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.
【저자키워드】 COVID-19, SARS-CoV-2, coronavirus, Virology, COVID-19, Coronavirus disease 2019, SARS-CoV-2, Severe acute respiratory syndrome coronavirus 2, ACE2, angiotensin-converting enzyme 2, virus entry, membrane fusion, TMPRSS2, Transmembrane serine protease 2, fusion protein, FBS, fetal bovine serum, DMEM, Dulbecco's modified Eagle's medium, MERS, Middle East respiratory syndrome, CoV, coronavirus, Viral protein, cath L, cathepsin L, EV, expression vector, hACE2, human ACE2, hpt, hours post transfection, MEF, mouse embryonic fibroblast, PBSN, PBS + 0.02% sodium azide, RIPA, radioimmunoprecipitation assay, S, spike protein, TTBS, Tris-buffered saline + Tween-20, 【초록키워드】 Mutation, lung, viral entry, Spike protein, stability, furin cleavage site, S2 subunit, binding, Evidence, therapeutic target, host cell, cathepsin L, acute respiratory syndrome, Factor, subunit, residue, cell entry, S1/S2, transmembrane serine protease, help, cleavage site, pathogenic, cell line, cytoplasmic tail, while, offer, SARS-CoV-2–infected patients, mammalian cell, protein cleavage, Proteolytic processing, Alter, S trimer, trimeric, responsible, identify, S1 and S2, examined, involved, addition, investigated, in viral, variety, modulate, processed, internal fusion peptide, 【제목키워드】 Mutation, Spike protein, cleavage, protein stability, cleavage site, Effect, the SARS-CoV-2,