FIG 2.

ACE2 variants bind viral spike proteins. (A) HeLa cells transduced with ACE2 variants were incubated with the recombinant S1 domain of the SARS-CoV-2, SARS-CoV, or HCoV-NL63 spike proteins C-terminally fused with Fc (1 μg/ml). Cells were then incubated with goat anti-human IgG (H+L) conjugated to Alexa Fluor 647 followed by flow cytometry analysis. Values are binding efficiencies defined as the percentage of ACE2-expressing cells (zsGreen1+) positive for S1-Fc. Values are means plus standard deviations (SD) (error bars) (n = 3). This experiment was independently repeated three times with similar results. (B) HeLa cells transduced with lentiviruses expressing FLAG-tagged human ACE2 variants were subjected to immunoblotting. Tubulin served as the loading control. This experiment was independently repeated three times with similar results. A representative blot is shown. (C) HeLa cells transduced with lentiviruses (pLVX-IRES-zsGreen1) expressing ACE2 variants as indicated were incubated with rabbit polyclonal antibody (Sino Biological Inc. China; catalog no. 10108-T24) against ACE2. The cells were washed and then stained with 2 μg/ml goat anti-rabbit IgG (H+L) conjugated with Alexa Fluor 568 and DAPI (1 μg/ml). The cell images were captured with a Zeiss LSM 880 confocal microscope. ACE2 on cell surface was shown in the merge images processed by ZEN3.2 software. This experiment was independently repeated three times with similar results, and the representative images are shown. (D) HeLa cells transduced with ACE2 variants were incubated with increasing doses (1 μg/ml, 5 μg/ml, or 10 μg/ml) of the recombinant S1 domain of SARS-CoV-2 (left), SARS-CoV (middle), or HCoV-NL63 (right) spike proteins fused to Fc. The other procedure and binding efficiency were performed as described in panel A. Values are means plus standard deviations (SD) (error bars) (n = 3). This experiment was independently repeated three times with similar results.