Molecular Biology of RV-C and its Asthma-related Receptor, CDHR3

项目摘要/摘要 哮喘是一种高度复杂的疾病，因为个体病因是遗传因素的综合结果 易感性、个人病史和无数的环境因素。该计划将收集和 评估与每个组件相关的数据。项目II提供了经验和技术来描述 病毒感染，特别是人鼻病毒(RV)感染的分子机制 塑造哮喘发作。与普通感冒有关，轮状病毒感染导致了50%-85%的哮喘 在最严重的呼吸道感染和 儿童的住院治疗。与RV-A和B分离株相比，RV-C物种中的病毒尤其 与儿童早期的疾病有关，他们的感染与随后的 哮喘。RV-C需要钙粘蛋白相关家族成员3(CDHR3)，一种不寻常的呼吸道特异蛋白，作为 它们的细胞进入受体。该基因的“A”等位基因(Tyr529蛋白变体)是已知最强的等位基因之一 基因与一种以严重的阵发性喘息为特征的儿童哮喘有关。相反，Cys529， 由“G”等位基因编码，在现代人类谱系中的患病率要高得多，不是哮喘 相互关联。我们的基本假设是CDHR3 Cys529变异不能在生化水平上表现出来 这种蛋白适当地或广泛地存在于细胞表面，使其携带者对RV-C感染更具折射性 以及病毒引起的哮喘加重。该项目的三个目标将检查结构， CDHR3在重组、细胞培养和天然组织中的生物化学和功能。预期中的 信息包括：(A)受体的低温EM结构测定：RV-C复合体，(B)低温EM结构 RV-C中和抗体和非中和抗体复合体的测定(C)生化 CDHR3细胞表面差异显示要求说明(Cys/Tyr529)，(D)生化 CDHR3糖基化位点、二聚化位点和病毒相互作用位点的描述。我们还将检查 原代组织和组织来源的分化细胞培养(例如ALI)，以验证和定位 CDHR3显示和确定遗传决定的等位基因(“A”和“G”)是如何赋予RV-C易感性的 感染。我们的技术和化验还将首次记录是否以及如何常见的环境 因素，如个人的微生物群含量，可能通过以下方式影响轮状病毒感染的程度和严重程度 改变RV受体的表达或改变细胞在其原始环境中的感染敏感性。

PROJECT SUMMARY/ABSTRACT Asthma is a highly complex disease because individual etiologies result from composite elements of genetic susceptibility, personal medical histories and a myriad of environmental factors. This Program will collect and evaluate data relevant to each component. Project II contributes experience and technologies to describe the molecular mechanisms by which virus infections, specifically by human rhinoviruses (RV), contribute and shape episodes of asthma. Canonically linked to the common cold, RV infections contribute 50-85% of asthma exacerbations and are the most frequently isolated viruses during the most severe respiratory infections and hospitalizations among children. More so than RV-A&B isolates, viruses in the RV-C species are particularly linked to illnesses in early childhood, and their infections are strongly linked to subsequent development of asthma. The RV-C require cadherin-related family member 3 (CDHR3), an unusual airway-specific protein, as their cell-entry receptor. The “A” allele of this gene (Tyr529 protein variant) is among the strongest known genetic correlates for a type of childhood asthma marked by severe episodic wheezing. Conversely, Cys529, encoded by the “G” allele, and of much higher prevalence in modern human lineages, is not an asthma correlate. Our fundamental hypothesis is that the CDHR3 Cys529 variant fails at the biochemical level to display this protein properly or extensively on cell surfaces, making their carriers more refractive to RV-C infections and virus-induced asthma-exacerbations. The three Aims of the Project will examine the structure, biochemistry and function of CDHR3 in recombinant, cell culture and native tissue formats. The expected information includes (a) a cryoEM structure determination of the receptor:RV-C complex, (b) cryoEM structure determinations of RV-C complexed with neutralizing and non-neutralizing antibodies, (c) biochemical descriptions of CDHR3 requirements for differential cell surface display (Cys/Tyr529), (d) biochemical descriptions of CDHR3 glycosylation sites, dimerization sites and virus interaction sites. We will also examine primary tissue, and tissue-derived differentiated cell cultures (e.g. ALI), to validate and localize the sites of CDHR3 display and determine how the genetics-determined allele (“A” vs “G”) confers susceptibility to RV-C infections. Our techniques and assays will also document for the first time, if and how common environmental factors, such as an individual's microbiome content, may influence the extent and severity of RV infections by altering RV receptor expression or a cell's infection susceptibility in its native primary context.