Receptor recognition and cell entry of coronaviruses

受体识别和病毒进入细胞是病毒感染周期的两个初始和基本步骤。它们是病毒宿主范围、组织嗜性和发病机制的重要决定因素，并且是人类干预的主要靶标。冠状病毒（CoV）对人类和其他动物构成严重的健康威胁。SARS-CoV和MERS-CoV已经感染了成千上万的人，具有显著的死亡率，而猪流行性腹泻CoV目前在仔猪中造成约100%的死亡率。病毒表面刺突蛋白通过其S1亚基与宿主受体结合并通过其S2亚基融合病毒和宿主膜来引导CoV进入宿主细胞。来自不同CoV的S1通过其一个或两个结构域（S1-NTD和S1-CTD）识别多种宿主受体，并且S1/S2边界被宿主蛋白酶切割以通过S2激活膜融合。我们之前的研究已经确定了一些CoV S1结构域本身或与其各自受体复合的晶体结构，并显示了蛋白水解如何调节一些CoV的细胞进入。我们的研究对目前关于CoV受体识别，细胞进入和跨物种传播的分子机制的知识做出了重要贡献。在R01的这种竞争性更新中，我们将继续研究CoV如何利用宿主受体和宿主蛋白酶进入细胞。这项建议有三个具体目标。目的1检测CoV S1-NTDs的受体结合。具体来说，我们将调查是否S1-NTDs从不同的冠状病毒属具有相同的结构折叠和进化起源的宿主半乳糖凝集素（半乳糖结合凝集素）。我们还将研究CoV S1-NTDs如何识别糖受体。这些研究将揭示CoV S1-NTDs的进化起源，增强对CoV糖识别的理解，并可能促进未来设计糖类似物和亚单位疫苗来抑制CoV感染。目的2关注CoV S1-CTD的受体结合。具体而言，我们将分析蝙蝠SARS样冠状病毒（SL-CoV）的S1-CTD与人类和其他动物的蛋白受体同源物之间的相互作用，并阐明蝙蝠SL-CoV如何通过其S1-CTD的进化变化传播给人类和其他动物，导致SARS流行。这些研究将为了解蝙蝠SL-CoV的出现潜力以及促进流行监测和控制提供关键信息。目的3研究CoV进入细胞。具体来说，我们将研究什么样的宿主蛋白酶激活CoV进入，以及CoV尖峰中的蛋白酶基序如何进化以调节CoV进入。这些研究将揭示宿主蛋白酶如何调节CoV进入，以满足其对宿主范围，组织嗜性和发病机制的特定需求，并可能有助于未来设计蛋白酶抑制剂来阻断CoV进入。总的来说，该提案研究了CoV的受体识别，细胞进入，跨物种传播和组织嗜性的分子和结构机制，这将导致病毒学的新原理。这项研究对于评估新冠病毒的潜在疾病以及预防、控制和治疗人类和其他动物的冠状病毒感染也很重要。

Receptor recognition and cell entry by viruses are two initial and essential steps in viral infection cycles. They are important determinants of viral host ranges, tissue tropisms and pathogenesis, and are primary targets for human intervention. Coronaviruses (CoVs) pose serious health threats to humans and other animals. SARS-CoV and MERS-CoV have infected thousands of people with significant fatality, whereas porcine epidemic diarrhea CoV is currently causing ~100% fatality in piglets. A virus-surface spike protein guides CoV entry into host cells by binding to its host receptor via its S1 subunit and fusing viral and host membranes via its S2 subunit. S1 from different CoVs recognizes a variety of host receptors through one or both of its domains (S1-NTD and S1-CTD), and the S1/S2 boundary is cleaved by host proteases for activation of membrane fusion by S2. Our previous research has determined a number of crystal structures of CoV S1 domains by themselves or in complex with their respective receptor, and also shown how proteolysis regulates the cell entry of some CoVs. Our research has contributed critically to the current knowledge about the molecular mechanisms for CoV receptor recognition, cell entry, and cross-species transmission. In this competitive renewal of R01, we will continue to investigate how CoVs exploit host receptors and host proteases for cell entry. This proposal has three specific aims. Aim 1 examines receptor binding by CoV S1-NTDs. Specifically, we will investigate whether S1-NTDs from different CoV genera have the same structural fold and evolutionary origin as host galectins (galactose-binding lectins). We will also examine how CoV S1-NTDs recognize sugar receptors. These studies will reveal the evolutionary origins of CoV S1-NTDs, enhance understanding of sugar recognition by CoVs, and may facilitate future design of sugar analogues and subunit vaccines to inhibit CoV infections. Aim 2 focuses on receptor binding by CoV S1-CTDs. Specifically, we will analyze the interactions between the S1-CTDs of bat SARS-like CoVs (SL-CoVs) and the protein receptor homologues from humans and other animals, and elucidate how bat SL-CoVs transmitted to humans and other animals to cause the SARS epidemic through evolutionary changes in their S1-CTDs. These studies will provide critical information for understanding emergence potential of bat SL-CoVs and for facilitating epidemic monitoring and control. Aim 3 investigates cell entry by CoVs. Specifically, we will investigate what host proteases activate CoV entry and how the proteases motifs in CoV spikes have evolved to modulate CoV entry. These studies will reveal how host proteases regulate CoV entry to meet their specific need for host range, tissue tropism and pathogenesis, and may facilitate future design of protease inhibitors to block CoV entry. Overall, this proposal investigates the molecular and structural mechanisms for receptor recognition, cell entry, cross-species transmission, and tissue tropism of CoVs, which will lead to novel principles in virology. This research is also important for evaluating the emerging disease potentials of CoVs and for preventing, controlling and treating CoV infections in humans and other animals.