Entry and pathogenesis of two human coronaviruses

人类冠状病毒(H-CoV)可引起从自限性毛细支气管炎到严重急性呼吸综合征(SARS)的各种呼吸道疾病。这项建议旨在将导致轻度疾病的NL63-CoV与SARS-CoV进行比较，重点是介导这些H-CoV进入宿主细胞的病毒尖峰(S)蛋白之间的差异。SARS和NL63的S蛋白是不同的，但与相同的宿主细胞受体血管紧张素转换酶2(ACE2)结合。在ACE2相互作用后，这两个S蛋白可能以不同的方式引导病毒进入和脱壳，这可能与病毒致病性的差异有关。目的1解决不同的H冠状病毒进入途径的问题，并扩展初步数据，表明整合素在NL63 S介导的进入过程中被使用。实验将确定NL63和其他H-CoV是否在肺上皮细胞的组织培养模型中使用特定的整合素，并将使用遗传学方法来识别参与H-CoV进入的新的辅助受体。 ACE2作为一种外肽酶和信号分子来调节肾素-血管紧张素系统和受损肺的炎症状态。S蛋白可能干扰这些中枢血管紧张素转换酶2的功能。目的实验2构建S片段、重组SARS/NL63 S蛋白和携带完整S蛋白的人乳头瘤病毒样颗粒，作为血管紧张素转换酶2的配体。S：将识别导致促炎宿主细胞反应的ACE2相互作用，以及在ACE2相互作用之后操作的病毒进入事件的宿主反应。这些与S相关的研究：血管紧张素转换酶2与炎症和肺部病理的相互作用构成了与PPG组件3的关键合作。 H-CoV进入、辅因子和致炎反应之间更广泛的生物学相关性要求使用完整的感染病毒和宿主动物模型。缺乏强大的体外和体内模型来评估NL63-CoV。在目标3中，我们计划选择NL63冠状病毒变异体，利用过量生产血管紧张素和整合素的细胞，在培养中旺盛地生长，并期望获得信息丰富的S突变体。将评估变种在小鼠中的生长情况，以建立人类疾病的模型。我们还将构建与PPG组件4合作的重组NL63/SARS嵌合病毒，以进一步将S蛋白与病毒生长、共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.