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（改编自研究者摘要）：嗜神经性小鼠 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， 持续的病毒感染可作为人类疾病的模型， 多发性硬化和亚急性硬化性全脑炎。 许多 小鼠感染的临床表现可以解释为 在病毒体刺突糖蛋白中，一种复杂的多功能分子 它介导病毒与细胞受体的结合以及 病毒和细胞膜。 除了它的重要性， 了解感染的发病机制，穗有独特的 这些特性使其有别于其他病毒融合蛋白。 的 这项建议的中心目标将是研究具有约束力和 这种重要蛋白质的融合特性， 功能是单独检查的。 为此，来自病毒的刺突基因已知在其受体- 通过序列分析比较结合性质。 病毒：受体 将开发结合测定法，并且这些结合测定法的相对亲和力将被确定。 将测量受体的病毒。 一种可溶的分泌形式 受体的表达、纯化及其对受体的亲和力。 将确定不同的尖峰。 构象变化 将分析伴随受体结合的尖峰。 一种能够测量膜融合能力的测定法， 受体结合功能将被开发并用于排名 刺突的融合性，预测继发性和 三级结构 PH诱导的结构变化与 将使用蛋白酶监测融合功能的激活， 抗肽抗体作为探针。 调查人员预计， 结果将为独特的聚变活性尖峰提供证据 构象 研究人员指出，该项目的结果 将导致更好地理解基本的初始步骤， 冠状病毒感染，并将推进我们对关系的认识 病毒糖蛋白结构和功能之间的联系