MOLECULAR DISSECTION OF THE CORONAVIRUS SPIKE

描述（改编自申请人的概述）： 鼠冠状病毒包括大量不同的 菌株，每种菌株感染不同的组织， 独特的疾病模式疾病包括肝炎，肠胃炎， 和慢性脑脊髓炎，后者作为人类的模型， 神经退行性疾病不同病毒对不同的 在很大程度上是由组织内的结构变化控制的。 病毒体刺突糖蛋白。刺突是一种复杂的寡聚体结构 介导病毒与特定细胞受体的结合， 病毒和细胞膜的融合。履行这些职能 将病毒基因组递送到细胞中，从而建立感染， 后续疾病。因为斯派克是几个重要步骤的核心 在病毒感染、嗜性和发病机制方面， 是描述刺突蛋白携带 基因组传递所需的功能。 研究者将调查自然发生的变异如何 冠状病毒刺突蛋白影响寡聚化的效率， 整合到病毒体中，以及最终的基因组递送功能。 各种刺突将由cDNA结合 假病毒体组装和病毒体掺入所需的组分 效率将被衡量。申请人将确定如何相同 结构变异改变了刺突结合其 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，他会试图触发转换 转化为融合活性构象融合构象 将通过脂质体结合试验进行鉴定， 构象变化将使用加标特异性单克隆抗体进行探测。 抗体的定点突变的使用将使他们能够识别 受体上的区域，需要刺激形成 融合构象。这些发现将被用来建立一个模型 描绘了冠状病毒进入细胞的各个阶段。这样的模型 有助于理解这一复杂的生物过程， 可用于揭示治疗性抗病毒药物的新靶点。

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.