Variations in Cell Surface ACE2 Levels Alter Direct Binding of SARS-CoV-2 Spike Protein and Viral Infectivity: Implications for Measuring Spike Protein Interactions with Animal ACE2 Orthologs.
Variations in Cell Surface ACE2 Levels Alter Direct Binding of SARS-CoV-2 Spike Protein and Viral Infectivity: Implications for Measuring Spike Protein Interactions with Animal ACE2 Orthologs.
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DOI：
10.1128/jvi.00256-22
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发表时间：
2022-09-14
影响因子：
5.4
通讯作者：
Dolan, Brian P.
中科院分区：
文献类型：
--
作者：
Kazemi, Soheila;Lopez-Munoz, Alberto Domingo;Holly, Jaroslav;Jin, Ling;Yewdell, Jonathan W.;Dolan, Brian P.
严重急性呼吸综合征冠状病毒2型（SARS-CoV-2）是2019冠状病毒病（COVID-19）的病原体，是世纪以来最严重的大流行病。当病毒刺突蛋白（S蛋白）与宿主细胞表面受体血管紧张素转换酶2（ACE 2）结合时，病毒获得进入宿主细胞的机会。研究试图通过在哺乳动物细胞中表达ACE 2直系同源物并测量病毒感染或S蛋白结合来理解SARS-CoV-2 S蛋白与脊椎动物ACE 2直系同源物的相互作用。通常，这些细胞仅瞬时表达ACE 2蛋白，并且细胞表面的ACE 2水平不能定量。在这里，我们描述了一种基于细胞的检测，使用稳定转染的细胞表达ACE 2蛋白的双顺反子载体与易于定量的报告蛋白，Thy1.1。我们发现，S蛋白受体结合域（RBD）的结合和SARS-CoV-2假病毒的感染都与细胞表面表达的人ACE 2的量成比例，这可以通过定量Thy1.1的水平来推断。我们还比较了不同的ACE 2直系同源物，这些同源物在稳定转染的细胞中表达，表达相同水平的Thy1.1。当对病毒感染性或RBD结合进行排名时，小鼠ACE 2对S蛋白具有弱至不可检测的亲和力，而人ACE 2具有最高水平的检测，猫ACE 2具有中间表型。稳定转染细胞的产生，其ACE 2水平可以被标准化用于交叉直系同源物比较，使我们能够创建可重复使用的细胞文库，用于测量新出现的SARS-CoV-2变体潜在感染不同动物的能力。SARS-CoV-2是一种人畜共患病毒，是世纪以来最严重的全球大流行病。了解病毒如何感染其他脊椎动物物种对于控制病毒传播和了解病毒的自然历史非常重要。在这里，我们描述了一种方法，以产生细胞稳定表达不同的直系同源物ACE 2，SARS-CoV-2的受体，在人类细胞系的表面。我们发现病毒刺突蛋白受体结合域（RBD）的结合和SARS-CoV-2假病毒对细胞的感染都与细胞表面的ACE 2水平成正比。该方法将允许创建表达相似水平的不同脊椎动物ACE 2直系同源物的稳定转染细胞的文库，其可重复用于鉴定可能易受SARS-CoV-2及其许多变体感染的脊椎动物物种。
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19), the most severe pandemic in a century. The virus gains access to host cells when the viral spike protein (S-protein) binds to the host cell surface receptor angiotensin-converting enzyme 2 (ACE2). Studies have attempted to understand SARS-CoV-2 S-protein interactions with vertebrate orthologs of ACE2 by expressing ACE2 orthologs in mammalian cells and measuring viral infection or S-protein binding. Often, these cells only transiently express ACE2 proteins, and the levels of ACE2 at the cell surface are not quantified. Here, we describe a cell-based assay that uses stably transfected cells expressing ACE2 proteins in a bicistronic vector with an easy-to-quantify reporter protein, Thy1.1. We found that both the binding of the S-protein receptor-binding domain (RBD) and infection with a SARS-CoV-2 pseudovirus are proportional to the amount of human ACE2 expressed at the cell surface, which can be inferred by quantifying the level of Thy1.1. We also compared different ACE2 orthologs, which were expressed in stably transfected cells expressing equivalent levels of Thy1.1. When ranked for either viral infectivity or RBD binding, mouse ACE2 had a weak to undetectable affinity for S-protein, while human ACE2 had the highest level detected, and feline ACE2 had an intermediate phenotype. The generation of stably transfected cells whose ACE2 level can be normalized for cross-ortholog comparisons allows us to create a reusable cellular library useful for measuring emerging SARS-CoV-2 variants’ abilities to potentially infect different animals. IMPORTANCE SARS-CoV-2 is a zoonotic virus responsible for the worst global pandemic in a century. An understanding of how the virus can infect other vertebrate species is important for controlling viral spread and understanding the natural history of the virus. Here, we describe a method to generate cells stably expressing different orthologs of ACE2, the receptor for SARS-CoV-2, on the surface of a human cell line. We find that both the binding of the viral spike protein receptor-binding domain (RBD) and infection of cells with a SARS-CoV-2 pseudovirus are proportional to the ACE2 levels at the cell surface. This method will allow the creation of a library of stably transfected cells expressing similar levels of different vertebrate ACE2 orthologs, which can be used repeatedly for identifying vertebrate species that may be susceptible to infection with SARS-CoV-2 and its many variants.
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