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必要的步骤。它们是病毒宿主范围、组织取向和致病机制的重要决定因素，也是人类干预的主要目标。冠状病毒(CoV)对人类和其他动物的健康构成严重威胁。SARS-CoV和MERS-CoV已经感染了成千上万的人，并造成了显著的死亡，而猪流行性腹泻冠状病毒目前在仔猪中造成了~100%的死亡。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冠状病毒进入宿主细胞。来自不同冠状病毒的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，S1/S2边界被宿主蛋白酶切割，由S2激活膜融合。我们以前的研究已经确定了冠状病毒S1结构域的一些晶体结构，以及它们与各自的受体形成的复合体，并显示了蛋白分解如何调节一些冠状病毒的细胞进入。我们的研究对目前关于冠状病毒受体识别、细胞进入和跨物种传播的分子机制的了解起到了至关重要的作用。在R01的这一竞争性更新中，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。这项提议有三个具体目标。目的1检测冠状病毒S1-NTDS与受体的结合。具体地说，我们将调查来自不同冠状病毒属的S1-NTD是否与宿主Galectins(半乳糖结合凝集素)具有相同的结构折叠和进化起源。我们还将研究冠状病毒S1-NTDS如何识别糖受体。这些研究将揭示冠状病毒S1-NTDS的进化起源，加强对冠状病毒对糖的识别的理解，并可能促进未来糖类似物和亚单位疫苗的设计以抑制冠状病毒感染。目的2研究冠状病毒S1-CTD与受体的结合。具体地说，我们将分析BAT类SARS冠状病毒(SL-CoV)的S1-CTD与人和其他动物的蛋白质受体同源物之间的相互作用，并阐明BAT SL-CoV是如何通过其S1-CTD的进化变化传播给人类和其他动物导致SARS疫情的。这些研究将为了解BAT SL-CoV的出现潜力和促进疫情监测和控制提供关键信息。AIM 3调查CoVS进入单元格的情况。具体地说，我们将研究哪些宿主蛋白酶激活冠状病毒进入，以及冠状病毒尖峰中的蛋白酶基序如何进化来调节冠状病毒进入。这些研究将揭示宿主蛋白酶如何调节冠状病毒进入，以满足其对宿主范围、组织嗜性和致病的特定需要，并可能促进未来设计蛋白酶抑制剂来阻止冠状病毒进入。总之，这项建议研究了冠状病毒受体识别、细胞进入、跨物种传播和组织趋向性的分子和结构机制，这将导致病毒学中的新原理。这项研究对于评估冠状病毒新出现的疾病潜力以及预防、控制和治疗人类和其他动物的冠状病毒感染也是重要的。

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.