Entry and pathogenesis of two human coronaviruses

人类冠状病毒（H-CoV）可引起从自限性细支气管炎到严重急性呼吸综合征（SARS）的呼吸道疾病。该提案旨在比较引起轻度疾病的NL 63-CoV与SARS-CoV，重点是介导这些H-CoV进入宿主细胞的病毒刺突（S）蛋白之间的区别。SARS和NL 63 S蛋白是不同的，但结合相同的宿主细胞受体，血管紧张素转换酶2（ACE 2）。在ACE 2相互作用之后，这两种S蛋白可能以可能与病毒致病性差异相关的方式差异地指导病毒进入和脱壳。目的1解决了这个问题的不同H-CoV的进入途径，并扩展了初步的数据表明，整合素在NL 63 S介导的进入。实验将确定特定的整合素是否被NL 63和其他H-CoV在肺上皮组织培养模型中使用，并且遗传方法将用于鉴定参与H-CoV进入的新的额外辅助受体。 ACE 2作为外肽酶和信号分子来调节肾素-血管紧张素系统和损伤肺的炎症状态。S蛋白可能干扰这些中心ACE 2功能。目的2构建S片段、重组SARS /NL 63 S蛋白和携带完整S蛋白的HCoV样颗粒，作为ACE 2配体。S：将鉴定导致促炎宿主细胞应答的ACE 2相互作用，还将辨别ACE 2相互作用后宿主对病毒进入事件的应答。这些研究涉及S：ACE 2与炎症和肺部病理学的相互作用，包括与PPG组件3的关键合作。 H-CoV进入辅因子和促炎反应的更广泛的生物学相关性需要使用完整的感染性病毒和宿主动物模型。缺乏用于评估NL 63-CoV的稳健的体外和体内模型。在目标3中，我们计划使用过量产生ACE 2和整合素的细胞，选择NL 63-CoV变体在培养物中旺盛生长，期望获得信息S突变体。将评估变体在小鼠中的生长情况，以建立人类疾病模型。我们还将与PPG组分4合作构建重组NL 63/ SARS嵌合病毒，以进一步将S蛋白与病毒生长、辅助受体使用和宿主细胞反应性相关联。

Human coronaviruses (H-CoVs) cause respiratory diseases ranging from self-limiting bronchiolitis to severe acute respiratory syndrome (SARS). This proposal aims to compare NL63-CoV, which causes mild disease, with SARS-CoV, focusing on distinctions between the viral spike (S) proteins that mediate the entry of these H-CoVs into host cells. The SARS and NL63 S proteins are divergent yet bind to the same host cell receptor, angiotensin converting enzyme 2 (ACE2). Subsequent to ACE2 interaction, these two S proteins may differentially direct virus entry and uncoating, in ways that may correlate with virus pathogenicity differences. Aim 1 addresses this question of distinct H-CoV entry pathways and extends preliminary data suggesting that integrins are used during NL63 S-mediated entry. Experiments will determine whether particular integrins are used by NL63 and other H-CoVs in tissue culture models of lung epithelia, and genetic approaches will be used to identify novel additional coreceptors participating in H-CoV entry. ACE2 operates as an ectopeptidase and as a signaling molecule to regulate the renin-angiotensin system and the inflammatory state of the injured lung. S proteins may disturb these central ACE2 functions. Aim 2 experiments will construct S fragments, recombinant SARS / NL63 S proteins, and HCoV-like particles bearing complete S proteins, for use as ACE2 ligands. S:ACE2 interactions leading to proinflammatory host cell responses will be identified, and the host responses to virus entry events operating subsequent to the ACE2 interaction will also be discerned. These studies relating S : ACE2 interactions with inflammation and lung pathology comprise a key collaboration with PPG component 3. The broader biological relevance of H-CoV entry cofactors and proinflammatory responses require the use of complete infectious viruses and host animal models. Robust in vitro and in vivo models to evaluate NL63-CoV are lacking. In Aim 3, we plan to select NL63-CoV variants for vigorous growth in culture, using cells overproducing ACE2 and integrins, with expectations for informative S mutants. Variants will be evaluated for growth in mice to establish models for the human disease. We will also construct recombinant NL63 / SARS chimeric viruses in collaboration with PPG component 4 to further correlate S proteins with virus growth, coreceptor usage and host cell responsiveness.