Close relatives of MERS-CoV in bats use ACE2 as their functional receptors

Abstract

Middle East respiratory syndrome coronavirus (MERS-CoV) and several bat coronaviruses use dipeptidyl peptidase-4 (DPP4) as an entry receptor^1-4. However, the receptor for NeoCoV-the closest known MERS-CoV relative found in bats-remains unclear⁵. Here, using a pseudotype virus entry assay, we found that NeoCoV and its close relative, PDF-2180, can efficiently bind to and use specific bat angiotensin-converting enzyme 2 (ACE2) orthologues and, less favourably, human ACE2 as entry receptors through their receptor-binding domains (RBDs) on the spike (S) proteins. Cryo-electron microscopy analysis revealed an RBD-ACE2 binding interface involving protein-glycan interactions, distinct from those of other known ACE2-using coronaviruses. We identified residues 337-342 of human ACE2 as a molecular determinant restricting NeoCoV entry, whereas a NeoCoV S pseudotyped virus containing a T510F RBD mutation efficiently entered cells expressing human ACE2. Although polyclonal SARS-CoV-2 antibodies or MERS-CoV RBD-specific nanobodies did not cross-neutralize NeoCoV or PDF-2180, an ACE2-specific antibody and two broadly neutralizing betacoronavirus antibodies efficiently inhibited these two pseudotyped viruses. We describe MERS-CoV-related viruses that use ACE2 as an entry receptor, underscoring a promiscuity of receptor use and a potential zoonotic threat.

Fig. 1. NeoCoV and PDF-2180 use ACE2 but not DPP4 for efficient entry.

a,b, Phylogenetic analysis of merbecoviruses (grey) based on whole-genome nucleotide sequences (a) and S₁ amino acid sequences (b). NL63 and 229E were set as outgroups. Host and receptor use are indicated. For the scale bars, 0.5 represents two nucleotide substitutions per site. c, Simplot analysis showing the whole-genome nucleotide sequence similarity of three merbecoviruses compared with MERS-CoV. The boundaries of regions encoding MERS-CoV proteins are indicated at the top. The box delineated by a dashed line underscores the divergence of the S₁ subunit. T/t, transition/transversion ratio. d, Pseudotyped S virus entry efficiency of six merbecoviruses in BHK-21 cells transiently expressing hACE2, hDPP4 or hAPN. The dashed lines indicate the baseline of background signals (mean values of vector-only groups). Data are mean ± s.e.m., representative of three independent experiments. n = 3 biologically replicates. Statistical analysis was performed using two-tailed unpaired Student’s t-tests. PSV, pseudotyped virus. e, The entry efficiency of NeoCoV S pseudotyped virus in HEK293T stable cell lines expressing different bat ACE2 orthologues as indicated by luciferase activity. Data are mean ± s.d. of biological triplicates examined over three independent infection assays. f,g, NeoCoV, PDF-2180 and MERS-CoV S-mediated cell–cell fusion analysis based on DSP assays in HEK293T cells stably expressing the indicated receptors. TPCK-trypsin, TPCK-treated trypsin treatment (10 μg ml⁻¹). eGFP intensity (f) and live-cell Renilla luciferase activity (g) are shown. Data are mean ± s.d. of n = 4 biologically independent cells. Three independent cell–cell fusion assays are shown. Statistical analysis was performed using two-tailed unpaired Student’s t-tests. For f, scale bars, 200 μm. h, Pseudotyped virus entry efficiency of six merbecoviruses in HEK293T cells stably expressing the indicated bat ACE2 or DPP4 orthologues. Data are representative results of three independent experiments and plotted by the mean of the biological triplicate. RLU, relative light units. **P < 0.01, ****P < 0.001. Source data

Fig. 2. The domain B (RBD) of NeoCoV and PDF-2180 S proteins are required for species-specific ACE2 binding.

