Structural bases of the functions of RNA-protein machines - Project 3

脊椎动物依靠先天免疫受体来感知入侵微生物的保守结构。的 这个项目的总体目标是在分子水平上了解先天免疫受体如何识别 黄病毒，以及这种识别如何转化为免疫反应。我们专注于先天免疫 通过DExD/H-box RNA解旋酶RIG-I和MDA 5识别细胞质中的病毒基因组RNA。Rigi 识别带有5 '-三磷酸的短RNA寡核苷酸，而MDA 5识别更长的双 单链RNA我们的第一个目标是测量RIG-I和MDA 5对RNA底物的结合亲和力。 各种长度和二级结构。我们将开始剖析信号转导的机制， RIG-I和MDA 5通过测量它们的ATP水解速率和它们的解旋酶活性来测定。 它们的首选RNA底物。在我们的建议的主要部分，我们将确定的晶体结构， RIG-I和MDA 5在存在和不存在其优选的RNA配体的情况下。这些晶体结构将 揭示了RNA结合引起的构象变化，这将使我们能够了解RNA结合是如何发生的。 RNA被翻译成先天免疫信号。为了测试我们基于结构的信号生成模型，我们 将设计突变以改变RIG-I和MDA 5的信号传导和配体结合特性。 响应病毒基因组RNA的细胞内信号传导由另一种DExD/H-box解旋酶调节， LGP 2，它直接与RIG-I和MDA 5相互作用。了解LGP 2如何调节RIG-I和MDA 5 通过信号传导，我们将确定与RIG-I或MDA 5复合的LGP 2的晶体结构。副粘 通过将MDA 5直接与它们的V蛋白结合来抑制MDA 5信号传导。为了了解分子 基于这种病毒免疫逃避，我们将确定与SV 5 V复合的MDA 5的晶体结构。 我们的结构方法将为病毒的产生和调控提供独特的机制见解， 通过RIG-I和MDA 5进行RNA感测。从长远来看，本研究将指导新型疫苗的合理设计 靶向RIG-I或MDA 5的佐剂和免疫调节治疗剂，从而提供新的策略， 预防和治疗病毒感染。

Vertebrates rely on innate immune receptors to sense conserved structures from invading microbes. The overall goal of this project is to understand at the molecular level how innate immune receptors recognize flaviviruses, and how this recognition is translated into an immune response. We focus on innate immune recognition of viral genomic RNA in the cytoplasm by the DExD/H-box RNA helicases RIG-I and MDA5. RIGI recognizes short RNA oligonucleotides bearing 5'-triphosphates, while MDA5 recognizes longer, double stranded RNAs. Our first goal is to measure the binding affinities of RIG-I and MDA5 for RNA substrates of various lengths and secondary structures. We will begin to dissect the mechanism of signal transduction of RIG-I and MDA5 by measuring their rates of ATP hydrolysis and their helicase activities in the presence of their preferred RNA substrates. In the major part of our proposal, we will determine the crystal structures of RIG-I and MDA5 in the presence and absence of their preferred RNA ligands. These crystal structures will reveal the conformational changes caused by RNA binding, which will allow us to understand how binding of RNA is translated into an innate immune signal. To test our structure-based model for signal generation, we will engineer mutations designed to alter the signaling and ligand-binding properties of RIG-I and MDA5. Intracellular signaling in response to viral genomic RNA is modulated by another DExD/H-box helicase, LGP2, which interacts directly with RIG-I and MDA5. To understand how LGP2 regulates RIG-I and MDA5 signaling, we will determine the crystal structure of LGP2 in complex with RIG-I or MDA5. Paramyxoviruses inhibit MDA5 signaling by binding MDA5 directly with their V proteins. In order to understand the molecular basis of this viral immune evasion, we will determine the crystal structure of MDA5 in complex with SV5 V. Our structural approach will provide unique mechanistic insights into the generation and regulation of viral RNA sensing by RIG-I and MDA5. In the long term, this work will guide the rational design of novel vaccine adjuvants and immunomodulatory therapeutics targeting RIG-I or MDA5, thus providing new strategies for the prevention and treatment of viral infections.