Fig. 2: The domain B (RBD) of NeoCoV and PDF-2180 S proteins are required for species-specific ACE2 binding.
From: Close relatives of MERS-CoV in bats use ACE2 as their functional receptors
a, Binding of NeoCoV RBD–hFc to bat ACE2 orthologues expressed on the surface of HEK293T cells analysed using a live-cell binding assay. Data are representative of three assays using independent preparations of proteins. Scale bars, 100 μm. b, Flow cytometry analysis of NeoCoV RBD–hFc binding to HEK293T cells expressing the indicated ACE2 orthologues. The ratio of positive cells compared with the vector control is indicated based on the threshold (dashed line). Data are mean values (n = 3 technical replicates), representative of three independent experiments. c, BLI assays analysing binding kinetics between NeoCoV RBD–hFc and PDF-2180-RBD–hFc with selected ACE2 ectodomains. The reported KD,app values correspond to avidities due to the use of dimeric ACE2 constructs. SSG, steady-state affinity determination. Unfitted curves are shown in Supplementary Fig. 4a. d, ELISA assay showing the binding efficiency of NeoCoV and PDF-2180 RBD–hFc proteins to soluble ACE2 ectodomain proteins. Data are representative of two assays using independent preparations of proteins. Data are mean ± s.d. (technical triplicates). OD450, optical density at 450 nm. e, The inhibitory activity of soluble ACE2 against NeoCoV S pseudotyped virus entry in HEK293T-Bat37ACE2 cells. Data are mean ± s.d. n = 3 biologically independent cells. f, Concentration-dependent inhibition of NeoCoV S-mediated entry by soluble Bat37ACE2 in HEK293T-Bat37ACE2 cells. Data points represent biological duplicates. g, Evaluation of the inhibitory effect of NeoCoV, PDF-2180 or MERS-CoV RBD–hFc proteins on NeoCoV S pseudotyped virus entry in HEK293T-Bat37ACE2 cells. Data are mean ± s.d. (biologically triplicates). h, Entry of the MERS-CoV S, NeoCoV S or NeoCoV S chimera containing the MERS-CoV RBD (residues 371–618) pseudotyped viruses into HEK293T cells stably expressing one of the indicated receptors. Data are mean ± s.d. n = 3 independently infected cells. For e–h, data are representative of two independent infection assays. Statistical analysis was performed using two-tailed unpaired Student’s t-tests (e, g and h); *P < 0.05, ***P < 0.005; NS, not significant.
