Extended Data Fig. 1: Development and optimization of a modular design strategy for CVR with a type I transmembrane topology.
a, Schematic illustration of the four miniprotein-based CVRs. b, Immunofluorescence analysis of ACE2/CVRs expression and authentic SARS-CoV-2 infection in HEK293T cells stably expressing the receptors. Upper: Receptor expression examined by C-terminal fused 3×Flag tags. Lower: SARS-CoV-2 infection efficiency as indicated by intracellular N proteins at 24 hpi. Data representative of two independent authentic SARS-CoV-2 infection assays with similar results. c, Cartoon illustrating the framework of the CVRs for TM evaluation. d, Immunofluorescence analysis of the expression of the 31 CVRs in HEK293T cells by detecting the C-terminal fused 3×Flag tags. e, SARS-CoV-2 PSV entry efficiency promoted by CVRs carrying different TMs. The detailed information on the TMs is summarized in Supplementary Table 2. Data are presented as mean ± s.d. (n = 3 wells of independently infected cells), representative of two independent experiments with similar results. f, Schematic diagram showing the LCB1-based CVRs with indicated TM or TMC substitutions. g, h, SARS-CoV-2 PSV entry in HEK293T cells transiently expressing the indicated CVRs examined by RLU (g) or GFP (h). Data are presented as mean ± s.d. (n = 3 wells of independently infected cells), Statistical analysis was performed using One-way ANOVA analysis followed by Dunnett’s test. Data represented were performed in at least two independent experiments with similar results. i, Immunofluorescence analysis of the subcellular distribution of LCB1-MXRA8 TMC-based CVRs with or without EPM transiently expressed in HEK293T cells. The white dashed boxes highlight the cell surface distribution with a higher magnification. j, SARS-CoV-2 PSV entry efficiency in HEK293T cells transiently expressing CVRs with or without EPM. Data are mean ± s.d. (n = 3 wells of independently infected cells) and analyzed with unpaired two-tailed Student’s t-tests, representative of two independent experiments with similar results. Scale bars: 100 μm in b, d, h, and i. *P < 0.05, **P < 0.01, ****P < 0.0001.
