Four experiments were performed to detect the binding between SARS-CoV-2 RBD and hACE2 receptor. First, we tested if stably transfected hACE2/293T cells expressed hACE2 by flow cytometry analysis. Since 293T cells alone did not express either hACE2 or hDPP4, they could not be recognized by anti-hACE2 or anti-hDPP4 antibodies (Fig. 2a (left panel)). Only hACE2/293T cells, but not hDPP4/293T cells, expressed hACE2, which was recognized by an anti-hACE2 antibody (Fig. 2a (middle panel)), whereas only hDPP4/293T cells, but not hACE2/293T cells, expressed hDPP4 and was, correspondingly, recognized by an anti-hDPP4 antibody (Fig. 2a (right panel)). These data confirmed the expression of hACE2 in hACE2/293T cells and the expression of hDPP4 in hDPP4/293T cells. Second, we used these hACE2/293T cells to detect the binding of SARS-CoV-2 RBD protein to cell-associated hACE2 by flow cytometry analysis and immunofluorescence staining. Similar to SARS-CoV RBD, SARS-CoV-2 RBD bound to hACE2/293T cells expressing hACE2 (Fig. 2b (left and middle panels)), but not to hDPP4/293T cells expressing hDPP4 (Fig. 2c (left and middle panels)). Furthermore, the binding between SARS-CoV-2 RBD and hACE2-expressing 293T cells was much stronger than the binding between SARS-CoV RBD and hACE2-expressing 293T cells (Fig. 2b (left and middle panels)). MERS-CoV RBD did not bind to hACE2-expressing 293T cells (Fig. 2b (right panel)), but rather bound to hDPP4-expressing 293T cells (Fig. 2c (right panel)). The results from immunofluorescence staining revealed positive signals for both hACE2 and hFc on hACE2/293T cells treated with SARS-CoV-2 RBD and SARS-CoV RBD, both of which contained a C-terminal hFc tag, whereas hACE2/293T cells treated with MERS-CoV RBD (containing a C-terminal hFc tag) showed positive signals for hACE2, but not for hFc, indicating that there is no binding of MERS-CoV RBD to the hACE2-expressing cells (Fig. 2d). These data suggest that SARS-CoV-2 RBD and SARS-CoV RBD can bind to cell-associated hACE2, but not to hDPP4. Third, we detected the binding of SARS-CoV-2 RBD to soluble hACE2 protein (sACE2) by ELISA. The results indicated that SARS-CoV-2 RBD bound to sACE2 in a dose-dependent manner and that the binding between SARS-CoV-2 RBD and sACE2 with 50% effective dose (EC50) of 1.07 μg/ml was stronger than that between SARS-CoV RBD and sACE2 (EC50: 1.66 μg/ml). In contrast, MERS-CoV RBD did not bind to sACE2 (Fig. 2e). While neither SARS-CoV-2 RBD nor SARS-CoV RBD bound to sDPP4, MERS-CoV RBD strongly bound to sDPP4 (EC50: 0.92 μg/ml) (Fig. 2f). These data suggest that both SARS-CoV-2 RBD and SARS-CoV RBD could bind to hACE2 in solution, but not to hDPP4 in solution. Fourth, flow cytometry analysis further indicated that the binding between SARS-CoV-2 RBD and cell-associated hACE2 receptor could be significantly blocked by sACE2 protein (Fig. 2g, i), but not by sDPP4 protein (Fig. 2h, i). Taken together, the above results confirm that the identified SARS-CoV-2 RBD could bind to both cell-associated and soluble hACE2 proteins.
Fig. 2 Detection of SARS-CoV-2 RBD binding to human ACE2 receptor. a Flow cytometry analysis of receptor expression in stable cell lines. (left panel) 293T cells alone expressed neither human ACE2 (hACE2) receptor (orange line) nor hDPP4 receptor (cyan line); (middle panel) hACE2-expressing 293T (hACE2/293T) cells expressed only hACE2 (orange line), but not hDPP4 (cyan line); (right panel) hDPP4-expressing 293T (hDPP4/293T) cells expressed only hDPP4 (cyan line), but not hACE2 (orange line). Mock-incubated cells (gray shading) were used as control. Representative images and median fluorescence intensity (MFI) ± standard error (s.e.m.) were shown (n = 4). b, c Flow cytometry analysis of SARS-CoV-2 RBD binding to cell-associated hACE2 receptor in hACE2/293T stable cell lines. SARS-CoV-2 RBD protein bound strongly to hACE2/293T cells (b (left panel, red line)), but not to hDPP4/293T cells (c (left panel, violet line)). SARS-CoV RBD protein bound to hACE2/293T cells (b (middle panel, red line)), but not to hDPP4/293T cells (c (middle panel, violet line)). MERS-CoV RBD protein did not bind to hACE2/293T cells (b (right panel, red line)), but rather bound to hDPP4/293T cells (c (right panel, violet line)). Human IgG Fc (hIgG-Fc, hereinafter hFc) protein-incubated cells (blue line) and mock-incubated cells (gray shading) were included as controls (b, c). Representative images and MFI ± s.e.m. were shown (n = 4). d Immunofluorescence detection of SARS-CoV-2 RBD binding to cell-associated hACE2 receptor in hACE2/293T cells. SARS-CoV-2 RBD protein (green) and SARS-CoV RBD protein (green), each of which was fused with a C-terminal hFc, were stained with FITC-labeled goat anti-human IgG antibody (1:500). hACE2 was stained with a goat-anti-hACE2 antibody (5 μg/ml) and Alexa-Fluor 647-labeled anti-goat antibody (red) (1:200). Fc-fused MERS-CoV RBD protein did not bind to hACE2, so only hACE2 (red), but not RBD (green), was detected in hACE2/293T cells. Nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI, blue). Scale bar: 10 μm. Representative images are shown. e Detection of dose-dependent binding of SARS-CoV-2 RBD protein to soluble hACE2 (sACE2) receptor by ELISA. The SARS-CoV-2 RBD binding to soluble hDPP4 (sDPP4) receptor (f), and the binding of both SARS-CoV RBD and MERS-CoV RBD proteins to sACE2 (e), or sDPP4 (f), were tested. Control: hFc protein. Data are presented as mean A450 ± s.e.m. (n = 4). 50% effective dose (EC50) was calculated for the binding between SARS-CoV-2 RBD (black) or SARS-CoV RBD (red) and hACE2 protein (e, sACE2), or the binding between MERS-CoV RBD and hDPP4 protein (sDPP4, green) (f). g–i Flow cytometry analysis of inhibition of SARS-CoV-2 RBD protein binding to hACE2/293T cells by sACE2. Binding of SARS-CoV-2 RBD to hACE2/293T cells (g, h, green line) was blocked by sACE2 (g, black line), but not by sDPP4 (h, red line). hFc protein-incubated cells (blue line) and mock-incubated cells (gray shading) were included as controls (g, h). Representative images are shown. i The blocking ability of sACE2 or sDPP4, as described above, was expressed as MFI ± s.e.m. (n = 4). Low MFI correlates with high blockage. Experiments were repeated twice and yielded similar results