Extended Data Fig. 2: Exploring factors that contribute to the receptor function of CVRs with different topologies or modules.
a, Schematic diagram showing CVRs carrying LCB1 or mNb1 displayed in either type I or type II transmembrane topology. b, Evaluation of SARS-CoV-2 or MERS-CoV PSV entry efficiency supported by the indicated CVRs with different transmembrane topologies in HEK293T cells. Data are mean ± s.d. of biological triplicates examined over three independent infection assays. Unpaired two-tailed Student’s t-tests. c, Assessment of CVR expression, SARS-CoV-2 RBD-mFc binding, and PSV entry efficiency supported by the CVRs carrying varying copies of TR23 repeats transiently expressed in HEK293T cells. Data are representative of three independent experiments. Scale bars: 100 μm. d, Schematic representation of the CVRs carrying different numbers of immunoglobulin (Ig) domains (left) or an Fc mutant with abolished dimerization ability. e, Western blot analysis of CVRs expression in HEK293T cells under either reducing or non-reducing conditions, respectively. f, Assessment of SARS-CoV-2 PSV entry efficiency in HEK293T cells transiently expressing the indicated CVRs. Data are mean ± s.d. (n = 3 wells of independently infected cells.) and analyzed by unpaired two-tailed Student’s t-tests. g, Schematic representation of the CVRs carrying different numbers of Ig-like domains (left) from mCEACAM1a. h, Western blot analysis of CVRs expression in HEK293T cells. i, SARS-CoV-2 PSV entry efficiency in HEK293T cells transiently expressing the indicated CVRs. Data are mean ± s.d. (n = 3 wells of independently infected cells). One-way ANOVA analysis followed by Dunnett’s test. j, Schematic representation of the CVRs carrying different SARS-CoV-2 RBD targeting miniproteins. k, l, Expression (k) and SARS-CoV-2 entry-supporting (l) ability of different CVRs in 293T cells. Data are mean ± s.d. (n = 3 wells of independently infected cells). One-way ANOVA analysis followed by Dunnett’s test. m, Schematic diagram showing CVRs carrying different types of VBDs, the two representative VBDs for each type are indicated. n, Immunofluorescence analyzing the expression of the indicated CVRs transiently expressed in HEK293T cells by detecting the C-terminal fused 3×Flag tags, and the SARS-CoV-2 RBD binding. Scale bars: 100 μm. o, PSV entry are supported by indicated CVRs transiently expressed in HEK293T cells. Data are mean ± s.d. (n = 3 wells of independently infected cells). Data representative of two independent transfections, expression verification, and infection assays with similar results for d-f, g-i, j-l, and m-o, respectively. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; NS, Not significant (P > 0.05).
