Molecular mechanisms for antiviral signaling and regulation by MDA5 and TRIM65

摘要 MDA5是一种保守的先天性免疫受体，在感染过程中检测病毒RNA并激活 抗病毒免疫反应。最近的研究表明，MDA5不仅可以在感染期间被激活， 而且在各种生理条件下也不会感染。这种“无菌”炎症可以 导致炎症性疾病的发病机制，但同时，可以在治疗上有益， 例如在癌症免疫治疗期间。在过去的几年里，我的实验室已经定义了分子框架 以了解MDA5如何识别病毒dsRNA并激活下游信号。我们发现 MDA5在与dsRNA结合时组装成细丝，并且有效地形成细丝是必需的 DsRNA结合和下游信号激活。然而，尽管取得了进展，但在我们的 了解MDA5是如何被激活的，以及它的活动是如何被调节的。也就是说，它的确切身份是什么 在病毒感染和无菌炎症条件下刺激MDA5的dsRNA，以及 MDA5细丝形成后的分子事件导致抗病毒信号激活。这样做的目的是 建议通过关注TRIM65来解决MDA5功能的这两个鲜为人知的方面， 泛素(Ub)E3连接酶对MDA5信号转导至关重要。 我们和其他人之前的研究表明，K63连锁的PolyUb链(K63-UBN)在 在MDA5介导的抗病毒信号中的作用。TRIM65被推测为E3连接酶，负责 MDA5的K63-UBN偶联。然而，事实是否如此，如果是的话，究竟是如何以及何时发生的 TRIM65对MDA5的作用尚不清楚。在初步分析中，我们发现TRIM65直接结合 MDA5，这种结合严格依赖于MDA5丝的形成。这一观察结果表明 TRIM65作为配体识别和信号激活的检查点发挥着核心作用。此外，我们 发现TRIM65下拉可用于特异性分离组装在激动剂上的MDA5丝 DsRNA，远离与丰富的单链RNA结合的MDA5的非活性复合体。这一发现有望写成一部小说。 识别MDA5配体的方法，这是该领域长期追求的里程碑。建立在这些基础上 进展，我们在这里建议解决关于MDA5功能的两个核心问题，即信号机制 (目标1)和RNA配体选择性(目标2)，从TRIM65的新角度。更确切地说，我们将 确定TRIM65激活和调节MDA5的结构和生化机制(目标1) 并开发了一种新的TRIM65下拉策略来确定MDA5的RNA配体。 我们相信，这项拟议的工作将展示E3连接酶如何直接参与自我 与非自我歧视和免疫信号过程的比较，并将为研究他人提供一个模型 免疫功能及以外的E3连接酶。此外，我们的研究还可能指导新的治疗方法 针对MDA5功能及其抗病毒信号通路的策略。

SUMMARY MDA5 is a conserved innate immune receptor that detects viral RNAs during infection and activates antiviral immune response. Recent studies have shown that MDA5 can be activated not only during infection, but also under various physiological conditions in the absence of infection. Such “sterile” inflammation can cause pathogenesis of inflammatory disorders, but at the same time, can be therapeutically beneficial, for example during cancer immunotherapies. Over the last few years, my lab has defined the molecular framework for understanding how MDA5 recognizes viral dsRNA and activates downstream signaling. We discovered that MDA5 assembles into filaments upon binding to dsRNA and that the filament formation is required for efficient dsRNA binding and downstream signal activation. Despite the progress, however, there are key gaps in our understanding of how MDA5 is activated and how its activity is regulated. That is, what is the exact identity of dsRNA that stimulates MDA5 both in the virus-infected and sterile inflammatory conditions, and what are the molecular events following MDA5 filament formation leading up to antiviral signal activation. The goal of this proposal is to address these two poorly understood aspects of MDA5 function by focusing on TRIM65, a ubiquitin (Ub) E3 ligase essential for MDA5 signaling. Previous studies from us and others showed that K63-linked polyUb chains (K63-Ubn) plays an important role in MDA5-mediated antiviral signaling. TRIM65 has been speculated to be the E3 ligase responsible for the K63-Ubn conjugation of MDA5. However, whether this is in fact the case, and if so, exactly how and when TRIM65 acts on MDA5 have been unclear. In our preliminary analysis, we found that TRIM65 directly binds MDA5, and this binding is strictly dependent on MDA5 filament formation. This observation suggests that TRIM65 plays a central role as a check-point for ligand discrimination and signal activation. Furthermore, we found that TRIM65 pull-down can be used for specific isolation of MDA5 filament assembled on agonist dsRNA, away from the inactive complexes of MDA5 bound to abundant ssRNAs. This finding promises a novel method for identifying MDA5 ligands, the long-sought-after milestone in the field. Building upon these progresses, we here propose to address two central questions on MDA5 functions, i.e. signaling mechanism (Aim 1) and RNA ligand selectivity (Aim 2), from the new perspective of TRIM65. More specifically, we will determine the structural and biochemical mechanisms by which TRIM65 activates and regulates MDA5 (Aim 1) and develop a novel TRIM65 pull-down strategy to identify the RNA ligands for MDA5. We believe that the proposed work would demonstrate how an E3 ligase can directly participate in the self vs. non-self discrimination and immune signaling processes, and would provide a model for investigating other E3 ligases in immune functions and beyond. Furthermore, our research may also guide new therapeutic strategies to target MDA5 functions and its antiviral signaling pathway.