Lung HRV: G-Protein Coupled Signaling Interactions in Asthma

描述（由申请方提供）：人鼻病毒（HRV）感染导致至少50%的哮喘急性发作。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衬里液/离子含量改变、ASM高反应性和ASM对β-激动剂舒张的抵抗，β-激动剂由这些细胞上的G蛋白偶联受体（GPCR）控制。这些哮喘反应的异质性被认为部分取决于HRV毒株（血清型）。有超过100种HRV毒株，但关于毒株的基因组差异如何影响哮喘急性发作表型知之甚少。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，这些血清型来自历史库。这揭示了以前未知的方面的HRV RNA和蛋白质结构，系统发育关系，重组，和广泛的多样性之间的典型血清型。它还提供了基于结构的序列比对，这是将额外的HRV整合到系统发育树中的支架。这项修订提案的广泛的长期目标是确定现代HRV株的基因组特征，这些特征有助于特定的哮喘气道GPCR表型及其异质性。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从200个现代临床分离株中确定HRV的全基因组序列。在目标2中，这些数据将被整合到我们基于结构的参考基因组支架中，以便定义菌株之间相似和不相似的基因组区域，为选择用于体外功能研究的HRV提供严格的机制。在目标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信号传导表型（子目标1）。并且，这些信号表型将使用贝叶斯技术与内部和外部验证相关联（子目标2）。这些研究将为那些诱发或不诱发AEC和ASM表型性状的HRV提供基因组基础，从而建立病毒诱导的哮喘急性发作异质性的一些机制。这些发现可能提供诊断和预后信息，以及管理哮喘急性发作最常见原因的药理学策略。 公共卫生相关性：人鼻病毒（HRV）感染导致约50%的哮喘发作（和COPD加重）。然而，由于未知的原因，HRV感染引起的哮喘发作的临床特征的性质和严重程度存在很大的差异。然而，现在很清楚，有许多不同的HRV毒株，这个建议将定义哪些HRV，以及它们的基因组的哪些部分，在呼吸道受体功能的变化，有助于在HRV感染的病理和哮喘发作的症状。

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.