Effect of clinical isolate or cleavage site mutations in the SARS-CoV-2 spike protein on protein stability, cleavage, and cell–cell fusion

Chelsea T. Barrett, Hadley E. Neal, Kearstin Edmonds, Carole L. Moncman, Rachel Thompson, Jean M. Branttie, Kerri Beth Boggs, Cheng Yu Wu, Daisy W. Leung, Rebecca E. Dutch

Research output: Contribution to journalArticlepeer-review

15 Scopus citations


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.

Original languageEnglish
Article number100902
JournalJournal of Biological Chemistry
Issue number1
StatePublished - Jul 1 2021

Bibliographical note

Funding Information:
vided 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. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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