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

Abstract

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.