HUMAN CORONAVIRUS 229E SPIKE AND RECEPTOR INTERACTIONS

尽管在过去的10年中已经确定了许多人类病毒的细胞受体，但我们还没有完全理解病毒与其受体的结合如何导致病毒基因组渗透到细胞中以启动复制。 我们鉴定了人氨肽酶N（hAPN），一种膜金属蛋白酶，作为人冠状病毒229 E的受体。 APN是唯一显示具有病毒受体活性的蛋白酶。 与hAPN结合的229 E病毒包膜糖蛋白是200 kDa刺突蛋白，S. 我们的目标是分析导致病毒感染的S和hAPN之间的分子相互作用。这是一个新的病毒受体系统的研究将提供新的见解包膜病毒如何结合和融合与细胞膜。人类冠状病毒导致15%至30%的所有年龄段的人类上呼吸道和鼻窦感染，以及儿童下呼吸道感染和哮喘恶化。 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229 E 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，表达突变蛋白，并探索突变对病毒-受体相互作用的影响。 我们将在杆状病毒载体中表达无锚的可溶性S和hAPN糖蛋白，并纯化蛋白至均一。 这些糖蛋白或由它们衍生的肽的结构将通过X射线晶体学进行分析。 我们将确定是否结合纯化的受体在中性或酸性pH值，或单独的酸性pH值可以导致构象变化的S蛋白的病毒粒子，可能与膜融合。 我们将选择和/或工程229 E病毒和VSV假型含有突变的S蛋白和表征的功能和抗原性的变化，导致突变。 将通过RT-PCR对人类临床标本的S基因进行测序，并克隆氨基酸序列显着不同的S蛋白，在真核细胞中表达，并将其与hAPN受体的相互作用与野生型229 E S蛋白进行比较。 除了为研究病毒-受体相互作用提供一种新的模型系统外，我们对229 E S糖蛋白和hAPN的研究可能导致开发新的抗病毒药物，阻断人类冠状病毒感染的初始阶段。

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.