Lung HRV: G-Protein Coupled Signaling Interactions in Asthma

描述（由申请人提供）：人鼻病毒（HRV）感染导致至少50%的哮喘加重。气道上皮细胞（AEC）感染引起炎症、生长和支气管痉挛因子以及其他自分泌/旁分泌介质的释放，导致全身性AEC和气道平滑肌（ASM）“促恶化”病理。这包括AEC衬里流体/离子含量的改变，ASM的高反应性，以及ASM对（-激动剂）松弛的抗性，（-激动剂）是由这些细胞上的g蛋白偶联受体（gpcr）控制的。这些哮喘反应的异质性在一定程度上被认为取决于HRV毒株（血清型）。有超过100种HRV毒株，但对毒株基因组差异如何影响哮喘加重表型知之甚少。我们最近完成了所有99个参考HRV-A和-B血清型的基因组测序。这揭示了以前未知的HRV RNA和蛋白质结构、系统发育关系、重组以及典型血清型之间的广泛多样性。它还提供了基于结构的序列比对，这是将其他hrv整合到系统发育树中的支架。这一修订建议的广泛长期目标是确定导致特定哮喘气道GPCR表型的现代HRV菌株的基因组特征及其异质性。这将通过三个目标来实现。在目标1中，我们将使用大规模平行测序方法确定来自200个现代临床分离株的hrv全基因组序列。在Aim 2中，这些数据将整合到我们基于结构的参考基因组支架中，以定义菌株之间相似和不同的基因组区域，为体外功能研究选择hrv提供严格的机制。在Aim 3中，将使用高通量方法在细胞培养模型中确定AEC和ASM背景下hrv的GPCR信号表型（Sub Aim 1）。并且，这些信号表型将通过内部和外部验证的贝叶斯技术与HRV基因组特征相关联（Sub Aim 2）。这些研究将为那些hrv是否引起AEC和ASM表型性状提供基因组基础，从而建立病毒诱导哮喘加重异质性的一些机制。这些发现可能提供诊断和预后信息，以及药物策略，以管理哮喘恶化的最常见原因。

DESCRIPTION (provided by applicant): Human rhinovirus (HRV) infection causes at least 50% of asthma exacerbations. Airway epithelial cell (AEC) infection evokes the release of inflammatory, growth and bronchospastic factors, and other autocrine/paracrine mediators leading to generalized AEC and airway smooth muscle (ASM) "pro-exacerbation" pathology. This includes altered AEC lining fluid/ion content, ASM hyperreactivity, and ASM resistance to relaxation by (-agonists, which are controlled by G-protein coupled receptors (GPCRs) on these cells. The heterogeneity of these asthmatic responses is, in part, thought to be dependent on the HRV strain (serotype). There are over 100 HRV strains, yet little is known about how genomic differences in strains impact asthma exacerbation phenotypes. We have very recently completed sequencing the genomes of all 99 reference HRV-A and -B serotypes from a banked historical repository. This revealed previously unknown aspects of HRV RNA and protein structure, phylogenetic relationships, recombination, and extensive diversity among the canonical serotypes. It also provided structure-based sequence alignments which are a scaffold for integration of additional HRVs into the phylogenetic tree. The broad long-term objectives of this revised proposal are to ascertain the genomic features of modern HRV strains that contribute to specific asthmatic airway GPCR phenotypes and their heterogeneity. This will be accomplished by three aims. In Aim 1, we will determine the complete genome sequences of HRVs from 200 modern clinical isolates using massively parallel sequencing methods. In Aim 2, this data will be integrated into our structure-based reference genomic scaffold so as to define genomic regions that are similar and dissimilar amongst the strains, providing a rigorous mechanism to select the HRVs for in vitro functional studies. In Aim 3, GPCR signaling phenotypes of HRVs in the context of AEC and ASM will be ascertained in cell culture models using high-throughput methods (Sub Aim 1). And, these signaling phenotypes will be correlated to HRV genome features using Bayesian techniques with internal and external validations (Sub Aim 2). Such studies will provide a genomic basis for those HRVs that do, and do not, evoke AEC and ASM phenotypic traits, and thus establish some of the mechanisms of heterogeneity of viral induced asthma exacerbations. These findings may provide diagnostic and prognostic information, and pharmacologic strategies, for managing the most common cause of asthma exacerbations. PUBLIC HEALTH RELEVANCE: Human rhinovirus (HRV) infection causes about 50% of asthma attacks (and COPD exacerbations). There is, though, substantial variability in the nature and severity of the clinical features of asthma attacks from HRV infections, for reasons that are not known. However, it is now clear that there are many distinct HRV strains, and this proposal will define which HRVs, and which parts of their genomes, impose changes in airway receptor function that contribute to the pathology and symptoms of asthmatic attacks during HRV infection.