Mechanism and Inhibition of SARS-CoV-2 Entry

这项研究的长期目标是了解引起2019年冠状病毒病(新冠肺炎)的严重急性呼吸综合征冠状病毒2(SARS-CoV-2)是如何进入细胞的，以及如何通过使用疗法来阻止这一过程。与其他包膜病毒一样，SARS-CoV-2细胞进入细胞始于细胞表面的接触，并在膜融合后释放病毒内容物时完成。在进入细胞的过程中，SARS-CoV-2刺突蛋白(S)与细胞受体血管紧张素转换酶(ACE2)结合。S的蛋白水解性激活是激活融合机制所必需的，这可以通过细胞表面或内体蛋白酶定位细胞表面和内体进入途径的模型来实现，该途径依赖于不同细胞类型的不同宿主细胞分子的参与。为了询问SARS-CoV-2的进入途径，我们开发了一套独特的工具，允许应用单一病毒粒子成像方法以无偏见的方式跟踪生产性进入途径，并帮助识别病毒进入过程中的宿主因素。通过使用嵌合水疱性口炎病毒(VSV)，其糖蛋白基因(G)被SARS-CoV-2的刺突(S)基因取代，这种成像变得容易。用单抗、可溶性受体和小分子抑制剂抑制SARS-CoV-2感染与抑制SARS-CoV-2临床分离株密切相关，证实嵌合体是研究SARS-CoV-2 S介导的侵袭的有效的BSL2替代物。这允许我们对VSV核糖核蛋白核心的核心蛋白进行遗传修饰，使颗粒在荧光显微镜下可见。通过将这种成像方法与遗传、化学和生物扰动相结合，我们将绘制VSV-SARS-CoV-2的进入路线，然后检查这些扰动对SARS-CoV-2临床分离株细胞感染的影响。我们将使用这种方法来确定目前正在进行临床试验的对策，包括单抗、可溶性血管紧张素转换酶2和两种小分子抑制剂阿皮利莫特和那法莫特如何阻止进入。使用全基因组功能丧失筛查，我们还将询问SARS-CoV-2在自然和扰动条件下进入的要求，以发现新的宿主蛋白，这些宿主蛋白在进入过程中被增选为潜在的治疗干预目标。这项工作的成功完成将确定导致产生SARS-CoV-2感染的进入途径，告知临床发展中的多个分子干扰这一过程的机制，并发现在进入途径中被增选的新宿主因素。

The long term goal of this study is to understand how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of coronavirus disease 2019 (COVID-19), enters cells and how to block that process through the use of therapeutics. Like other enveloped viruses, SARS-CoV-2 cell entry begins with engagement at the cell-surface and is completed on release of the viral contents following membrane fusion. During the process of cell entry, the SARS-CoV-2 spike protein (S) engages the cellular receptor, angiotensin converting enzyme (ACE2). Proteolytic activation of S is required to activate the fusion machinery which can be achieved by cell surface or endosomal proteases positing a model of cell surface and endosomal entry routes that depend on engagement of different host-cell molecules that vary among cell types. To interrogate the entry pathway of SARS-CoV-2 we developed a set of unique tools that permit application of single virion imaging approaches to track productive entry routes in an unbiased way and to help identify host factors coopted during viral entry. This imaging is facilitated by the use of a chimeric vesicular stomatitis virus (VSV) in which its glycoprotein gene (G) was replaced with the spike (S) gene of SARS-CoV-2. Inhibition of VSV-SARS-CoV-2 infection with monoclonal antibodies, soluble receptor and small molecule inhibitors correlates closely with inhibition of a clinical isolate of SARS-CoV-2, corroborating that the chimera is an effective BSL2 surrogate to study SARS-CoV-2 S-mediated entry. This permits us to genetically modify a core protein of the VSV ribonucleoprotein core to render the particles visible by fluorescent microscopy. By combining this imaging approach, with genetic, chemical and biological perturbations, we will map the entry routes of VSV-SARS-CoV-2 and then examine the effect of those perturbations on infection of cells with a clinical isolate of SARS-CoV-2. We will use this approach to determine how countermeasures currently in clinical trials including monoclonal antibodies, soluble ACE2, and two small molecule inhibitors apilimod and nafamostat block entry. Using genome-wide loss-of-function screens we will also interrogate the requirements for entry of SARS-CoV-2, under native and perturbed conditions to uncover new host proteins that are coopted during entry as potential additional targets for therapeutic intervention. Successful completion of this work will define the entry pathways that lead to productive SARS-CoV-2 infection, inform the mechanism by which multiple molecules in clinical development interfere with that process and unearth new host factors that are coopted during the entry pathway.