Role of the Respiratory Syncytial Virus Fusion Protein in Airway Mucus Induction

描述(申请人提供)：我们的长期目标是阐明呼吸道合胞病毒(RSV)导致肺功能障碍的机制。在美国，RSV每年治疗10万名婴儿。肺粘液是呼吸道合胞病毒病的标志。粘液与上皮细胞碎片混合在一起会阻塞呼吸道。呼吸道合胞病毒感染引起呼吸道粘液的机制尚不清楚。这项建议的目的是确定粘液诱导的RSV毒株的病毒融合(F)蛋白在发病机制中的作用，并确定中性粒细胞和嗜碱性粒细胞在RSV诱导的粘液中的作用。与实验室RSV株不同，RSV株19和2-20株可引起小鼠粘液和毛细支气管炎。利用RSV反向遗传系统，我们证明了19号品系的F蛋白在粘液诱导中起作用。我们确定了19F蛋白中的5个氨基酸参与粘液诱导。我们将使用F突变病毒来定义19号品系的F残基，这些残基对体内粘液的表达很重要。RSV 2-20对小鼠具有毒力。我们推测RSV 2-20F对呼吸道粘液和毒力有贡献.我们将利用2-20F嵌合RSV来确定2-20F在发病机制中的作用。19和2-20株比实验室RSV株在体内引起更大的上皮细胞病变效应(CPE)和脱屑。我们将使用F突变体来确定RSV 19F和2-20F株在体内和体外对上皮细胞CPE、合胞体和脱皮的影响。我们将使用融合实验来确定RSV 19、2-20株和LAB株F蛋白在体外培养的上皮细胞中的融合原性。我们假设19F和2-20F在体内和体外都能诱导更大的合胞体和融合。这项研究还将确定中性粒细胞和嗜碱性粒细胞在RSV毛细支气管炎中的作用。RSV 19株和2-20株在损伤小鼠肺上皮细胞的同时，可引起小鼠肺内中性粒细胞增多。我们假设中性粒细胞在19和2-20致病机制中对病毒清除和粘液表达有贡献。19号线诱导的粘液依赖于IL-13，一种TH2细胞因子。IL-4是主要的TH2驱动细胞因子。我们发现在RSV感染中，嗜碱性粒细胞是表达IL-4的主要细胞类型。我们推测嗜碱性粒细胞参与了RSV 19和2-20诱导的IL-13和粘液的表达。这些RSV毛细支气管炎模型将促进我们对RSV株依赖发病机制的理解，并可能导致急需的治疗方法。

DESCRIPTION (provided by applicant): Our long term goal is to elucidate mechanisms by which respiratory syncytial virus (RSV) causes lung dysfunction. In the USA, RSV hospitalizes >100K infants/year. Pulmonary mucus is a hallmark of RSV disease. Mucus mixed with epithelial cell debris blocks the airways. Mechanisms by which RSV infection induces airway mucus are not known. Goals of this proposal are to define the role of the viral fusion (F) proteins of mucus-inducing RSV strains in pathogenesis and to define the roles of neutrophils and basophils in RSV-induced mucus. In contrast to laboratory RSV strains, RSV strains line 19 and 2-20 induce mucus and bronchiolitis in mice. Using a RSV reverse genetics system, we showed that the F protein of line 19 plays a role in mucus induction. We identified five amino acids in the line 19 F protein as candidates for involvement in mucus induction. We will use F mutant viruses to define line 19 F residues that are important for mucus expression in vivo. RSV 2- 20 is virulent in mice. We hypothesize that RSV 2-20 F contributes to airway mucus and virulence. We will define the role of 2-20 F in pathogenesis using 2-20F-chimeric RSV. Line 19 and 2-20 cause greater epithelial cell cytopathic effect (CPE) and desquamation in vivo than lab RSV strains. We will use F mutants to define effects of RSV line 19 F and 2-20 F on epithelial cell CPE, syncytia, and desquamation in vivo and in vitro. We will define the fusogenicity of RSV line 19, 2-20, and lab strain F proteins in epithelial cells in vitro using a fusion assay. We hypothesize that line 19 F and 2-20 F induce greater syncytia and fusion in vivo and in vitro. The study will also determine the roles of neutrophils and basophils in RSV bronchiolitis. Concurrently wih epithelial cell damage, RSV line 19 and 2-20 induce neutrophilia in the lungs of mice. We hypothesize that neutrophils contribute to viral clearance and mucus expression in line 19 and 2-20 pathogenesis. Line 19- induced mucus is dependent on IL-13, a TH2 cytokine. IL 4 is the primary TH2-driving cytokine. We showed that basophils are the major IL 4-expressing cell type in RSV-infection. We hypothesize that basophils contribute to RSV line 19- and 2-20-induced IL-13 and mucus expression. These RSV bronchiolitis models will advance our understanding of RSV strain-dependent pathogenesis and may lead to much-needed therapies.