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御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反应。这类受体的一个重要家族是病毒RNA受体RIG-I和MDA5，它们与其共同的接头MAVS合作，激活I型干扰素反应。rig - 1 /MDA5和MAVS之间的相互作用是抗病毒免疫反应启动的一个重要步骤，通常受到宿主和入侵病毒的多层调控。尽管具有重要意义，rig - 1和MDA5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过程中发生的蛋白质聚集或寡聚化的挑战。在此，我们建议使用一种创新的“混合”方法来研究rig - 1、MDA5和MAVS的信号激活过程，该方法系统地将结构和生化分析与细胞功能验证相结合。特别是，我们将重点关注两个关键步骤：(i) rig - 1和MDA5的信号域（串联caspase激活募集域，2CARD）的同源寡聚化，这发生在它们的病毒RNA识别过程中；（ii） MAVS CARD的丝形成，这发生在它与rig - 1 /MDA5 2CARD寡聚物相互作用时。我们将从一个由孤立的信号域（即2CARD和CARD）组成的模型系统开始，以了解RIG-I和MDA5 2CARDs寡聚（目标1）以及2CARD寡聚物如何触发MAVS CARD细丝形成（目标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 - 1和MDA5的背景下，信号域之间的寡聚化和相互作用是如何被调节的（目的3）。该建议建立在我们的新发现的基础上，包括MDA5和rig - 1的细丝形成（Peisley等，PNAS, 2010 & 2011; Mol Cell, 2013）， MDA5:dsRNA复合物的第一个晶体结构（Wu等，Cell, 2013）以及最近未发表的rig - 1 2CARD四聚体的结构（Aim 1A）和MAVS CARD细丝（Aim 2A）。这些发现为解决rig - 1和MDA5信号激活过程中尚未解决的关键问题提供了前所未有的机会，这些问题既包括该领域长期存在的争议，也包括我们的发现带来的新问题。我们期望所提出的研究将揭示“组装介导”信号传导机制的新分子原理，这是先天免疫和细胞死亡中信号转导的新兴范式。此外，我们的机制理解可以为利用rig - 1 /MDA5/MAVS通路治疗免疫疾病和开发抗病毒或抗癌疫苗疗法提供新的治疗策略。

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.