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

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.

新冠病毒刺突蛋白（S）是唯一一种负责病毒与宿主细胞结合以及细胞进入所需的膜融合事件的病毒蛋白。除了促进病毒进入所需的融合外，S还能驱动细胞 - 细胞融合，这是在新冠病毒感染患者的肺部观察到的一种致病效应。虽然有几项研究已经调查了病毒颗粒进入所涉及的S蛋白的要求，但对S蛋白稳定性以及参与S蛋白介导的细胞 - 细胞融合的因素的研究仍然有限。我们证明，S蛋白必须在S1/S2边界处被加工才能介导细胞 - 细胞融合，并且S2亚基内潜在切割位点的突变会改变S1/S2边界处的S蛋白加工，从而阻止细胞 - 细胞融合。我们还确定了内部融合肽和细胞质尾部内调节S蛋白介导的细胞 - 细胞融合的氨基酸残基。此外，我们检测了多种哺乳动物细胞系（包括一种与新冠病毒可能的自然宿主物种相关的蝙蝠细胞系）中S蛋白的稳定性和蛋白质切割动力学，并提供证据表明蛋白水解加工改变了S蛋白三聚体的稳定性。因此，这项工作为S蛋白的稳定性、蛋白水解加工以及介导S蛋白介导的细胞 - 细胞融合的因素提供了见解，所有这些都有助于更全面地理解这个备受关注的治疗靶点。