Extended Data Fig. 8: Generation and characterization of VBDs used for CVRs customized for various coronaviruses.
a, Workflow demonstrating the customization of nanobody-based CVRs for specific coronaviruses. b, Coronavirus CTD or S1 binding in HEK293T cells transiently expressing the corresponding CVRs. Dashed lines indicate thresholds for positive ratio calculation. Data are presented as mean ± s.d. (n = 3 wells of biologically independent cells), representative of two independent experiments. c, The pan-sarbecovirus entry-promoting ability of CVR-Nb27 was evaluated by six different sarbecoviruses in 293T cells. Data are presented as mean ± s.d. (biological quadruples of infected cells), representative of two independent infection assays. Unpaired two-tailed Student’s t-tests. d, BLI analyses of binding kinetics of immobilized RBD and S1-hFc (for 229E) of the indicated coronaviruses with the indicated monomeric nanobodies. e, a summary of binding kinetics of nanobodies bound to the immobilized virus antigens. f, g, Expression (f) and entry-supporting efficiency (g) of the CVRs with or without EPM transiently expressed in the HEK293T cells. EPM: endocytosis prevention motif. Data are mean ± s.d. (n = 3 independently infected cells). Unpaired two-tailed Student’s t-tests. Experiments were performed twice with similar results, and representative data were shown. h-k, HKU1 specific 2D1-CVR exhibited a comparable receptor function to TMPRSS2. The expression (h) and HKU1-PSV entry promoting efficiency (h, i) of the two receptors were examined. The amplification of propagation-competent rVSV-HKU1-GFP in Caco2 cells expressing 2D1-CVR and TMPRSS2 was demonstrated by GFP (j) and RNA accumulation (k). Scale bars: 100 μm for f, h, and j. Data are mean ± s.d. (n = 3 biologically independent wells of cells) analyzed by unpaired two-tailed Student’s t-tests for i. Data presented are RNA copies of two independently infected cells with each point representing the mean of technical duplicates (RT-qPCR) for k. Experiments presented were independently performed twice with similar results for h-j and single time for k. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; NS, Not significant (P > 0.05).
