MOLECULAR DISSECTION OF THE CORONAVIRUS SPIKE

描述(改编自申请者摘要)： 小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，从而导致 一种独特的疾病模式。疾病包括肝炎、胃肠炎、 和慢性脑脊髓炎，后者是人类 神经退行性疾病。不同病毒对不同病毒的趋向性 组织在很大程度上受组织内结构变化的控制 病毒粒子刺激性糖蛋白。尖峰是一种复杂的低聚结构 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。履行这些职能 将病毒基因组送入细胞，从而建立感染和 随后的疾病。因为扣球是几个基本步骤的中心 在病毒感染、趋向性和致病机制方面，长期目标 是描述SPEKE蛋白携带的分子机制 完成了基因组传递所需的功能。 研究人员将调查自然发生的变异是如何 冠状病毒刺突蛋白影响齐聚效率， 整合到病毒粒子中，以及最终的基因组传递功能。 不同的刺激物将由cdna与 假病毒粒子组装和病毒粒子掺入所需的组件 将衡量效率。申请者将决定如何相同 结构变化改变了穗与其结合的动力学 细胞受体和棘波介导的膜融合。使用可溶的 细胞受体的形式，他会尝试触发转换 结合成融合活性的构象。融合构象 将通过脂质体结合试验进行鉴定，并推测 构象变化将使用SPEK特异性单抗进行探测 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 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，此外还可以 用于揭示治疗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.