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。这个 这个项目的总体目标是在分子水平上了解先天免疫受体是如何识别 黄病毒，以及这种识别如何转化为免疫反应。我们关注的是先天免疫 DExD/H-box RNA解旋酶RIG-I和MDA5识别细胞质中的病毒基因组RNA。里吉 识别含有5‘-三磷酸的短RNA寡核苷酸，而MDA5识别较长的、双 搁浅的RNA。我们的第一个目标是测量RIG-I和MDA5与RNA底物的结合亲和力 各种长度和次级结构。我们将开始剖析细胞信号转导的机制。 测定RIG-I和MDA5的ATP水解率和解旋酶活性 他们喜欢的核糖核酸底物。在我们提案的主要部分，我们将确定 RIG-I和MDA5在其首选的RNA配体存在和不存在的情况下。这些晶体结构将 揭示了RNA结合引起的构象变化，这将使我们了解结合 RNA被翻译成一种先天免疫信号。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MDA5的信号和配体结合特性。 应答病毒基因组RNA的细胞内信号受另一种DExD/H-box解旋酶调节， LGP2，它直接与RIG-I和MDA5交互。了解LGP2如何调节RIG-I和MDA5 通过信号传递，我们将确定LGP2与RIG-I或MDA5的络合物的晶体结构。副粘病毒 通过直接将MDA5与其V蛋白结合来抑制MDA5信号转导。为了理解分子 在这种病毒免疫逃避的基础上，我们将确定MDA5与SV5V的复合体的晶体结构。 我们的结构化方法将为病毒的产生和监管提供独特的机械性见解 RIG-I和MDA5的RNA传感。从长远来看，这项工作将指导新型疫苗的合理设计 针对RIG-I或MDA5的佐剂和免疫调节疗法，从而为 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.