Fig. 4: Evaluation of the critical binding sites determining the species-specific restriction of SARS-CoV and SARS-CoV-2 binding and entry. | Nature Ecology & Evolution

Fig. 4: Evaluation of the critical binding sites determining the species-specific restriction of SARS-CoV and SARS-CoV-2 binding and entry.

From: ACE2 receptor usage reveals variation in susceptibility to SARS-CoV and SARS-CoV-2 infection among bat species

Fig. 4

a, Swap mutagenesis assay to investigate the role of critical residues on bat ACE2 orthologues for tropism determination. Residues involved in RBD (according to the structure between SARS2-RBD and human ACE2, Protein Data Bank 6M0J) interaction are shown in the table. Residues that changed in the mutagenesis assay are marked in red. b, The expression level of the bat ACE2 orthologues and related mutants in transduced 293T cells was determined by an immunofluorescence assay recognizing the 3×FLAG-tag. Scale bar, 200 μm. c,d, Binding efficiency of SARS2-RBD-hFc and SARS2-RBD-hFc on 293T cells expressing bat ACE2 and related mutants. Cells were incubated with 5 μg ml−1 of recombinant proteins at 37 °C for 1 h and then washed and incubated with a secondary antibody recognizing human Fc. Immunostaining (c) and flow cytometry (d) were conducted to show binding efficiency. Scale bar, 200 μm. e,f, Ability of the indicated ACE2 and related mutants to support the entry of coronavirus pseudotypes. The 293T cells expressing the indicated ACE2 and their mutants were infected with SARS-CoV and SARS-CoV-2 pseudotypes expressing GFP (e) and luciferase (f). Infection was analysed at 20 h post-infection. Scale bar, 200 μm. Data are presented as the mean with s.d. (n = 2). g, 293T cells infected by the SARS-CoV-2 live virus at an MOI = 0.01; the infection was examined at 48 h post-infection through N protein (red) immunostaining. Nuclei were stained with Hoechst 33342 (blue). Scale bar, 200 μm. h,i, Comparison of the interface between Bat33/SARS-CoV-2-RBD and Bat34/SARS-CoV-2-RBD. Bat33 and its complexed RBD are coloured cyan and gold, respectively (h); Bat34 and its complexed RBD are coloured wheat and green, respectively (i). The mutated residues in ACE2 and the corresponding residues in SARS-CoV-2-RBD are shown and labelled. The red dotted lines between residues indicate hydrogen or ionic bonds.

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