Effect of mutations in the SARS‐CoV‐2 spike protein on protein stability, cleavage, and cell‐cell fusion function

Neal, Hadley; Barrett, Chelsea; Edmonds, Kearstin; Moncman, Carole; Thompson, Rachel; Branttie, Jean; Boggs, Kerri Beth; Wu, Cheng‐Yu; Leung, Daisy; Dutch, Rebecca

The 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. 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 cell‐cell fusion. Additionally, we examine 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 highly sought‐after therapeutic target.