Fig. 4: VHH R3DC23 binds the spike protein at a membrane proximal site in the HR2 region.

a Complete SARS-CoV-2 spike trimer model (6VXX_1_1_1), red and blue sticks: amino acids at which substitutions were observed in the escape viruses; red and blue helices: region corresponding to the two peptides in the spike that were protected from HDX by R3DC23. Helices in black: transmembrane domain. b HR2 amino acid sequence of the indicated viruses. Dots: amino acids that are identical to the SARS-CoV-2 HR2. Red: positions at which substitutions were observed in the escape variants, blue: N-glycosylation site. The sequence in normal font: the part of the HR2 that contains the heptad repeat motif, gray italic: the flexible linker between the HR2 and transmembrane region, red and blue braces: two spike peptides that were protected by R3DC23 as revealed by HDX-MS. c, d Neutralization of replication-competent VSV pseudotyped with SARS-CoV-2 spike viral escape variants by R3DC23 on VeroE6/TMPRSS2 cells (c) and Vero E6 (d) cells. The graphs show the mean ± SEM (N = 4 replicates of the same dilution series) level of normalized GFP MFI. e Binding of R3DC23 to cells expressing the Wuhan (WT), N1192D, L1197P, L1200P, Q1201R, or Q1201K spike variants. f Kinetics of viral replication of replication-competent VSV-GFP reporter viruses pseudotyped with SARS-CoV-2 Wuhan (parental, WT) or the selected escape variants as measured by life cell imaging of infected Vero E6 cells. The graph shows the mean ± SEM (N = 5 wells) GFP⁺ area per well of infected cells at the indicated time points post-infection. g Left: the HR2 coiled-coil structure (PDB: 2FXP) as determined by NMR. Red: substitutions observed in the escape viruses, green: glycans conjugated at N1194 as modeled in 6VXX_1_1_1. Right: top view of part of the HR2 (K1191-K1205) coiled-coil, red: substitutions observed in the escape viruses, blue: N1194 on one protomer. The first GlcNac residues of the N-glycans to N1194 are shown in green⁶⁹. h Binding of R3DC23, GBP (10 μg/mL), or S309 (1 μg/mL) to cells expressing SARS-CoV-2 spike protein variants. Source data are provided as a source data file.