The S1/S2 boundary of SARS-CoV-2 spike protein modulates cell entry pathways and transmission

Zhu, Yunkai; Feng, Fei; Hu, Gaowei; Wang, Yuyan; Yu, Yin; Zhu, Yuanfei; Xu, Wei; Cai, Xia; Sun, Zhiping; Han, Wendong; Ye, Rong; Chen, Hongjun; Ding, Qiang; Cai, Qiliang; Qu, Di; Xie, Youhua; Yuan, Zhenghong; Zhang, Rong

The global spread of SARS-CoV-2 is posing major public health challenges. One unique feature of SARS-CoV-2 spike protein is the insertion of multi-basic residues at the S1/S2 subunit cleavage site, the function of which remains uncertain. We found that the virus with intact spike (Sfull) preferentially enters cells via fusion at the plasma membrane, whereas a clone (Sdel) with deletion disrupting the multi-basic S1/S2 site instead utilizes a less efficient endosomal entry pathway. This idea was supported by the identification of a suite of endosomal entry factors specific to Sdel virus by a genome-wide CRISPR-Cas9 screen. A panel of host factors regulating the surface expression of ACE2 was identified for both viruses. Using a hamster model, animal-to-animal transmission with the Sdel virus was almost completely abrogated, unlike with Sfull. These findings highlight the critical role of the S1/S2 boundary of the SARS-CoV-2 spike protein in modulating virus entry and transmission.