Re-defining RIG-I-like helicases as viral RNA receptors with effector functions

摘要 脊椎动物的抗病毒先天免疫系统通常被认为由两个截然不同的组组成 蛋白质：检测病毒感染并激活干扰素的模式识别受体(PRR) 信号通路，以及直接对抗病毒复制的效应器。因此，以前关于 RIG-I和MDA5等PRR主要集中在病毒双链RNA中的功能 (DsRNA)检测和下行信令。在刻画和解剖方面所做的努力有限 它们在这些规范的PRR功能之外的潜在作用。我们最近发现RIG-I和MDA5 利用它们的解旋酶结构域和ATP水解驱动的受体动力学来取代来自 DsRNA，可以对广谱的RNA病毒执行不依赖于信号的抗病毒功能。 RIG-I/MDA5的非规范效应器样功能的这一概念也得到了以下出版物的支持 其他实验室(韦伯、高冈和TenOever)，尽管更深层次的分子机制仍有待研究 被解剖了。这些发现挑战了传统观点，即PRR被动地结合预先暴露的配体和 它们的抗病毒活性完全依赖于它们诱导免疫信号的能力。 在这项建议中，我们研究了RIG-I和RIG-I蛋白置换活性的分子机制 MDA5可以发挥信号非依赖性的抗病毒功能。特别是，我们研究了以下两个非 使用生化和细胞分析相结合的排他性假设。在AIMS 1-2中，我们测试 假设RIG-I和MDA5取代病毒dsRNA结合蛋白可能导致 病毒dsRNA的整体可及性，从而促进其他依赖dsRNA的抗病毒蛋白在 宿主，如PKR、OAS1和ADAR1。在目标3中，我们探索了蛋白质置换的假设 RIG-I/MDA5的活性可作用于病毒复制或转录复合体或衣壳蛋白， 这可能导致对核心病毒生命周期步骤的直接抑制。 虽然这个建议探索了RIG-I/MDA5的新功能，但我们之前在这些受体上的工作，都是他们的 正则函数和非正则函数，为本文的研究提供了坚实的基础。此外，我们的 蛋白质-RNA复合体的生化和细胞分析方面的专业知识将进一步帮助我们实现这些目标 在这项提案中概述了。我们认为，这项调查不仅将重新界定RIG-I和 MDA5作为一类具有“效应器样”功能的新型受体，也将为 相关蛋白在抗病毒防御中的作用。

ABSTRACT The vertebrate antiviral innate immune system is often considered to be composed of two distinct groups of proteins: pattern recognition receptors (PRRs) that detect viral infection and activate the interferon (IFN) signaling pathway, and effectors that directly act against viral replication. Accordingly, previous studies on PRRs, such as RIG-I and MDA5, have been primarily focused on their functions in viral double-stranded RNA (dsRNA) detection and downstream signaling. Limited effort has been made in characterizing and dissecting their potential roles outside these canonical PRR functions. We have recently discovered that RIG-I and MDA5 utilize their helicase domains and ATP hydrolysis-driven receptor dynamics to displace viral proteins from dsRNA, and can perform signaling-independent antiviral functions against a broad spectrum of RNA viruses. This notion of the non-canonical, effector-like functions of RIG-I/MDA5 was also supported by publications from other laboratories (Weber, Takaoka and tenOever), although the deeper molecular mechanisms remain to be dissected. These findings challenge the conventional views that PRRs passively bind pre-exposed ligands and that their antiviral activities are solely dependent on their ability to induce immune signaling. In this proposal, we examine molecular mechanisms by which the protein displacement activity of RIG-I and MDA5 can exert signaling-independent antiviral functions. In particular, we examine the following two non- exclusive hypotheses using a combination of biochemical and cellular assays. In Aims 1-2, we test the hypothesis that displacement of viral dsRNA-binding proteins by RIG-I and MDA5 could lead to increase in overall accessibility of viral dsRNA, thereby promoting functions of other dsRNA-dependent antiviral proteins in the host, such as PKR, OAS1 and ADAR1. In Aim 3, we explore the hypothesis that the protein displacement activity of RIG-I/MDA5 could be exerted on the viral replication or transcription complexes or capsid proteins, which may lead to direct inhibition of core viral life cycle steps. While this proposal explores the new function of RIG-I/MDA5, our previous work on these receptors, both their canonical and non-canonical functions, provide a solid foundation for the proposed research. In addition, our expertise on biochemical and cellular analysis of protein-RNA complexes will further help us achieve the goals outlined in this proposal. We believe that this investigation would not only re-define the functions of RIG-I and MDA5 as a novel class of receptors with “effector-like” functions, but would also bring unique perspectives on functions of related proteins in antiviral defense.