Bats carry diverse severe acute respiratory syndrome-related coronaviruses (SARSr-CoVs). The suspected interspecies transmission of SARSr-CoVs from bats to humans has caused two severe CoV pandemics, the SARS pandemic in 2003 and the recent COVID-19 pandemic. The receptor utilization of SARSr-CoV plays the key role in determining the host range and the interspecies transmission ability of the virus. Both SARS-CoV and SARS-CoV-2 use angiotensin-converting enzyme 2 (ACE2) as their receptor. Previous studies showed that WIV1 strain, the first living coronavirus isolated from bat using ACE2 as its receptor, is the prototype of SARS-CoV. The receptor-binding domain (RBD) in the spike protein (S) of SARS-CoV and WIV1 is responsible for ACE2 binding and medicates the viral entry. Comparing to SARS-CoV, WIV1 has three distinct amino acid residues (442, 472, and 487) in its RBD. This study aimed at exploring whether these three residues could alter the receptor utilization of SARSr-CoVs. We replaced the three residues in SARS-CoV (BJ01 strain) S with their counterparts in WIV1 S, and then evaluated the change of their utilization of bat, civet, and human ACE2s using a lentivirus-based pseudovirus infection system. To further validate the S-ACE2 interactions, the binding affinity between the RBDs of these S proteins and the three ACE2s were verified by flow cytometry. The results showed that the single amino acid substitution Y442S in the RBD of BJ01 S enhanced its utilization of bat ACE2 and its binding affinity to bat ACE2. On the contrary, the reverse substitution in WIV1 S (S442Y) significantly attenuated the pseudovirus utilization of bat, civet and human ACE2s for cell entry, and reduced its binding affinity with the three ACE2s. These results suggest that the S442 is critical for WIV1 adapting to bats as its natural hosts. These findings will enhance our understanding of host adaptations and cross-species infections of coronaviruses, contributing to the prediction and prevention of coronavirus epidemics.

蝙蝠携带多种严重急性呼吸系统综合症相关冠状病毒 (SARSr-CoV)。疑似 SARSr-CoV 从蝙蝠到人类的种间传播导致了两次严重的 CoV 大流行，即 2003 年的 SARS 大流行和最近的 COVID-19 大流行。SARSr-CoV的受体利用在决定病毒的宿主范围和种间传播能力方面起着关键作用。SARS-CoV 和 SARS-CoV-2 都使用血管紧张素转换酶 2 (ACE2) 作为它们的受体。此前的研究表明，WIV1株是SARS-CoV的原型，它是第一个从蝙蝠身上分离出来的以ACE2为受体的活冠状病毒。SARS-CoV 和 WIV1 的刺突蛋白 (S) 中的受体结合域 (RBD) 负责 ACE2 结合并控制病毒进入。与 SARS-CoV 相比，WIV1 具有三个不同的氨基酸残基（442，472 和 487）在其 RBD 中。本研究旨在探索这三个残基是否可以改变 SARSr-CoV 的受体利用。我们用 WIV1 S 中的对应物替换了 SARS-CoV（BJ01 株）S 中的三个残基，然后使用基于慢病毒的假病毒感染系统评估了它们对蝙蝠、果子狸和人类 ACE2 的利用变化。为了进一步验证 S-ACE2 相互作用，通过流式细胞仪验证了这些 S 蛋白的 RBD 与三种 ACE2 之间的结合亲和力。结果表明，BJ01 S 的 RBD 中的单个氨基酸取代 Y442S 增强了其对蝙蝠 ACE2 的利用及其与蝙蝠 ACE2 的结合亲和力。相反，WIV1 S (S442Y) 中的反向替换显着减弱了假病毒利用蝙蝠、果子狸和人类 ACE2 进入细胞，并降低其与三种 ACE2 的结合亲和力。这些结果表明，S442 对于 WIV1 适应蝙蝠作为其自然宿主至关重要。这些发现将加强我们对冠状病毒宿主适应和跨物种感染的理解，有助于冠状病毒流行的预测和预防。