Structure & Function of the Interhelical Domain of Coronavirus Spike Glycoprotein

摘要 SARS冠状病毒等新型病毒的突然出现可能会造成严重的疾病和经济损失，这就要求迅速开发新型抗病毒药物。为了开发治疗SARS的新方法，我将研究SARS-CoV刺突糖蛋白(S)独特的长螺旋间区(IH)的结构和功能。受体诱导的S在病毒粒子上的构象变化导致病毒被膜与宿主细胞膜融合并进入病毒。我将从IH结构域中识别和优化多肽，这些多肽可以阻止S的这些构象变化，防止感染。 提出研究建议，总结研究方法。在目标1中，我将通过互换SARS冠状病毒和小鼠冠状病毒MHV S蛋白的IH结构域来检验它们之间的功能保守性。编码带有SARS冠状病毒IH结构域的嵌合MHV S蛋白的基因将通过靶向重组进入MHV基因组，并由此产生的病毒进行传染性测试。表达具有MHV IH结构域的嵌合SARS-CoV S的逆转录病毒假型将被检测是否感染表达受体的细胞。嵌合蛋白与 IH结构域中的较小交换以及截断和定点突变将在IH结构域中进行工程设计，以确定在病毒进入过程中具有功能重要性的区域，这些区域可以作为药物开发的靶点。在目标2中，我将使用结构和生化方法(例如圆二色谱、分析 超速离心法和X射线结晶学)。我们将对野生型、嵌合型和突变型S蛋白的可溶性胞外区进行纯化，并比较它们的结构和生化性质。这将进一步深入了解IH结构域在受体诱导的构象变化导致病毒进入中的重要作用。由此产生的结构和生化知识将有助于开发针对IH结构域的新型抗病毒药物。在目标3中，我将从SARS冠状病毒和MHV S的IH结构域中筛选和优化阻断感染的多肽。我会用化学交联法和质谱法来确定S体内抑制肽结合的位置。这些数据，加上来自AIM 2的结构信息，将使 我对这些多肽进行修饰，以优化对病毒入侵的抑制。优化后的多肽将成为开发抗病毒药物的先导化合物，以防止SARS冠状病毒和其他冠状病毒的入侵。这种快速开发抗病毒多肽的策略可以应用于通过基因组测序发现具有1型病毒融合糖蛋白的任何新出现的病毒。

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.