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

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J Biol Chem


. 2021 Jun 19;100902.
doi: 10.1016/j.jbc.2021.100902. Online ahead of print.
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 ¹ , Hadley E Neal ¹ , Kearstin Edmonds ¹ , Carole L Moncman ¹ , Rachel Thompson ¹ , Jean M Branttie ¹ , Kerri Beth Boggs ¹ , Cheng-Yu Wu ¹ , Daisy W Leung ² , Rebecca E Dutch ³



Affiliations

• PMID: 34157282
• DOI: 10.1016/j.jbc.2021.100902

Abstract

The trimeric 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 TMPRSS2 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. Additionally, 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.

Keywords: COVID-19; Fusion protein; SARS-CoV-2; coronavirus; membrane fusion; viral protein; virology; virus entry.