The fusion peptide of SARS-CoV-2 spike protein is functionally important for membrane fusion during virus entry and is part of a broadly neutralizing epitope. However, sequence determinants at the fusion peptide and its adjacent regions for pathogenicity and antigenicity remain elusive. In this study, we perform a series of deep mutational scanning (DMS) experiments on an S2 region spanning the fusion peptide of authentic SARS-CoV-2 in different cell lines and in the presence of broadly neutralizing antibodies. We identify mutations at residue 813 of the spike protein that reduced TMPRSS2-mediated entry with decreased virulence. In addition, we show that an F823Y mutation, present in bat betacoronavirus HKU9 spike protein, confers resistance to broadly neutralizing antibodies. Our findings provide mechanistic insights into SARS-CoV-2 pathogenicity and also highlight a potential challenge in developing broadly protective S2-based coronavirus vaccines.

SARS-CoV-2 刺突蛋白的融合肽对于病毒进入过程中的膜融合具有重要的功能，并且是广泛中和表位的一部分。然而，融合肽及其邻近区域的致病性和抗原性的序列决定因素仍然难以捉摸。在这项研究中，我们在不同细胞系中并在广泛中和抗体存在的情况下，对跨越真实 SARS-CoV-2 融合肽的 S2 区域进行了一系列深度突变扫描 (DMS) 实验。我们鉴定了刺突蛋白残基 813 处的突变，该突变减少了 TMPRSS2 介导的进入并降低了毒力。此外，我们还发现蝙蝠β冠状病毒HKU9刺突蛋白中存在的F823Y突变赋予了对广泛中和抗体的抗性。我们的研究结果提供了对 SARS-CoV-2 致病性的机制见解，也凸显了开发具有广泛保护性的 S2 冠状病毒疫苗的潜在挑战。