Evolution of ACE2 in bats inhabiting urban or rural areas
We collected ACE2 orthologues from 46 bat species across the phylogeny (Fig. 1 and Supplementary Table 1). These species contained 28 species that roost or forage in urban areas near humans and 18 species more restricted to rural areas and hence likely to have minimal contact with humans (Supplementary Table 2). In total, we examined 46 species representing 11 bat families (Supplementary Table 3). After aligning the protein sequences of bat ACE2 orthologues, we examined 25 critical residues involved in the binding of the surface spike glycoprotein (S protein) of SARS-CoV-2 (ref. 9) (Extended Data Fig. 1). Genetic variations were observed in nearly all these 25 sites, which may have led to different abilities to support entry of SARS-CoV and SARS-CoV-2 (ref. 9). Furthermore, we detected at least 22 amino acid sites that are putatively under positive selection (Supplementary Table 4), which is indicative of heterogeneous selection pressure across sites. Notably, four of these positively selected sites are in the binding region of ACE2 to the SARS-CoV-2 S protein (Supplementary Table 4).
The labels of bat species in our experiments are indicated. Expression levels determined by western blot (Fig. 2a) are shown with asterisk symbols compared with human ACE2: the triple asterisk indicates high expression, the double asterisk indicates medium expression and the single asterisk indicates low but detectable expression. The ability of bat ACE2 to support SARS-CoV and SARS-CoV-2 pseudovirus entry is shown with different signs (Fig. 3a,b): infection data are presented as percentage mean values of bat ACE2 supporting infection compared with the infection supported by human ACE2. Infection efficiency <5% is indicated with a minus sign, between 5 and 50% with a plus sign and >50% with a double plus sign. Bat phylogeny was taken from previous studies28,29,30.
Interaction between bat ACE2 orthologues and SARS-CoV or SARS-CoV-2 receptor binding domain
Efficient binding between the S protein and the ACE2 receptor is essential for SARS-CoV and SARS-CoV-2 entry. This binding is mainly mediated by the interaction between the critical residues on the receptor-binding domain (RBD) and ACE2. To characterize the receptor function of ACE2 orthologues in a range of diverse bat species, we generated a stable cell library consisting of cell lines expressing the respective 46 bat ACE2 orthologues through lentiviral transduction of 293T cells lacking ACE2 expression10. All bat ACE2 orthologues were exogenously expressed at a comparable level after puromycin selection, as indicated by western blot and immunofluorescence assays detecting the C-terminal 3×FLAG-tag (Fig. 2a,b).
a, Western blot detected the expression levels of ACE2 orthologues on 293T stable cells by targeting the C-terminal 3×FLAG-tag. Glyceraldehyde 3-phosphate dehydrogenase was employed as a loading control. b, Visualization of the intracellular bat ACE2 expression level by immunofluorescence assay detecting the C-terminal 3×FLAG-tag. Scale bar, 100 μm. c,d, Assessment of the interaction between different ACE2 orthologues and SARS-CoV-RBD-hFc (c) or SARS-CoV-2-RBD-hFc (d) proteins. Species that do not support efficient binding are underlined. 293T cells stably expressing the different bat ACE2 orthologues were incubated with 5 μg ml−1 of the recombinant proteins at 37 °C for 1 h; binding efficiency was examined by Alexa Fluor 488 goat anti-human IgG via fluorescence assay. Scale bar, 200 μm.
To analyse the interaction, we produced recombinant SARS-CoV or SARS-CoV-2 RBD human immunoglobulin G (IgG) Fc fusion proteins (RBD-hFc), previously reported to be sufficient to bind human ACE2 efficiently11,12. Protein binding efficiency was tested on the bat ACE2 cell library by means of immunofluorescence or flow cytometry targeting the human Fc. As expected, binding was almost undetectable on mock 293T cells but a strong binding signal was detected in the 293T cells expressing human ACE2 (Fig. 2c,d). Consistent with previous reports13,14, SARS-CoV-2 RBD showed higher binding to human ACE2 than SARS-CoV, which can also be observed on many bat ACE2 orthologues (Fig. 2c,d). Previous reports have shown that only a small fraction of ACE2 orthologues from tested mammalian species could not bind with SARS-CoV-2 S protein (n = 6 of 49 species7; n = 5 of 17 species15). However, our study revealed that many bat species (n = 32 and n = 28 of 46 species) do not support efficient binding with SARS-CoV-RBD and SARS-CoV-2-RBD, respectively (Fig. 2c,d). The overall profiles of bat ACE2 to bind to SARS-CoV and SARS-CoV-2 RBD are generally comparable; a few showed contrasting modes of binding preferences (Fig. 2c,d). For instance, Bat22 could bind to SARS-CoV but not SARS-CoV-2, whereas Bat14, 21 and 40 could bind to SARS-CoV-2 but not SARS-CoV (Fig. 2c,d). Flow cytometry analysis showed consistent results (Extended Data Fig. 2).
