HUMAN CORONAVIRUS 229E SPIKE AND RECEPTOR INTERACTIONS

尽管在过去的10年里已经确定了许多人类病毒的细胞受体，但我们还没有完全了解病毒与其受体的结合是如何导致病毒基因组渗透到细胞中启动复制的。我们发现人氨基肽酶N(HAPN)是一种膜金属蛋白酶，是人类冠状病毒229E的受体。APN是唯一具有病毒受体活性的蛋白水解酶。与hAPN结合的229E病毒包膜糖蛋白是200 kDa的刺突蛋白S。我们的目标是分析S与hAPN之间导致病毒感染的分子相互作用。对这一新型病毒受体系统的研究将对包膜病毒如何与细胞膜结合和融合提供新的见解。在所有年龄段的人类中，15%至30%的上呼吸道和鼻窦感染是由人类冠状病毒引起的，在儿童中，下呼吸道感染和哮喘恶化是由人类冠状病毒引起的。目前还没有疫苗或药物可用于治疗或预防由人类冠状病毒引起的疾病。我们将对229E S糖蛋白和hAPN进行结构和功能分析。我们将在S基因和hAPN基因中引入突变，表达突变的蛋白，并探讨突变对病毒-受体相互作用的影响。我们将在杆状病毒载体中表达无锚定的、可溶性的S糖蛋白和hAPN糖蛋白，并对其进行纯化。这些糖蛋白或从它们衍生的多肽的结构将通过X射线结晶学进行分析。我们将确定纯化的受体在中性或酸性pH下结合，或单独在酸性pH下结合是否能导致S蛋白在病毒粒子上的构象变化，这可能与膜融合有关。我们将选择和/或工程229E病毒和含有突变S蛋白的VSV假型，并表征这些突变导致的功能和抗原变化。利用RT-PCR法对人临床标本中的S基因进行测序，并克隆氨基酸序列差异较大的S蛋白，在真核细胞中表达，并与野生型229E S蛋白进行比较。除了为研究病毒-受体相互作用提供了一个新的模型系统，我们对229E S糖蛋白和hAPN的研究可能会导致新的抗病毒药物的开发，从而阻断人类冠状病毒感染的早期阶段。

Although cellular receptors for many human viruses have been identified in the past 10 years, we do not yet fully understand how binding of a virus to its receptor leads to penetration of the viral genome into the cell to initiate replication. We identified human aminopeptidase N (hAPN), a membrane metalloprotease, as the receptor for human coronavirus 229E. APN is the only protease shown to have virus receptor activity. The 229E viral envelope glycoprotein that binds to hAPN is the 200 kDa spike protein, S. Our goal is to analyze the molecular interactions between S and hAPN that lead to virus infection. The study of this is a novel virus-receptor system will provide new insight into how enveloped viruses bind to and fuse with cellular membranes. Human coronaviruses cause 15 to 30 percent of upper respiratory tract and sinus infections in humans of all ages, and lower respiratory tract infections and exacerbations of asthma in children. No vaccines or drugs are available to treat or prevent diseases caused by human coronaviruses. We will do structural and functional analyses of the 229E S glycoprotein and hAPN. We will introduce mutations into the S gene and the hAPN gene, express the mutant proteins, and explore the effects of the mutations on virus-receptor interactions. We will expressed anchorless, soluble S and hAPN glycoproteins in baculovirus vectors and purify the proteins to homogeneity. The structures of these glycoproteins, or peptides derived from them, will be analyzed by X-ray crystallography. We will determine whether binding of the purified receptor at neutral or acid pH, or acid pH alone can lead to conformational changes in the S protein on virions that may be associated with membrane fusion. We will select and/or engineer 229E viruses and VSV pseudotypes containing mutant S proteins and characterize the functional and antigenic changes that result from the mutations. The S gene will be sequenced by RT-PCR from human clinical specimens, and S proteins that differ significantly in amino acid sequence will be cloned, expressed in eukaryotic cells and their interactions with the hAPN receptor will compared with the wild type 229E S protein. In addition to providing a novel model system for studying virus-receptor interactions, our research on 229E S glycoprotein and hAPN may lead to development of new anti-viral drugs that block the initial stages of human coronavirus infection.