MOLECULAR DISSECTION OF THE CORONAVIRUS SPIKE

描述(改编自研究人员摘要)：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， 持续的病毒感染是人类疾病的模型，例如 多发性硬化症和亚急性硬化性全脑炎。许多人 小鼠感染的临床表现是通过变化来解释的 在病毒粒子刺突糖蛋白中，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融合 病毒和细胞膜。除了它在以下方面的重要性 了解感染的发病机制，尖峰具有独一无二的 使其有别于其他病毒融合蛋白的特性。这个 这项提案的中心目标将是研究具有约束力的 这一重要蛋白质在每种检测中的融合特性 功能将单独进行检查。 为此，来自已知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 将开发结合分析方法，这些方法的相对亲和力 病毒受体(S)将被检测。一种可溶的、可分泌的形式 的受体将被表达、纯化及其与 将确定不同的峰值。分子中的构象变化 将分析伴随着受体结合的尖峰信号。 一种能够测量膜融合能力的独立于 受体结合功能将被开发并用于对 穗的融合与次生和次生变异性的预测变化 三级结构。相关的pH诱导的结构变化 融合功能的激活将使用蛋白酶和 以抗肽抗体为探针。调查人员预计， 结果将为一种独特的融合活性尖峰提供证据 构象。调查员说，这个项目的结果是 将使我们更好地理解 冠状病毒感染，并将促进我们对关系的了解 病毒糖蛋白结构和功能之间的关系。

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.