A genome-wide CRISPR screen identifies host factors that regulate SARS-CoV-2 entry

Yangyong Zhu ,Gaowei Hu,Hongjun Chen,Qiliang Cai,Wendong Han,Xiujun Cai ,Yuyan Wang,Zhiping Sun,Zhenghong Yuan,Yan Yin ,Xinxin Huang,Youhua Xie,Qiang Ding,Fan Feng ,Yuanfei Zhu,Wei Xü ,Di Qu,Rong Ye,Rong Zhang

doi:10.1038/s41467-021-21213-4

Abstract

The global spread of SARS-CoV-2 is posing major public health challenges. One feature of SARS-CoV-2 spike protein is the insertion of multi-basic residues at the S1/S2 subunit cleavage site. Here, we find 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 utilizes an endosomal entry pathway. Using Sdel as model, we perform a genome-wide CRISPR screen and identify several endosomal entry-specific regulators. Experimental validation of hits from the CRISPR screen shows that host factors regulating the surface expression of angiotensin-converting enzyme 2 (ACE2) affect entry of Sfull virus. Animal-to-animal transmission with the Sdel virus is reduced compared to Sfull in the hamster model. These findings highlight the critical role of the S1/S2 boundary of SARS-CoV-2 spike protein in modulating virus entry and transmission and provide insights into entry of coronaviruses.

Highlights

The global spread of SARS-CoV-2 is posing major public health challenges
In the culture of A549 lung epithelial cells, we demonstrated that the deletion at the S1/S2 boundary of spike protein of SARS-CoV-2 resulted in a switch from the plasma membrane to endosomal fusion pathway for entry
In Vero cells expressing no or minimal TMPRSS2, Sfull virus enters via endosomal pathway, making the multi-basic residues dispensable, which results in its deletion, presumably due to an adaptive advantage

Summary

Introduction

The global spread of SARS-CoV-2 is posing major public health challenges. One feature of SARS-CoV-2 spike protein is the insertion of multi-basic residues at the S1/S2 subunit cleavage site. We find that the virus with intact spike (Sfull) preferentially enters cells via fusion at the plasma membrane, whereas a clone (Sdel) with deletion disrupting the multibasic S1/S2 site utilizes an endosomal entry pathway. Animal-to-animal transmission with the Sdel virus is reduced compared to Sfull in the hamster model These findings highlight the critical role of the S1/S2 boundary of SARS-CoV2 spike protein in modulating virus entry and transmission and provide insights into entry of coronaviruses. We isolated a clone of SARS-CoV-2 that has the deletion disrupting the multi-basic residues at the S1/ S2 cleavage site in spike protein This virus preferentially utilized the endosomal entry pathway in A549 cells expressing the receptor ACE2, which provide ideal virus and cell models to dissect the entry. We determined the impact of endosomal entry-specific virus on the pathogenesis and transmission in a hamster model

Methods

Results

Conclusion