SARS-CoV-2 spike engagement of ACE2 primes S2′ site cleavage and fusion initiation
Edited by Stephen Goff, Department of Biochemistry and Molecular Biophysics and Department of Microbiology and Immunology, Columbia University Irving Medical Center, New York, NY; received June 25, 2021; accepted November 15, 2021
Significance
The SARS-CoV-2 spike protein is responsible for host receptor recognition, membrane fusion, and viral infection. Understanding the cellular and inhibiting the molecular mechanisms of spike-driven viral entry is a research priority in curbing the ongoing pandemic and preventing future coronavirus outbreaks. Here, we highlight that the generation of SARS-CoV-2 S2′ fragments, a proteolytic event occurring within the S2 subunit, is a molecular switch coupled to membrane fusion. Downstream of host receptor recognition, spike-driven syncytia formation requires the presence of an S2′ cleavage site at arginine 815 but not 685. Hence, the proteolytic processing of spike at the S2′ site upon its engagement of host ACE2 may serve as a potential antiviral target against the current SARS-CoV-2 and related coronavirus strains.
Abstract
The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has resulted in tremendous loss worldwide. Although viral spike (S) protein binding of angiotensin-converting enzyme 2 (ACE2) has been established, the functional consequences of the initial receptor binding and the stepwise fusion process are not clear. By utilizing a cell–cell fusion system, in complement with a pseudoviral infection model, we found that the spike engagement of ACE2 primed the generation of S2′ fragments in target cells, a key proteolytic event coupled with spike-mediated membrane fusion. Mutagenesis of an S2′ cleavage site at the arginine (R) 815, but not an S2 cleavage site at arginine 685, was sufficient to prevent subsequent syncytia formation and infection in a variety of cell lines and primary cells isolated from human ACE2 knock-in mice. The requirement for S2′ cleavage at the R815 site was also broadly shared by other SARS-CoV-2 spike variants, such as the Alpha, Beta, and Delta variants of concern. Thus, our study highlights an essential role for host receptor engagement and the key residue of spike for proteolytic activation, and uncovers a targetable mechanism for host cell infection by SARS-CoV-2.
Data Availability
All study data are included in the article and/or SI Appendix. Plasmids were deposited at the AddGene with following information: Deposit number: 80522, pVAX1-SARS-CoV-2-S, Plasmid #173049, https://www.addgene.org/173049/; pVAX1-SARS-CoV-2 R685A S, Plasmid #180194, https://www.addgene.org/180194/; pVAX1-SARS-CoV-2 R815A S HA-tag, Plasmid #180195, https://www.addgene.org/180195/; pVAX1-SARS-CoV-2 delRRAR S HA-tag, Plasmid #180196, https://www.addgene.org/180196/.
Acknowledgments
We thank Qiuhong Guo, Mengmeng Cui, Lulu Bai, and Weiqian Dai for their experimental support; we are grateful to Zhong Huang, Xiaozhen Liang, Haikun Wang, and Lanfeng Wang for their key reagents used in this work. This study is supported by grants from the International Postdoctoral Exchange Fellowship (Grant 251371) and the Prevention and Control of COVID-19 Program (Grant 2020T130119ZX) of the Postdoctoral Science Foundation of China, the special scientific research fund for COVID-19 prevention and control from Zhejiang University, the National Key R&D Program of China (Grants 2018YFA0507300 and 2020YFC0845900), the Strategic Priority Research Program (Grant XDB29030303) and the International Partnership Program (Grant 153831KYSB20190008) of the Chinese Academy of Sciences, and the Natural Science Foundation of China (Grants 81830049, 81761128012, and 31870153), as well as by the Shanghai Municipal Science and Technology Major Project (Grants 2019SHZDZX02 and 20431900402) and Research Leader Program (Grant 20XD1403900), and the CAS president's international fellowship initiative (Grant 2020VBA0023).
Supporting Information
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Copyright © 2021 the Author(s). Published by PNAS. This open access article is distributed under Creative Commons Attribution License 4.0 (CC BY).
Data Availability
All study data are included in the article and/or SI Appendix. Plasmids were deposited at the AddGene with following information: Deposit number: 80522, pVAX1-SARS-CoV-2-S, Plasmid #173049, https://www.addgene.org/173049/; pVAX1-SARS-CoV-2 R685A S, Plasmid #180194, https://www.addgene.org/180194/; pVAX1-SARS-CoV-2 R815A S HA-tag, Plasmid #180195, https://www.addgene.org/180195/; pVAX1-SARS-CoV-2 delRRAR S HA-tag, Plasmid #180196, https://www.addgene.org/180196/.
Submission history
Accepted: November 15, 2021
Published online: December 20, 2021
Published in issue: January 5, 2022
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Acknowledgments
We thank Qiuhong Guo, Mengmeng Cui, Lulu Bai, and Weiqian Dai for their experimental support; we are grateful to Zhong Huang, Xiaozhen Liang, Haikun Wang, and Lanfeng Wang for their key reagents used in this work. This study is supported by grants from the International Postdoctoral Exchange Fellowship (Grant 251371) and the Prevention and Control of COVID-19 Program (Grant 2020T130119ZX) of the Postdoctoral Science Foundation of China, the special scientific research fund for COVID-19 prevention and control from Zhejiang University, the National Key R&D Program of China (Grants 2018YFA0507300 and 2020YFC0845900), the Strategic Priority Research Program (Grant XDB29030303) and the International Partnership Program (Grant 153831KYSB20190008) of the Chinese Academy of Sciences, and the Natural Science Foundation of China (Grants 81830049, 81761128012, and 31870153), as well as by the Shanghai Municipal Science and Technology Major Project (Grants 2019SHZDZX02 and 20431900402) and Research Leader Program (Grant 20XD1403900), and the CAS president's international fellowship initiative (Grant 2020VBA0023).
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This article is a PNAS Direct Submission.
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The authors declare no competing interest.
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SARS-CoV-2 spike engagement of ACE2 primes S2′ site cleavage and fusion initiation, Proc. Natl. Acad. Sci. U.S.A.
119 (1) e2111199119,
https://doi.org/10.1073/pnas.2111199119
(2022).
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