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

描述(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，它们与它们的共同适配器MAV合作，激活I型干扰素反应。RIG-I/MDA5和MAV之间的相互作用代表着启动抗病毒免疫反应的承诺步骤，通常受到宿主和入侵病毒的多层调控。尽管很重要，RIG-I和MDA5与MAV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。这在一定程度上是由于分析蛋白质聚集或寡聚的挑战，这发生在信号激活过程中。我们建议使用一种创新的“混合”方法来研究RIG-I、MDA5和MAV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-I和MDA5的TE信号域(串联caspase激活招募域，2CARD)的同源寡聚，这是在病毒RNA识别时发生的；(Ii)MAVS卡片的丝状形成，发生在它与RIG-I/MDA5 2CARD寡聚体相互作用时。我们将从一个由分离的信号域(即2CARD和CARD)组成的模型系统开始，以了解RIG-I和MDA5 2CARDS如何齐聚(目标1)以及2CARD寡聚体如何触发MAVS卡丝形成(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，如何在全长RIG-I和MDA5的背景下调节信号结构域之间的寡聚和相互作用(目标3)。这一建议建立在我们的新发现的基础上，包括MDA5和RIG-I的细丝形成(Peisley al，PNAS，2010和2011；Mol Cell，2013)，MDA5：dsRNA复合体的第一个晶体结构(Wu等人，Cell，2013)，以及最近未发表的RIG-I 2CARD四聚体结构(在目标1A中)和MAVS卡片细丝(在目标2A中)。这些发现提供了前所未有的机会来解决RIG-I和MDA5信号激活过程中的关键悬而未决的问题，这既是该领域长期争论的问题，也是我们的发现带来的新问题。我们预计，这项拟议的研究将揭示“组装介导的”信号机制背后的新的分子原理，这是一种在先天性免疫和细胞死亡中进行信号转导的新兴范例。此外，我们对机制的理解可以为利用RIG-I/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.