Vagal airway sensory nerve activation by beta-coronavirus spike protein

项目摘要 感觉神经，特别是伤害性C纤维的激活是大多数呼吸系统的特征。 病毒。这种激活的证据在C纤维激活的经典结果中被发现 包括打喷嚏、喉咙痛、咳嗽和反射性分泌物。除了引起麻烦 在病毒感染的症状，这些神经的激活允许病毒逃离身体， 传播到其他宿主，即伤害感受器激活放大病毒在社区中的传播。此外，本发明还提供了一种方法， 气道迷走神经C-纤维的激活可导致强烈的反射性支气管收缩和过度的 可能导致哮喘恶化的分泌物，特别是儿童。鉴于 与人类疾病的相关性，令人惊讶的是，关于病毒感染如何诱导C纤维 活化和敏化。从理论上讲，病毒感染导致C纤维激活， 机制等第一种是上皮细胞的病毒感染导致介体的产生 刺激C纤维末梢第二种是病毒本身直接激活神经。 第二种机制可能取决于特定的病毒类型。该提案的重点是 这第二种（直接）激活机制与冠状病毒有关。我假设 冠状病毒刺突蛋白直接与C纤维末端相互作用， 使伤害感受C纤维敏感。我的初步数据，使用三个正交的方法，支持 刺突蛋白直接激活（引起动作电位放电）约40- 小鼠气道中50%的迷走神经C纤维。我的第一个目标是描述迷走神经C纤维的亚型 这些蛋白被刺突蛋白激活，并评估刺突蛋白是否缺乏明显的活性， 激活，导致C-纤维末端的敏化，即使它们对其他刺激更敏感。 激活刺激。我的第二个目标是机制。我假设这种互动 包括刺突蛋白中的半乳糖肌动蛋白-3折叠，并且独立于刺突蛋白发生 受体ACE 2或Toll样受体。无论近端结合靶点如何，我将讨论我们的 假设激活继发于TRPV 1和/或TRPA 1通道的开放。这些目标 将使用单细胞RT-PCR分析气道特异性 伤害性C纤维、细胞外和膜片钳电生理学以及双光子活体成像 技术.这些研究的结果预计将为研究一种新的机制提供见解。 冠状病毒诱导气道C纤维活化。

PROJECT SUMMARY Activation of sensory nerves, in particular nociceptive C-fibers, is a feature of most respiratory viruses. Evidence of such activation is found in the classical consequences of C-fiber activation including sneezing, sore throat, coughing, and reflex secretions. As well as causing the troubling symptoms of viral infection, the activation of these nerves allows viruses to escape the body and be transmitted to other hosts, i.e. nociceptor activation amplifies viral spread in a community. In addition, activation of airway vagal C-fibers can lead to strong reflex bronchoconstriction and excessive secretions that likely contribute to the exacerbation of asthma particularly in children. Given the relevance to human disease, surprisingly little is known about how virus infection induces C-fiber activation and sensitization. In theory, viral infection leads to C-fiber activation by two general mechanisms. The first is that viral infection of epithelial cells leads to the production of a mediator(s) that stimulates the C-fiber terminals. The second is that the virus itself directly activates the nerves. This second mechanism will likely be dependent on the specific virus type. This proposal focuses on this second (direct) mechanism of activation as it relates to coronaviruses. I hypothesize that the coronavirus spike protein interacts directly with C-fiber terminals in a manner that activates and sensitizes the nociceptive C-fibers. My preliminary data, using three orthogonal approaches, support the conclusion that the spike protein directly activates (evokes action potential discharge) about 40- 50% of vagal C-fibers in mouse airways. My first aim is to characterize the subtype of vagal C-fibers that are activated by spike protein and also to assess whether the spike protein, short of overt activation, leads to the sensitization of C-fiber terminals, i.e. renders them more sensitive to other activating stimuli. My second aim focuses on the mechanism. I hypothesize that this interaction involves the galactin-3 fold in the spike protein, and occurs independently of the spike protein receptor ACE2 or toll-like receptors. Irrespective of the proximal binding target, I will address our hypothesis that activation is secondary to the opening of TRPV1 and or TRPA1 channels. These aims will be addressed using single cell RT-PCR analysis of mRNA expression in airway specific nociceptive C-fibers, extracellular and patch-clamp electrophysiology, and 2-photon live imaging techniques. The results of the studies are expected to provide insights into a novel mechanism of coronavirus induced airway C-fiber activation.