Structure & Function of the Interhelical Domain of Coronavirus Spike Glycoprotein

结构

• 批准号: 7690435
• 负责人: Kathryn V Holmes
• 金额: $ 8.56万
• 依托单位: COLORADO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-05-01 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7690435
• 关键词: Binding; Biochemical; Biological Assay; Cell membrane; Cells; Chemicals; Chimeric Proteins; Circular Dichroism; Coronavirus; Data; Development; Disease; Economics; Engineering; Genes; Genetic; Genetic Recombination; Genome; Glycoproteins; Infection; Infection prevention; Knowledge; Lead; Location; Mass Spectrum Analysis; Methodology; Molecular Conformation; Mus; Mutation; Peptides; Property; Proteins; RNA; Research; Retroviridae; Role; SARS coronavirus; Severe Acute Respiratory Syndrome; Site; Structure; Tertiary Protein Structure; Testing; Viral; Viral Fusion-GP; Virion; Virus; X-Ray Crystallography; abstracting; analytical ultracentrifugation; coronavirus spike glycoprotein; crosslink; drug development; genome sequencing; insight; mutant; novel; novel strategies; novel virus; prevent; receptor

Abstract The sudden emergence of novel viruses such as SARS-CoV may cause severe disease and economic losses that call for rapid development of novel anti-virals. To develop new approaches for the treatment of SARS, I will study the structure and function of the unique long interhelical (IH) domain of the spike glycoprotein (S) of SARS-CoV. Receptor-induced conformational changes in S on virions lead to fusion of the viral envelope with host cell membranes and virus entry. I will identify and optimize peptides from the IH domain that block these conformational changes in S and prevent infection. Proposed research and summarized methodology. In Aim 1 I will test the functional conservation between the IH domains of the S proteins of SARS-CoV and murine coronavirus MHV by interchanging their IH domains. The gene encoding the chimeric MHV S protein with the SARS-CoV IH domain will be introduced into the MHV genome by targeted RNA recombination, and the resulting viruses tested for infectivity. Retrovirus pseudotypes expressing the chimeric SARS-CoV S with the MHV IH domain will be assayed for infection of receptor-expressing cells. Chimeric proteins with smaller swaps in the IH domains, and truncations and site-directed mutations will be engineered in the IH domains to identify regions of functional importance in virus entry that can serve as targets for drug development. In Aim 2 I will characterize receptor-induced conformational changes in the IH domains of SARS-CoV and MHV spike proteins using structural and biochemical approaches (e.g. circular dichroism, analytical ultracentrifugation and X-ray crystallography). Soluble ectodomains of the wild type, chimeric and mutant S proteins will be purified, and their structural and biochemical properties will be compared. This will provide further insight into the important roles of the IH domain in the receptor-induced conformation changes leading to virus entry. The resulting structural and biochemical knowledge will aid in the development of novel anti-virals that target the IH domain. In Aim 3 I will identify and optimize peptides from the IH domain of SARS-CoV and MHV S that block infection. I will determine the location in S where the inhibitory peptides bind using chemical cross-linking followed by mass spectrometry. These data, together with the structural information from Aim 2 will enable me to modify the peptides in order to optimize inhibition of virus entry. The optimized peptides will be lead compounds for development of anti-virals to prevent entry of SARS-CoV and other coronaviruses. This strategy for rapid development of anti-viral peptides can be applied to any emerging virus that is found by sequencing of the genome to have a type 1 viral fusion glycoprotein.

抽象的 新型病毒（例如SARS-COV）的突然出现可能会导致严重的疾病和经济损失，要求快速发展新型反病毒病。为了开发用于治疗SARS的新方法，我将研究SARS-COV的尖峰糖蛋白的独特长间内（IH）结构域的结构和功能。受体诱导的s对病毒体上的构象变化导致病毒包膜与宿主细胞膜和病毒的进入。我将从IH结构域中识别并优化肽，这些肽阻断了S中的这些构象变化并防止感染。 拟议的研究和总结方法。在AIM 1中，我将通过互换其IH结构域来测试SARS-COV S蛋白的IH结构域与Murine冠状病毒MHV之间的功能保护。编码嵌合MHV蛋白与SARS-COV IH结构域编码的基因将通过靶向RNA重组引入MHV基因组中，并测试了导致的病毒感染性。用MHV IH结构域表达嵌合SARS-COV的逆转录病毒假病毒型，将分析以表达受体的细胞感染。带有嵌合蛋白 IH域中的较小掉期，将在IH域中设计截断和定向突变，以确定可以作为药物开发目标的病毒进入中功能重要性的区域。在AIM 2中，我将使用结构和生化方法来表征受体诱导的SARS-COV和MHV SPIKE蛋白IH结构域中的构象变化（例如，圆形二科运动，分析性 超速离心和X射线晶体学）。将纯化野生型，嵌合和突变体S蛋白的可溶性外生构，并将其结构和生化特性进行比较。这将进一步了解IH结构域在受体诱导的构象变化中的重要作用，从而导致病毒进入。由此产生的结构和生化知识将有助于开发针对IH领域的新型抗病毒。在AIM 3中，我将从SARS-COV和MHV S阻断感染的MHV的IH结构域中识别并优化肽。我将确定抑制性肽使用化学交联结合，然后进行质谱法结合的位置。这些数据以及AIM 2的结构信息将启用 我要修改肽，以优化对病毒进入的抑制作用。优化的肽将是开发抗病毒的铅化合物，以防止SARS-COV和其他冠状病毒进入。这种快速开发抗病毒肽的策略可以应用于通过测序基因组具有1型病毒融合糖蛋白的任何新出现的病毒。