Extended Data Fig. 3: Investigating receptor function of CVRs with VBDs connected in various ways.
a-c, Illustration (a), RBD binding efficiency (b), and PSV entry-supporting efficiency (c) of a SARS-CoV-2/MERS-CoV bi-specific CVR transiently expressed in HEK293T cells. Data are presented as mean ± s.d. (biological triplicates of infected cells), representative of three independent experiments with similar results. Unpaired two-tailed Student’s t-test. d-f, Illustration (d), expression (e), and PSV entry-supporting efficiencies (f) of CVRs carrying single or trimeric VBD. Data are mean ± s.d. (biological triplicates of infected cells), analyzed by unpaired two-tailed Student’s t-test. Representative of two independent experiments. g-i, Schematic illustration of bispecific adapter protein (g) and MERS-CoV PSV entry efficiency in BHK-21-hACE2 cells in the presence of indicated concentrations of adapter proteins (h11B11-mNb1) throughout the infection. Entry efficiency is examined by GFP intensity (h) or RLU (i). Data are mean ± s.d. (biological triplicates of infected cells), representative of three independent infection assays. j-l, Schematic illustration of FcγR (CD32a) mediated antibody-dependent coronavirus entry (j). CD32a expression, antibody (CB6) binding (k), and SARS-CoV-2 PSV entry (l) into HEK293T-CD32a cells pretreated with indicated concentration (con.) of the CB6 antibodies. Data are mean ± s.d. (n = 4 wells of biologically independent cells), examined over two independent experiments. m-o, Entry of pre-attached PSV promoted by soluble neutralizing antibody (Nb27-hFc) or bi-specific neutralizing antibody with membrane-associating ability (Nb27-hFc-h11B11). Schematic illustration (m), Nb27-hFc promoted entry (n), and Nb27-hFc-h11B11 promoted PSV entry (o) in Caco2 cells with virus pre-attachment by 1500 rpm centrifugation at 4 °C for 1 h. Data are mean ± s.d. (biological triplicates of infected cells). Data are representative of three independent infection assays. Scale bars: 100 μm for b, e, and k, and 200 μm for h. One-way ANOVA analysis followed by Dunnett’s test for i, l, n, and o. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; NS, Not significant (P > 0.05).
