Functional and antigenic characterization of SARS-CoV-2 spike fusion peptide by deep mutational scanning

Abstract

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.

Fig. 1. Deep mutational scanning of SARS-CoV-2 bFP and FPPR.

The fitness values of individual mutations at residues 808–855 of SARS-CoV-2 S were measured by deep mutational scanning in A Calu-3 cells and B Vero cells and are shown as heatmaps. Wild-type (WT) amino acids are indicated by black circles. “_” indicates nonsense mutations. Mutations in gray were excluded from our data analysis due to low frequency in the plasmid mutant library. Red indicates superior fitness, white is similar to WT, and blue has reduced fitness.

Fig. 2. Mutations at residue 813 influence the protease utilization during cell entry.

A Vero cell entry of VSVpps bearing various SARS-CoV-2 S constructs was measured by the relative light unit (RLU) in a luciferase assay. B Vero-TMPRSS2 cell entry of VSVpps bearing various SARS-CoV-2 S constructs. Each bar represents the mean of four biological replicates. Each data point represents one biological replicate. Deviations from the WT were analyzed by two-tailed t-tests. “ns” indicates no significance (i.e., p-value > 0.05). C, D The effects of C E64D (cathepsin inhibitor) or D camostat (TMPRSS2 inhibitor) on Vero cell entry of VSVpps bearing various SARS-CoV-2 S constructs are shown. Curves depicted in C are significantly different (p = 0.0088, two-way ANOVA). The mean and standard error of the mean (SEM) of four independent biological replicates are depicted.

Fig. 3. S813V mutation reduces virulence in vivo.

A, B Vero, Vero-TMPRSS2, and Vero-TMPRSS2/ACE2 cells were separately infected with WT, S813V, or S813K viruses from the same aliquot for each virus. The numbers of plaques obtained from A Vero-TMPRSS2 cells or B Vero-TMPRSS2/ACE2 cells were normalized to those obtained from Vero cells. Bar represents the mean of seven biological replicates. Each data point represents one biological replicate. P-values were computed by two-tailed t-tests. C Vero cells or D Vero-TMPRSS2 cells were infected with WT, S813V, or S813K mutants at a multiplicity of infection of 0.01. Virus titers were determined for each variant at the indicated time point. Each data point represents the geometric mean of three biological replicates, and the error bar represents the geometric standard deviation (SD). Representative data from two independent experiments are shown. Deviations from the WT were analyzed by two-tailed t-tests. E, F Percentage of initial weight change of C57BL/6 mice infected with E 1000 PFU or F 5000 PFU of WT, S813V, or S813K mutants. Data points in the weight curve represent the mean, and error bars represent the SEM. Deviations from the WT were analyzed by two-tailed t-tests. “*” indicates p-value < 0.05. p = 0.0128, 0.00738, 0.0139, 0.0360 for WT vs. S813V at 2, 3, 4, and 10 dpi, respectively in (E); p = 0.0291, 0.0291, 0.0216 for WT vs. S813K at 2, 3, and 4 dpi respectively in (E). n = 8, 5, and 9 for WT, S813K, and S813V, respectively in (E); p = 0.0203, 0.00358, 0.00168, 0.0208 for WT vs. S813K at 2, 4, 5 and 6 dpi respectively in (F). n = 8, 10 and 9 for WT, S813K and S813V respectively in (F). G Kaplan–Meier survival curves are shown for C57BL/6 mice infected with 5000 PFU of S813V or S813K mutants. “ns” indicates not significant (i.e., p-value > 0.05). H, I Virus titers in the lungs of mice infected with 5000 PFU of WT, S813V, or S813K mutants were measured at the indicated time point on H Vero cells and I Vero-TMPRSS2/ACE2 cells. Statistical significance was determined by two-tailed t-tests. Bars represent geometric means. dpi days post-infection.

Fig. 4. Structural analysis of the mutational tolerance of SARS-CoV-2 bFP and FPPR.

A Mutational tolerance of each residue in Calu-3 cells is shown on the NMR structure of the bFP and FPPR (PDB 7MY8). A disulfide bond (yellow in panel B) is present in the FPPR between Cys840 and Cys851. B The mutational tolerance of each residue in Calu-3 cells is shown. The locations of helices 1–3 in the NMR structure of the bFP and FPPR (PDB 7MY8) are indicated. The side chains of Leu828, Cys840, Asp848, and Cys851 are shown in stick representation.

Fig. 5. F823Y weakens the binding of bFP antibodies.

A, B Relative resistance for each mutation against A 230 μg/mL COV44-62 or B 330 μg/mL COV44-79 in Vero cells is shown as heatmaps. Relative resistance for WT is set as 0. Mutations with a fitness value of less than 0.75 in the absence of antibodies are shown as gray. Amino acids corresponding to the WT sequence are indicated by the black dots. “_” indicates nonsense mutations. C, D The neutralization activities of C COV44-62 and D COV44-79 against VSVpp bearing WT or F823Y S are shown. The mean and SEM of three biological replicates are depicted. E, F The structural effects of F823Y on the binding of E COV44-62 (PDB 8D36) and F COV44-79 (PDB 8DAO) were modeled using FoldX.