The trimeric severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein (S) is the sole viral protein responsible for both viral binding to a host cell and the membrane fusion event needed for cell entry. In addition to facilitating fusion needed for viral entry, S can also drive cell-cell fusion, a pathogenic effect observed in the lungs of SARS-CoV-2-infected patients. While several studies have investigated S requirements involved in viral particle entry, examination of S stability and factors involved in S cell-cell fusion remain limited. A furin cleavage site at the border between the S1 and S2 subunits (S1/S2) has been identified, along with putative cathepsin L and transmembrane serine protease 2 cleavage sites within S2. We demonstrate that S must be processed at the S1/S2 border in order to mediate cell-cell fusion and that mutations at potential cleavage sites within the S2 subunit alter S processing at the S1/S2 border, thus preventing cell-cell fusion. We also identify residues within the internal fusion peptide and the cytoplasmic tail that modulate S-mediated cell-cell fusion. In addition, we examined S stability and protein cleavage kinetics in a variety of mammalian cell lines, including a bat cell line related to the likely reservoir species for SARS-CoV-2, and provide evidence that proteolytic processing alters the stability of the S trimer. This work therefore offers insight into S stability, proteolytic processing, and factors that mediate S cell-cell fusion, all of which help give a more comprehensive understanding of this high-profile therapeutic target.

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Published in The Journal of Biological Chemistry, v. 297, issue 1, 100902.

© 2021 The Authors

This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/).

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Funding Information

Financial support was provided by the CCTS CURE Alliance pilot award from the University of Kentucky, National Institute of Allergy and Infectious Diseases, National Institutes of Health grant R01AI051517, and National Institutes of Health grant 2P20 RR02017 to R. E. D.

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The datasets generated during and/or analyzed during the current study are available upon request from the corresponding author, Rebecca Dutch, on reasonable request.

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Supplemental figures S1–S3