Severe acute respiratory syndrome coronavirus (SARS-CoV), SARS-CoV-2, and human coronavirus (hCoV)-NL63 utilize ACE2 as the functional receptor for cell entry, which leads to zoonotic infection. Horses (Equus caballus) attracted our attention because the spike protein receptor-binding domains (RBDs) of SARS-CoV-2 and SARS-CoV-2-related coronaviruses bind equine ACE2 (eACE2) with high affinity. Here we show that eACE2 binds the RBDs of these three coronaviruses and also SARS-CoV-2 variants but with lower affinities compared with human ACE2 (hACE2). Structural analysis and mutation assays indicated that eACE2-H41 accounts for the lower binding affinity of eACE2 to the RBDs of SARS-CoV-2 variants (Alpha, Beta, and Gamma), SARS-CoV, and hCoV-NL63. Pseudovirus infection assays showed that the SARS-CoV-2 Delta strain (B.1.617.2) displayed a significantly increased infection efficiency in eACE2-expressing HeLa cells. Our results reveal the molecular basis of eACE2 binding to the RBDs of SARS-CoV, SARS-CoV-2, and hCoV-NL63, which provides insights into the potential animal transmission of these ACE2-dependent coronaviruses.

严重急性呼吸系统综合症冠状病毒 (SARS-CoV)、SARS-CoV-2 和人类冠状病毒 (hCoV)-NL63 利用 ACE2 作为细胞进入的功能性受体，从而导致人畜共患感染。马 ( Equus caballus) 引起了我们的注意，因为 SARS-CoV-2 和 SARS-CoV-2 相关冠状病毒的刺突蛋白受体结合域 (RBD) 以高亲和力结合马 ACE2 (eACE2)。在这里，我们显示 eACE2 结合这三种冠状病毒和 SARS-CoV-2 变体的 RBD，但与人类 ACE2 (hACE2) 相比亲和力较低。结构分析和突变分析表明，eACE2-H41 是 eACE2 与 SARS-CoV-2 变体（Alpha、Beta 和 Gamma）、SARS-CoV 和 hCoV-NL63 的 RBD 结合亲和力较低的原因。假病毒感染测定表明，SARS-CoV-2 Delta 菌株 (B.1.617.2) 在表达 eACE2 的 HeLa 细胞中表现出显着提高的感染效率。我们的结果揭示了 eACE2 与 SARS-CoV、SARS-CoV-2 和 hCoV-NL63 的 RBD 结合的分子基础，