Structure & Function of the Interhelical Domain of Coronavirus Spike Glycoprotein

摘要 新型病毒如SARS-CoV的突然出现可能会导致严重的疾病和经济损失，这要求快速开发新型抗病毒药物。为了开发新的治疗SARS的方法，我将研究SARS-CoV刺突糖蛋白（S）独特的长螺旋间（IH）结构域的结构和功能。受体诱导的病毒体上S的构象变化导致病毒包膜与宿主细胞膜的融合和病毒进入。我将从IH结构域中鉴定和优化肽，以阻断S中的这些构象变化并防止感染。 拟议的研究和总结的方法。目的1通过交换SARS-CoV和鼠冠状病毒MHV的S蛋白的IH结构域，验证它们之间的功能保守性。将通过靶向RNA重组将编码具有SARS-CoV IH结构域的嵌合MHV S蛋白的基因引入MHV基因组中，并测试所得病毒的感染性。将测定表达具有MHV IH结构域的嵌合SARS-CoV S的逆转录病毒假型对受体表达细胞的感染。嵌合蛋白与 IH结构域中较小的交换、截短和定点突变将在IH结构域中被工程化，以鉴定在病毒进入中具有重要功能的区域，这些区域可以作为药物开发的靶点。在目标2中，我将使用结构和生物化学方法（例如圆二色谱、分析方法和生物化学方法）表征SARS-CoV和MHV刺突蛋白IH结构域中受体诱导的构象变化。 超离心和X射线晶体学）。将纯化野生型、嵌合型和突变型S蛋白的可溶性胞外域，并将比较它们的结构和生物化学性质。这将提供进一步深入了解IH结构域在受体诱导的构象变化导致病毒进入的重要作用。由此产生的结构和生物化学知识将有助于开发靶向IH结构域的新型抗病毒药物。在目标3中，我将从SARS-CoV和MHV S的IH结构域中鉴定和优化阻断感染的肽。我将使用化学交联和质谱法确定抑制肽结合在S中的位置。这些数据，加上目标2的结构信息， 我修饰的肽，以优化抑制病毒进入。优化的肽将是开发抗病毒药物的先导化合物，以防止SARS-CoV和其他冠状病毒的进入。这种快速开发抗病毒肽的策略可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兴病毒。

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.