Structural Studies of the Coronavirus Life Cycle

摘要 冠状病毒是导致SARS和MERS疾病的零星出现的病毒 疫情爆发。目前还没有针对这些病毒的直接治疗方法，也没有一种疫苗可以 诱导广泛的保护免受感染。然而，病毒生命周期的几个阶段是有希望的 治疗干预的靶点。细胞进入是由大的糖蛋白尖峰介导的，它结合 以宿主受体，并介导病毒和宿主膜的融合。冠状病毒的能力 适应新物种或逃避免疫系统归因于病毒尖峰蛋白。一次 在细胞内，病毒rna合成复合体由16种非结构蛋白(Nsp)组装而成。 转录、编辑和修改病毒RNA并重塑ER膜以创建RNA复制 工厂。病毒结构蛋白的表达涉及RNA合成复合体的进行 不连续的链合成以产生一组截短5‘端开放的病毒mRNAs 读框。不连续的链合成对于产生新的病毒粒子和 了解其机制将有助于揭示相关的病毒过程，如病毒重组 产生血清型或寄主取向改变的穗状突变体。 在K99阶段，我将接受冷冻电子显微镜方面的培训，以补充我的 在X射线结晶学方面的专业知识。我会用冷冻电子显微镜来检查 冠状病毒刺突蛋白与宿主受体结合并为融合做准备时的构象 当我过渡到颁奖的R00阶段时，宿主蛋白水解酶的过程。这些研究建立在 安德鲁·沃德博士实验室最近对HKU1冠状病毒刺突蛋白的结构测定 这是我贡献的。这种尖峰结构不仅证明了拟议的 实验结果，也为新的刺激性蛋白功能假说提供了依据。 同样在K99阶段，我将利用埃里卡·萨菲尔博士的实验室的专业知识来 建立RNA解旋酶测定法以评估病毒NSP13解旋酶的功能。我要用这些 提供机械的、生化的证据来识别RNA模板的检测方法 NSP13解旋酶停滞，并可能导致诱导RNA合成复合体进行 不连续的链合成。在R00阶段，我将补充这些研究 生物化学、X射线结晶学和低温电子显微镜鉴定分子机制 NSP13通过它识别RNA底物并与RNA合成复合体通信。 这些拟议的研究将阐明抗病毒治疗的新靶点。

SUMMARY Coronaviruses are sporadically emerging viruses responsible for SARS and MERS disease outbreaks. There are currently no direct treatments for these viruses, nor is there a vaccine which induces broad protection from infection. However, several stages in the virus life cycle are promising targets for therapeutic intervention. Cell entry is mediated by the large glycoprotein spike, which binds to host receptors and mediates fusion of the viral and host membranes. The ability of coronaviruses to adapt to new species or escape from the immune system is attributed to the viral spike protein. Once inside the cell, the viral RNA synthesis complex is assembled from 16 non-structural proteins (NSP) which transcribe, edit and modify viral RNAs and remodel ER membranes to create RNA replication factories. Expression of the viral structural proteins involves the RNA synthesis complex carrying out discontinuous strand synthesis to produce a nested set of viral mRNAs with truncations of the 5' open reading frames. Discontinuous strand synthesis is essential for the production of new virions and understanding its mechanisms will shed light on related viral processes such as viral recombination to generate spike variants with altered serotypes or host tropisms. During the K99 phase, I will obtain training in cryo-electron microscopy to complement my expertise in X-ray crystallography. I will use cryo-electron microscopy to examine the distinct conformations of the coronavirus spike protein as it binds host receptors and is primed for the fusion process by host proteases as I transition to the R00 phase of the award. These studies build on the recent structure determination of the HKU1-CoV spike protein from Dr. Andrew Ward's laboratory to which I contributed. Not only does this spike structure demonstrate the feasibility of the proposed experiments, but also provides a basis for new hypotheses of spike protein function. Also during the K99 phase, I will utilize the expertise of Dr. Erica Saphire's laboratory to develop RNA helicase assays to assess the function of the viral NSP13 helicase. I will use these assays to provide mechanistic, biochemical evidence to identify RNA templates upon which the NSP13 helicase stalls and may lead to induction of the RNA synthesis complex to carryout discontinuous strand synthesis. During the R00 phase, I will complement these studies with biochemistry, X-ray crystallography and cryo-electron microscopy to identify molecular mechanisms by which NSP13 recognizes RNA substrates and communicates with the RNA synthesis complex. These proposed studies will illuminate novel targets for antiviral therapy.