Like SARS-CoV and MERS-CoV, SARS-CoV-2 also originates from bats.22–24 Next, we detected the binding affinity of the identified SARS-CoV-2 RBD to bat ACE2 (bACE2) and compared this binding with that of SARS-CoV RBD. We transiently transfected a bACE2-expressing plasmid into 293T cells and included a hACE2-expressing plasmid as a control, followed by detection of fluorescence intensity 48 h later. Results indicated that SARS-CoV-2 RBD bound strongly to 293T-expressed bACE2 with intensity similar to that of its binding to 293T-expressed hACE2 (Fig. 3a, c), and that this binding occurred in a dose-dependent manner (Fig. 3e, f). In addition, the binding affinity between SARS-CoV-2 RBD and 293T-expressed bACE2 (EC50: 0.08 μg/ml) or hACE2 (EC50: 0.14 μg/ml) was significantly higher than that between SARS-CoV RBD and 293T-expressed bACE2 (EC50: 0.96 μg/ml) or hACE2 (EC50: 1.32 μg/ml) (Fig. 3b, d–f). Nevertheless, MERS-CoV RBD bound neither bACE2- nor hACE2-expressing 293T cells (Fig. 3). These data suggest that SARS-CoV-2 RBD can bind to both bACE2 and hACE2 with significantly stronger binding than that of SARS-CoV RBD to either bACE2 or hACE2, supporting the bat origin of SARS-CoV-2. These results may partially explain why SARS-CoV-2 is more transmissible than SARS-CoV.
Fig. 3 Comparison of SARS-CoV-2 RBD protein binding to human and bat ACE2 receptors. Flow cytometry analysis of SARS-CoV-2 RBD binding to hACE2 and bat ACE2 (bACE2) receptors in 293T cells transiently expressing hACE2 or bACE2. 293T cells were transiently transfected with hACE2 or bACE2 plasmid and incubated with SARS-CoV-2 RBD protein at various concentrations for analysis. SARS-CoV RBD and MERS-CoV RBD proteins were used as controls. Representative images of SARS-CoV-2 RBD protein (2.5 μg/ml) binding to bACE2/293T (a, black line), or hACE2/293T (c, black line), cells were shown. Binding of SARS-CoV RBD protein (2.5 μg/ml) to bACE2/293T (b, red line), or hACE2/293T (d, red line), cells were used as a comparison. MERS-CoV RBD protein (green line) and mock-incubated (gray shading) cells (a–d) were included as controls. e, f Dose-dependent binding of SARS-CoV-2 RBD protein to bACE2/293T (e), or hACE2/293T (f), cells by flow cytometry analysis. Significant differences between binding of SARS-CoV-2 RBD (black) and SARS-CoV RBD (red) to cell-associated bACE2 receptor (e), or hACE2 receptor (f) were identified based on the EC50 values. The data are presented as mean ± s.e.m. (n = 4). Experiments were repeated twice and yielded similar results