Fig. 8: R3DC23 does not affect S1 shedding, folding of the HR1 coiled-coil but hampers 6HB formation.

a Transition of S-2P from its open-interface conformation to the prefusion conformation (left) or vice versa (right). The fraction prefusion S−2P as determined by hydrogen-exchange pulse-labeling is plotted as a function of incubation time prior to the deuterium pulse²⁷. b Anti-S1 Western blot analysis of the growth medium (SN) and cell lysates (LYS) of HEK293T cells expressing the spike protein (SC2 D614G S) or not (HEK EV) incubated sequentially with the indicated VHHs/antibodies. The shown western blot is representative for two experiments. The right panel shows the mean ratio (N = 2) of S1 detected in the supernatant to S1 detected in the cell lysate. c Schematic overview of membrane fusion (top) and proposed steps where R3DC23 interferes with membrane fusion (bottom). RBD: green, S1: turquoise, FP: fusion peptide (brown), HR1: heptad repeat 1 (pink), CH: central helix (blue), CD: connector domain (orange), HR2: heptad repeat 2 (red), MPR: membrane proximal region (light gray), TM+cytosolic: transmembrane domain and cytosolic domain (dark gray). Created in BioRender. Saelens, X. (2025) https://BioRender.com/5i3waej. d Binding of SUMO-HR2 that was added to spike-expressing cells before stimulation with VHH72_S56A-Fc to induce S1 shedding in the absence/presence of huR3DC23-Fc (N = 8 samples pooled from three independent experiments, line indicates the mean). e Binding of SUMO-HR2 (left) and control SUMO-HR1 peptides (right) to spike-expressing cells that were on beforehand stimulated with VHH72_S56A-Fc to undergo S1 shedding in the absence or presence of huR3DC23-Fc (N = 9 samples pooled from three independent experiments, line indicates the mean) (*P < 0.05 and ****P < 0.0001). f Binding of respectively HR2(SARS-CoV-2)-foldon (left) and HR2(MERS)-foldon (right) pretreated with either R3DC23 or control nanobody (GBP) to cells displaying the extended intermediate of spikes that have shed S1 (N = 6 samples pooled from two independent experiments; in each condition, line indicates the mean) (*P < 0.05, **P < 0.005, ****P < 0.0001). P-values are shown in gray above graphs. Statistical tests are specified in the Methods section. Source data are provided as a source data file.