filament formation of MDA5 and RIG-I (Peisley al, PNAS, 2010 & 2011; Mol Cell, 2013), the first crystal structure of the MDA5:dsRNA complex (Wu et al, Cell, 2013) and the recent, unpublished structures of the RIG-I 2CARD tetramer (in Aim 1A) and the MAVS CARD filament (in Aim 2A). These findings provide unprecedented opportunities to address key unresolved issues on the signal activation process of RIG-I and MDA5, both long-debated issues in the field and new questions arising from our discoveries. We expect that the proposed research would reveal novel molecular principles underlying the "assembly-mediated" signaling mechanism, an emerging paradigm for signal transduction in innate immunity and cell death. Furthermore, our mechanistic understanding could provide novel therapeutic strategies to harness the RIG-I/MDA5/MAVS pathways in treatment of immune disorders and development of antiviral or anticancer vaccine therapies.