MOLECULAR DISSECTION OF THE CORONAVIRUS SPIKE

冠状病毒刺突的分子解剖

• 批准号: 6126251
• 负责人: Thomas Miller Gallagher
• 金额: $ 22.63万
• 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 2002-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6126251
• 关键词: Coronaviridae; antireceptor antibody; conformation; gene mutation; genetic polymorphism; glycoproteins; membrane fusion; monoclonal antibody; protein biosynthesis; protein structure function; receptor binding; site directed mutagenesis; tissue /cell culture; virus genetics; virus infection mechanism; virus protein; virus receptors; virus replication

DESCRIPTION (Adapted from Applicant's Summary): The murine coronaviruses include a large collection of different strains, with each strain infecting different tissues and thus causing a unique disease pattern. Diseases include hepatitis, gastroenteritis, and chronic encephalomyelitis, the latter serving as a model for human neurodegenerative disease. Tropism of the different viruses for distinct tissues is controlled in large part by structural variations within the virion spike glycoprotein. The spike is a complex oligomeric structure that mediates virus binding to specific cellular receptors as well as fusion of viral and cellular membranes. Fulfillment of these functions delivers viral genomes into cells, thereby establishing infection and subsequent disease. Because spike is central to several essential steps in viral infection, tropism and pathogenesis, the long-term objective is to describe the molecular mechanisms by which spike proteins carry out the functions required for genome delivery. The investigator will investigate how naturally occurring variation in the coronavirus spike protein impacts the efficiency of oligomerization, incorporation into virions, and ultimate genome delivery functions. Various spikes will be synthesized from cDNA in conjunction with components required for pseudovirion assembly and virion incorporation efficiencies will be measured. The applicant will determine how the same structural variations alter the kinetics of spike binding to its cellular receptor and spike-mediated membrane fusion. Using soluble forms of the cellular receptor, he will attempt to trigger conversion of the spikes into a fusion-active conformation. Fusion conformations will be identified by liposome binding assays, and putative conformational changes will be probed using spike-specific monoclonal antibodies. The use of site-directed mutants will allow them to identify regions on the receptors that are required to stimulate the formation of fusion conformations. These findings will be used to build a model depicting various stages of coronavirus entry into cells. Such models help to understand this complex biological process and additionally can be used to reveal new targets for therapeutic antiviral agents.

描述（改编自申请人的摘要）： 鼠冠状病毒包括大量不同的病毒 菌株，每种菌株感染不同的组织，从而导致 独特的疾病模式。疾病包括肝炎、胃肠炎、 和慢性脑脊髓炎，后者是人类的模型 神经退行性疾病。不同病毒的趋向性不同 组织在很大程度上是由内部结构变化控制的 病毒体刺突糖蛋白。刺突是一种复杂的寡聚结构 介导病毒与特定细胞受体的结合以及 病毒和细胞膜的融合。实现这些功能 将病毒基因组传递到细胞中，从而建立感染并 后续疾病。因为尖峰是几个基本步骤的核心 在病毒感染、趋向性和发病机制方面，长期目标 是描述刺突蛋白携带的分子机制 找出基因组传递所需的功能。 研究者将调查自然发生的变异是如何发生的 冠状病毒刺突蛋白影响寡聚化的效率， 整合到病毒体中，以及最终的基因组传递功能。 各种刺突将从 cDNA 中合成，并结合 假病毒粒子组装和病毒粒子掺入所需的组件 将衡量效率。申请人将决定如何相同 结构变化改变了尖峰与其结合的动力学 细胞受体和刺突介导的膜融合。使用可溶性 细胞受体的形式，他将尝试触发转换 尖峰转化为融合活性构象。融合构象 将通过脂质体结合测定来鉴定，并且假定 将使用尖峰特异性单克隆抗体来探测构象变化 抗体。使用定点突变体将使他们能够识别 受体上刺激形成所需的区域 的融合构象。这些发现将用于构建模型 描绘了冠状病毒进入细胞的各个阶段。此类车型 有助于理解这一复杂的生物过程，此外还可以 可用于揭示治疗性抗病毒药物的新靶点。