MOLECULAR DISSECTION OF THE CORONAVIRUS SPIKE

冠状病毒刺突的分子解剖

• 批准号: 2269555
• 负责人: Thomas Miller Gallagher
• 金额: $ 10万
• 依托单位: LOYOLA UNIVERSITY CHICAGO
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-12-15 至 1998-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2269555
• 关键词: Coronaviridae; antireceptor antibody; chromatography; conformation; gene expression; glycoproteins; immunoaffinity chromatography; membrane fusion; molecular biology; monoclonal antibody; mutant; nucleic acid sequence; polymerase chain reaction; protein purification; protein structure function; receptor binding; structural genes; tissue /cell culture; transfection /expression vector; vaccinia virus; virion; virus genetics; virus infection mechanism; virus protein; virus receptors

DESCRIPTION (Adapted from investigator's abstract): Neurotropic murine coronaviruses cause a spectrum of central nervous system diseases ranging from acute encephalitis to chronic demyelinating disorders. The latter, persistent viral infection serves as a model for such human diseases as multiple sclerosis and subacute sclerosing panencephalitis. Many of the clinical manifestations of the murine infection are explained by changes in the virion spike glycoprotein, a complex multifunctional molecule which mediates virus binding to cell receptors as well as fusion of viral and cellular membranes. In addition to its importance in understanding the pathogenesis of the infections, the spike has unique properties which set it apart from other viral fusion proteins. The central objective of this proposal will be to study the binding and fusion properties of this important protein in assays in which each function is examined separately. To this end, spike genes from viruses known to differ in their receptor- binding properties will be compared by sequence analysis. Virus:receptor binding assays will be developed, and the relative affinity of these viruses for receptor(s) will be measured. A soluble and secreted form of the receptor will be expressed, purified and its affinity for the different spikes will be determined. Conformational changes in the spikes that accompany receptor binding will be analyzed. An assay capable of measuring membrane fusion capacity independent of receptor binding function will be developed and used to rank the fusogenicity of spikes with predicted alterations in secondary and tertiary structure. Ph-induced structural changes that are associated with activation of fusion function will be monitored using proteases and antipeptide antibodies as probes. The investigator expects that the results will provide evidence for a unique fusion-active spike conformation. The investigator states that the results of this project will lead to a greater understanding of the essential initial steps in coronavirus infection, and will advance our knowledge of relationships between viral glycoprotein structure and function.

描述（改编自调查员的摘要）：神经性鼠 冠状病毒引起一系列中枢神经系统疾病 从急性脑炎到慢性脱髓鞘疾病。 后者， 持续性病毒感染是人类疾病等模型 多发性硬化症和亚急性硬化脑炎。 许多 鼠感染的临床表现通过变化来解释 在病毒体峰值糖蛋白中，复杂的多功能分子 介导病毒与细胞受体结合以及融合 病毒和细胞膜。 除了它的重要性 了解感染的发病机理，尖峰具有独特的 将其与其他病毒融合蛋白区分开的特性。 这 该提案的核心目标是研究结合和 该重要蛋白在每个测定中的融合特性 分别检查功能。 为此，来自已知的受体不同的病毒的尖峰基因 - 结合特性将通过序列分析进行比较。 病毒：受体 将开发绑定测定法，并相对亲和力 将测量受体的病毒。 可溶性和分泌的形式 该受体将被表达，纯化及其对 将确定不同的尖峰。 构象变化 将分析伴随受体结合的尖峰。 能够测量膜融合能力的测定 受体结合函数将被开发并用于对 峰值的融合性，次级和 三级结构。 pH引起的结构变化 随着融合功能的激活，将使用蛋白酶监测，并 抗肽抗体作为探针。 调查人员期望 结果将为独特的融合活性尖峰提供证据 构象。 研究者指出该项目的结果 将导致对基本初始步骤的更多了解 冠状病毒感染，并将提高我们对人际关系的了解 在病毒糖蛋白结构和功能之间。