Structural Studies of the Coronavirus Life Cycle

冠状病毒生命周期的结构研究

• 批准号: 10046412
• 负责人: Robert N Kirchdoerfer
• 金额: $ 24.9万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-12-18 至 2021-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10046412
• 关键词: Address; Antiviral Agents; Antiviral Therapy; Award; Binding; Binding Proteins; Biochemical; Biochemistry; Biological Assay; Birds; Cells; Complement; Complex; Coronavirus; Coronavirus spike protein; Cryoelectron Microscopy; Crystallography; Data; Development; Disease Outbreaks; Dissociation; Elements; Epidemic; Estrogen receptor positive; Event; Genetic Recombination; Genetic Transcription; Genome; Glycoproteins; Goals; Health; Human; Immune system; In Vitro; Infection; Intervention; Laboratories; Lead; Learning; Life Cycle Stages; Light; Mediating; Membrane; Messenger RNA; Middle East Respiratory Syndrome; Middle East Respiratory Syndrome Coronavirus; Modeling; Molecular; Molecular Conformation; Mutate; Nature; Nonstructural Protein; Open Reading Frames; Peptide Hydrolases; Phase; Polymerase; Positioning Attribute; Process; Production; Proteins; RNA; RNA Helicase; RNA Synthesis Induction; RNA chemical synthesis; RNA replication; Replication Error; Research Personnel; SARS coronavirus; Sequence Homologs; Serotyping; Severe Acute Respiratory Syndrome; Site; Specificity; Structure; Surface; Therapeutic Intervention; Time; Training; Transcription Process; Traumeel S; Tropism; Vaccine Design; Vaccines; Variant; Viral; Viral Physiology; Viral Structural Proteins; Virion; Virus; X-Ray Crystallography; experimental study; helicase; novel; novel coronavirus; programs; protein function; receptor; receptor binding; skills; structural biology; targeted treatment; viral RNA; ward

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.

总结 冠状病毒是导致SARS和MERS疾病的零星出现的病毒 爆发目前没有针对这些病毒的直接治疗方法，也没有疫苗， 诱导广泛的保护免受感染。然而，病毒生命周期中的几个阶段是有希望的 治疗干预的目标。细胞进入是由大糖蛋白刺介导的， 宿主受体并介导病毒和宿主膜的融合。冠状病毒能够 适应新的物种或逃避免疫系统归因于病毒刺突蛋白。一旦 在细胞内，病毒RNA合成复合物由16种非结构蛋白（NSP）组装而成。 它转录、编辑和修饰病毒RNA，重塑内质网膜以产生RNA复制 工厂病毒结构蛋白的表达涉及RNA合成复合物， 不连续链合成，以产生具有5'开放链截短的病毒mRNA的嵌套组。 阅读帧。不连续链合成对于新病毒粒子的产生至关重要， 了解其机制将有助于了解相关的病毒过程，如病毒重组， 产生具有改变的血清型或宿主向性的刺突变体。 在K99阶段，我将获得冷冻电子显微镜方面的培训，以补充我的 X射线晶体学的专家我会用低温电子显微镜检查 冠状病毒刺突蛋白的构象，因为它结合宿主受体，并为融合做好准备 过程中由宿主蛋白酶作为我过渡到R 00阶段的奖励。这些研究建立在 安德鲁·沃德博士实验室最近对HKU 1-CoV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所提出的方法的可行性， 实验，而且还提供了一个新的假设的刺突蛋白功能的基础。 同样在K99阶段，我将利用埃里卡萨菲尔博士实验室的专业知识， 开发RNA解旋酶测定以评估病毒NSP 13解旋酶的功能。我会用这些 分析以提供机制、生物化学证据来鉴定RNA模板， NSP 13解旋酶停滞，并可能导致诱导RNA合成复合物进行 不连续链合成。在R 00阶段，我将补充这些研究， 生物化学、X射线晶体学和冷冻电子显微镜，以确定分子机制 NSP 13通过其识别RNA底物并与RNA合成复合物通讯。 这些拟议的研究将阐明抗病毒治疗的新靶点。