Overall, the RBD-hFc binding assays demonstrated that bat ACE2 orthologues showed different affinity and selectivity levels to SARS-CoV and SARS-CoV-2, indicating that the ACE2 receptors of many bat species may not support efficient SARS-CoV and SARS-CoV-2 infection.
Receptor function of bat ACE2 orthologues to support the entry of SARS-CoV and SARS-CoV-2 using pseudotyped and live viruses
To further evaluate the receptor function of different bat ACE2 orthologues, we employed a vesicular stomatitis virus (VSV)-based rhabdoviral pseudotyping system to mimic the coronavirus spike protein-mediated single-round entry15. SARS-CoV and SARS-CoV-2 pseudotypes were generated by assembling the coronavirus spike proteins and replication-deficient VSV with the VSV glycoprotein gene replaced with a fluorescence protein (VSV-dG-GFP) or a firefly luciferase (VSV-dG-Luc) reporter15. Both viruses showed minimal background infection on 293T cells, but efficient infection on 293T-human ACE2 cells (Extended Data Fig. 3). The susceptibility of the 293T cells expressing bat ACE2 orthologues was then examined with SARS-CoV and SARS-CoV-2 pseudotypes. The results showed that bat ACE2 orthologues have varying abilities to support coronavirus entry and different preferences for SARS-CoV and SARS-CoV-2. (Fig. 3a,b and Extended Data Fig. 4). Pseudotypes with green fluorescent protein (GFP) reporter showed similar results (Extended Data Fig. 5). Notably, we found that 24, 21 and 16 of the 46 bat species showed almost no entry for SARS-CoV, SARS-CoV-2 and both viruses, respectively (Figs. 1 and 3a,b and Supplementary Table 5), suggesting that these species are not likely to be potential hosts of either or both coronaviruses. The bat species showing no viral entry include those that occur in urban areas and those more restricted to rural areas (Fig. 1), suggesting that there is no correlation between proximity to humans and probability of being natural hosts of SARS-CoV or SARS-CoV-2. Although horseshoe bats were suggested as potential natural hosts of SARS-CoV and SARS-CoV-2 (refs. 1,2,3), only one of the three species examined (Rhinolophus sinicus) supported SARS-CoV entry; this species was suggested as the potential host of SARS-CoV3,16. None of these tested horseshoe bats showed entry for SARS-CoV-2 (Figs. 1 and 3). These results unambiguously indicate that ACE2 receptor usage is species-dependent.
a,b, Ability of bat ACE2 orthologues to support the entry of SARS-CoV and SARS-CoV-2 pseudovirus. 293T cells expressing bat ACE2 orthologues in a 96-well plate were infected with VSV-dG-Luc pseudotyped with SARS-CoV (a) and SARS-CoV-2 (b) spike proteins, respectively. Intracellular luciferase activity was determined at 20 h post-infection. RLU, relative light unit. c, 293T cells expressing bat ACE2 orthologues were inoculated with the SARS-CoV-2 live virus at an MOI = 0.01. N protein (red) in the infected cells was detected through immunofluorescence assay at 48 h post-infection. Scale bar, 200 μm. Samples expressing the indicated ACE2 orthologues that showed almost no entry for SARS-CoV-2 live virus are underlined. Data shown are representative results from 3 independent experiments and are presented as the mean ± s.d. (n = 3 for a and n = 2 for b).
The SARS-CoV-2 S protein used in this study for pseudotyping contains a D614G mutation, which is currently a dominant variation17. The D614G mutation remarkably improved the in vitro infectivity of SARS-CoV-2 but may not significantly affect the receptor interaction since it is not in the RBD18. Indeed, we identified a very similar susceptibility profile using an original strain without D614G (Extended Data Fig. 4). We further demonstrated that the pseudotyped entry assay mimics the entry of live viruses through a SARS-CoV-2 infection assay (Fig. 3c). As expected, the profile of SARS-CoV-2 N protein expression is highly consistent with the results from the VSV-dG-based pseudotyped virus entry assay, except for some ACE2 that showed relatively higher infection efficiency (for example, Bat43–46) compared with the pseudovirus infection assay, which may be attributed to the different virus strains used (Fig. 3c). In addition, the live virus infection resulted in the phenotype of plaque formation, while the pseudotypes showed evenly distributed, single-round infection (Extended Data Fig. 5), which also partially explains why some bat ACE2 showed higher infection in the live virus infection assay.
When comparing the RBD-hFc binding and pseudotyped entry profiles, we found that binding and susceptibility are not always consistent, although the phenotypes were reproducible. For instance, some species (Bat12, 13, 14) were able to bind to SARS-CoV-2 RBD-hFc efficiently but could not support infection of the same virus, indicating that high binding affinity does not guarantee efficient viral entry (Figs. 2 and 3). In contrast, some species (Bat3–8) were defective or less efficient in SARS-CoV RBD-hFc binding but supported the entry of the same virus to some degree (Figs. 2 and 3). We hypothesize that such minimal binding may be sufficient for viral entry mediated by those ACE2 orthologues; alternatively, additional residues outside the traditional RBD region might be required for efficient interaction. These hypotheses should be tested in the future. Together, our results demonstrated dramatic variation of susceptibility to SARS-CoV and SARS-CoV-2 infection among bat species, suggesting that SARS-CoV and SARS-CoV-2 can selectively use some bat ACE2 as functional receptors for viral entry and many—if not most—bat ACE2 are not favoured by one or both viruses.
