Fig. 4
Interpretation of N235/MN235-mediated neutralization mechanism. a The overall features of N235 bound to the Omicron sub-variant BA.1 S trimer. The NTD/N235 complex was superimposed onto the S trimer (PDB code: 7wz1), which contains one “up” RBD and two “down” RBDs. The S monomer that clashed with N235 is colored in slate. b Comparison of mean fluorescence intensity (MFI). The GFP-fused S proteins were transiently expressed on the surface of BHK-21 cells and stained with His-tagged N235 or CV3-13 Fab in 10, 30, and 100 μg/mL, respectively. The complex proteins were then incubated with RBD-targeting antibody CB6, human ACE2 (hACE2), S2-targeting antibody 76E1, and NTD-targeting antibody 4A8. Experiments were performed twice, and one representative was displayed. c Comparisons of MFI. The GFP-fused S proteins were transiently expressed on the surface of BHK-21 cells and stained with RBD-targeting antibody CB6, S2-targeting antibody 76E1, and NTD-targeting antibody 4A8 before incubation with His-tagged N235 or CV3-13 Fab in 10, 30, and 100 μg/mL, respectively. The vertical axis represents the level of S1 shedding, calculated by dividing the MFI of cells with surface-expressed S treated with CB6, hACE2, 4A8 and 76E1 by the MFI of the 76E1-treated group. Cells were gated based on the FSC-A and SSC-A (P1) as shown in Supplementary Fig. 11c. d Western blot analysis of S1 subunits. The S proteins were transiently expressed on the surface of 293T cells and incubated with His-tagged N235 or CV3-13 Fab in 1, 10, and 100 μg/mL, respectively. PBS was negative control. The supernatants were collected after 1 h and detected via Western blot assay using an anti-SARS-CoV-2 S1 polyclonal antibody. Experiments were performed twice, and one representative was displayed. e Possible mechanism of N235-mediated neutralization: N235 binding triggers shedding off S1 subunits from S trimers, rending viruses non-infectious
