冠状病毒(CoV)的经常性溢出对公共卫生和全球经济构成了严重威胁。蝙蝠严重急性呼吸系统综合症(SARS)-类似的CoVsRsSHC014和WIV1,目前在蝙蝠种群中传播,为人类的出现做好了准备。三聚体刺突(S)糖蛋白,负责受体识别和膜融合,在跨物种传播和感染中起着至关重要的作用。这里,我们在封闭状态下确定了RsSHC014S蛋白的低温电子显微镜(EM)结构,处于关闭状态的WIV1S蛋白为2.8µ,和中间状态为4.0µ。在中间状态下,一个受体结合域(RBD)处于“下”位置,而其他两个RBD表现出较差的密度。我们还解决了与人ACE2(hACE2)结合的WIV1S蛋白的复杂结构,这为将来WIV1在人类中的出现提供了结构基础。通过生化实验,我们发现,尽管RBD与人和麝猫ACE2具有很强的结合亲和力,但RsSHC014而非WIV1的假病毒未能感染过表达人或麝猫ACE2的293T细胞.诱变分析表明,Y623H取代,位于SD2地区,显著提高了RsSHC014假病毒的细胞进入效率,这可能是通过促进刺突糖蛋白的开放构象来实现的。我们的发现强调了有效的RBD提升和紧密的RBD-hACE2结合对病毒感染的必要性,并强调了刺突糖蛋白623位点对蝙蝠SARS样CoV的感染性的重要性。
目的:蝙蝠SARS样CoVRsSHC014和WIV1可以使用hACE2进入细胞而无需进一步适应,表明它们在人群中出现的潜在风险。S糖蛋白,负责受体识别和膜融合,在跨物种传播和感染中起着至关重要的作用。在这项研究中,我们确定了RsSHC014和WIV1的S糖蛋白的低温EM结构。详细的比较揭示了刺突蛋白内的动态结构变化。我们还阐明了WIV1S-hACE2的复杂结构,为WIV1在人类中的潜在出现提供了结构证据。尽管RsSHC014和WIV1具有相似的hACE2结合亲和力,它们表现出明显的假病毒细胞进入行为。通过诱变和低温EM分析,我们发现除了结构变化之外,SD2区域的623位点是刺突感染性的另一个重要结构决定因素。
The recurrent spillovers of coronaviruses (CoVs) have posed severe threats to public health and the global economy. Bat severe acute respiratory syndrome (SARS)-like CoVs RsSHC014 and WIV1, currently circulating in bat populations, are poised for human emergence. The trimeric spike (S) glycoprotein, responsible for receptor recognition and membrane fusion, plays a critical role in cross-species transmission and infection. Here, we determined the cryo-electron microscopy (EM) structures of the RsSHC014 S protein in the closed state at 2.9 Å, the WIV1 S protein in the closed state at 2.8 Å, and the intermediate state at 4.0 Å. In the intermediate state, one receptor-binding domain (RBD) is in the \"down\" position, while the other two RBDs exhibit poor density. We also resolved the complex structure of the WIV1 S protein bound to human ACE2 (hACE2) at 4.5 Å, which provides structural basis for the future emergence of WIV1 in humans. Through biochemical experiments, we found that despite strong binding affinities between the RBDs and both human and civet ACE2, the pseudoviruses of RsSHC014, but not WIV1, failed to infect 293T cells overexpressing either human or civet ACE2. Mutagenesis analysis revealed that the Y623H substitution, located in the SD2 region, significantly improved the cell entry efficiency of RsSHC014 pseudoviruses, which is likely accomplished by promoting the open conformation of spike glycoproteins. Our findings emphasize the necessity of both efficient RBD lifting and tight RBD-hACE2 binding for viral infection and underscore the significance of the 623 site of the spike glycoprotein for the infectivity of bat SARS-like CoVs.
OBJECTIVE: The bat SARS-like CoVs RsSHC014 and WIV1 can use hACE2 for cell entry without further adaptation, indicating their potential risk of emergence in human populations. The S glycoprotein, responsible for receptor recognition and membrane fusion, plays a crucial role in cross-species transmission and infection. In this study, we determined the cryo-EM structures of the S glycoproteins of RsSHC014 and WIV1. Detailed comparisons revealed dynamic structural variations within spike proteins. We also elucidated the complex structure of WIV1 S-hACE2, providing structural evidence for the potential emergence of WIV1 in humans. Although RsSHC014 and WIV1 had similar hACE2-binding affinities, they exhibited distinct pseudovirus cell entry behavior. Through mutagenesis and cryo-EM analysis, we revealed that besides the structural variations, the 623 site in the SD2 region is another important structural determinant of spike infectivity.