MOLECULAR DISSECTION OF THE CORONAVIRUS SPIKE

描述(申请人提供)：被包膜的、含有RNA的小鼠冠状病毒包括大量的毒株。每种菌株感染不同的组织，从而导致一种不同的疾病，如肝炎、胃肠炎或慢性脑脊髓炎；后者是人类神经退行性疾病的模型。这项提议将阐明冠状病毒进入机制，并在这样做的过程中将解释非常相似的菌株如何导致如此截然不同的疾病。冠状病毒趋向性的一个关键决定因素是尖峰蛋白(S)，这是一种执行基本病毒进入功能的蛋白质。S蛋白与癌胚抗原相关细胞黏附分子受体结合，引起构象变化，最终导致病毒-细胞膜融合。 第一个目标是刻画S和CEACAM相互作用后的结构变化。我们将特别关注S蛋白的新的生化特征，例如它们的硫醇-二硫键反应能力，因为它们与病毒进入所需的结构变化有关。我们期望为蛋白质介导膜融合反应提供新的见解。 第二个目标将确定胆固醇如何作为辅助因子支持冠状病毒进入。我们将开发病毒融合的体外检测方法，并用它们来识别S介导的融合过程中胆固醇依赖的阶段。 第三个目标将把我们对冠状病毒进入的生化研究与对疾病的活体调查联系起来。这将通过在S-CEACAM结合和S诱导的膜融合的体外受控测量中比较不同神经毒力的病毒来完成。我们的结果将把冠状病毒的生化特性与它们的致病机制联系起来。我们的集体发现将扩大目前关于病毒进入的知识，并有助于为未来的抗病毒药物开发奠定基础。

DESCRIPTION (provided by applicant): The enveloped, RNA-containing murine coronaviruses include a large collection of strains. Each strain infects different tissues and thus causes a distinct disease such as hepatitis, gastroenteritis, or chronic encephalomyelitis; the latter serves as a model for human neurodegenerative diseases. This proposal will elucidate coronavirus entry mechanisms, and in doing so will explain how very similar strains cause such dramatically different diseases. A key determinant of coronavirus tropism is the spike (S), a protein that carries out essential virus entry functions. S proteins bind to carcinoembryonic antigen-related cell adhesion molecule (CEACAM) receptors, which cause conformational changes culminating in virus-cell membrane fusion. The first aim will characterize the structural changes following interactions between S and CEACAM. We will specifically focus on novel biochemical features of the S proteins, such as their thiol-disulfide reactivities, as they relate to structural changes required for virus entry. We expect to provide new insights into protein-mediated membrane fusion reactions. The second aim will determine how cholesterol operates as a cofactor to support coronavirus entry. We will develop in vitro assays for virus fusion and use them to identify cholesterol-dependent stages of the S-mediated fusion process. The third aim will connect our biochemical studies of coronavirus entry with in vivo investigations of disease. This will be accomplished by comparing viruses of variable neurovirulence in controlled in vitro measurements of S-CEACAM binding and S-induced membrane fusion. Our results will relate the biochemical properties of the coronaviruses with their pathogenesis. Our collective findings will expand current knowledge of virus entry, and help set the stage for future antiviral drug developments.