Evaluation of critical residues in bat ACE2 orthologues affecting viral binding and entry efficiency or specificity
We comprehensively analysed the relationship between critical RBD binding sites in bat ACE2 sequences and their ability to support SARS-CoV and SARS-CoV-2 RBD binding and viral entry. Several critical residues were identified that may play critical roles in the determination of species specificity (Extended Data Fig. 1). According to the sequence alignment, two species pairs (Bat33 and Bat34 and Bat38 and Bat40) were selected to demonstrate the role of critical residues in RBD binding and viral entry because they were phylogenetically close but showed contrasting phenotypes for supporting RBD binding and viral entry. Specifically, Bat34 and Bat38 do not support SARS-CoV and SARS-CoV-2 RBD binding and infection, while Bat33 supports efficient binding and infection of both viruses and Bat40 supports infection of both viruses and to a lesser degree SARS-RBD binding (Figs. 2 and 3). We compared their protein sequences and highlighted the residues that may affect RBD interaction. For example, substitutions I27K, N31G and K42E were observed when comparing Bat33 with Bat34, while Q24L, E30K, K35Q and G354N were present between Bat38 and Bat40 (Fig. 4a). We hypothesized that the discrepancy in binding and infection phenotype is determined by their differences in critical residues for RBD interaction. To test this hypothesis, we designed a residue swap mutagenesis assay to investigate the role of critical residues on RBD binding and virus entry (Fig. 4a). We generated four swap mutations and corresponding 293T stable cell lines to test whether these substitutions could achieve gain-of-function and loss-of-function. All bat ACE2 orthologues and related mutants were expressed at a comparable level after lentiviral transduction, as indicated by the immunofluorescence of the C-terminal 3×FLAG-tag (Fig. 4b). Recombinant SARS-CoV and SARS-CoV-2 RBD-hFc proteins were applied to the cells expressing different ACE2 and binding efficiency was evaluated by immunofluorescence (Fig. 4c) and flow cytometry assays (Fig. 4d). As expected, the swap of critical residues on the selected four bat ACE2 changed their receptor function to the opposite, except for Bat38 mutant, which remained unable to bind SARS-CoV RBD-hFc (Fig. 4c,d). GFP (Fig. 4e) and luciferase levels (Fig. 4f) from the pseudotyped virus entry assay and the N protein staining from the live SARS-CoV-2 infection assay (Fig. 4g) further confirmed our hypothesis at the viral entry level. Structure modelling of bat ACE2/SARS-CoV-2-RBD complexes showed that the substitutions of I27K and N31G between Bat33 and Bat34 lead to a reduced packing interaction and the substitution of K42E disrupts the hydrogen bond with Y449, which may be related to the difference of susceptibility between Bat33 and Bat34 (Fig. 4h,i and Extended Data Fig. 6). In comparison, the substitutions of Q24L and E30K between Bat38 and Bat40 destroyed the favourable hydrophilic interactions with N487 and K417, respectively (Extended Data Fig. 6).
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.
In addition, two bat cell lines, the lung epithelial cell line Tb 1 Lu of Tadarida brasiliensis (Bat31) and the kidney epithelial cell line of Pteropus alecto (Bat2), were used to validate our findings derived from human HEK293T cells. Endogenous ACE2 expression was almost undetectable in these two cell lines, accounting for at least 1,000 folds lower than the susceptible Vero-E6 cells (Extended Data Fig. 7a). Therefore, these cells cannot support the entry of SARS-CoV and SARS-CoV-2. We successfully generated Tb 1 Lu stable cell lines expressing human ACE2 and bat ACE2 (Bat2, 3, 31, 32) since the transduction efficiency of Tb 1 Lu is much higher than that of PakiT03 cells (Extended Data Fig. 7b). As expected, Tu 1 Lu were susceptible to both SARS-CoV and SARS-CoV-2 when human ACE2 or some bat ACE2 orthologues (Bat2, 3 and 31) were expressed, yet remained non-susceptible when an ACE2 of a closely related species (Bat32) was expressed (Extended Data Fig. 7c–e). Furthermore, we conducted SARS-CoV and SARS-CoV-2 pseudovirus entry assays on the two bat cell lines transiently transfected with various bat ACE2 (Bat2, 3, 31, 32, 33, 34, 38, 40) and their mutants (mutant Bat33, 34, 38 and 40m). The results were consistent with those derived from human cells, further confirming that ACE2 is the main receptor for the species-specific entry of SARS-CoV and SARS-CoV-2 in these bat cells (Extended Data Fig. 7f,g).
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