a, Binding of NeoCoV RBD–hFc to bat ACE2 orthologues expressed on the surface of HEK293T cells analysed using a live-cell binding assay. Data are representative of three assays using independent preparations of proteins. Scale bars, 100 μm. b, Flow cytometry analysis of NeoCoV RBD–hFc binding to HEK293T cells expressing the indicated ACE2 orthologues. The ratio of positive cells compared with the vector control is indicated based on the threshold (dashed line). Data are mean values (n = 3 technical replicates), representative of three independent experiments. c, BLI assays analysing binding kinetics between NeoCoV RBD–hFc and PDF-2180-RBD–hFc with selected ACE2 ectodomains. The reported K_D,app values correspond to avidities due to the use of dimeric ACE2 constructs. SSG, steady-state affinity determination. Unfitted curves are shown in Supplementary Fig. 4a. d, ELISA assay showing the binding efficiency of NeoCoV and PDF-2180 RBD–hFc proteins to soluble ACE2 ectodomain proteins. Data are representative of two assays using independent preparations of proteins. Data are mean ± s.d. (technical triplicates). OD₄₅₀, optical density at 450 nm. e, The inhibitory activity of soluble ACE2 against NeoCoV S pseudotyped virus entry in HEK293T-Bat37ACE2 cells. Data are mean ± s.d. n = 3 biologically independent cells. f, Concentration-dependent inhibition of NeoCoV S-mediated entry by soluble Bat37ACE2 in HEK293T-Bat37ACE2 cells. Data points represent biological duplicates. g, Evaluation of the inhibitory effect of NeoCoV, PDF-2180 or MERS-CoV RBD–hFc proteins on NeoCoV S pseudotyped virus entry in HEK293T-Bat37ACE2 cells. Data are mean ± s.d. (biologically triplicates). h, Entry of the MERS-CoV S, NeoCoV S or NeoCoV S chimera containing the MERS-CoV RBD (residues 371–618) pseudotyped viruses into HEK293T cells stably expressing one of the indicated receptors. Data are mean ± s.d. n = 3 independently infected cells. For e–h, data are representative of two independent infection assays. Statistical analysis was performed using two-tailed unpaired Student’s t-tests (e, g and h); *P < 0.05, ***P < 0.005; NS, not significant. Source data

Fig. 3. Cryo-EM structures of NeoCoV RBD and PDF-2180 RBD in a complex with Bat37ACE2.

a,b, Cryo-EM density map (left) and ribbon representation (right) of the NeoCoV RBD–Bat37ACE2 complex (a) and the PDF-2180 RBD–Bat37ACE2 complex (b). NeoCoV RBD, PDF-2180 RBD and Bat37ACE2 are coloured red, orange and cyan, respectively. c, Structural comparison between the NeoCoV RBD–Bat37ACE2 complex (left, PDB: 7WPO) and MERS-CoV RBD-hDPP4 complex (right, PDB: 4KR0). The NeoCoV RBD, MERS-CoV RBD, NeoCoV RBM, MERS-CoV RBM, Bat37ACE2 and hDPP4 are coloured red, green, yellow, grey, cyan and purple, respectively. d, Magnified view of the NeoCoV RBD–Bat37ACE2 complex interface. All of the structures are shown as ribbon representations with key residues rendered as sticks. Salt bridges and hydrogen bonds are shown as red and yellow dashed lines, respectively. e–g, The contribution of critical NeoCoV RBD residues to receptor binding (e) and pseudotyped virus entry (f) in HEK293T-Bat37ACE2 cells. g, The effect of mutations on S expression (lysate) and virion incorporation (supernatant) in HEK293T cells. h–j, The contribution of critical Bat37ACE2 residues on NeoCoV RBD binding (h), pseudotyped virus entry (i) and the impact of mutations on ACE2 expression (j) in HEK293T cells. For e–j, data are representative of two independent infections assays. Two independent preparations of pseudoviruses were used for the assays shown in e–g. Data are mean ± s.d. for f (biological triplicates of infected cells) and i (biological quadruples of infected cells). Statistical analysis was performed using two-tailed unpaired Student’s t-tests. For e and h, scale bars, 100 μm. Source data

Fig. 4. Molecular determinants affecting hACE2 recognition by NeoCoV and PDF-2180.

a, RBD binding modes and receptor (hACE2 or Bat37ACE2) footprints of the four indicated ACE2-using coronaviruses. b, The overlap of the ACE2 footprints. The heat map indicates the per-residue frequency of participation to the virus–ACE2 interfaces identifying residues 329–330 as a virus-binding hot spot. c, Schematic of the hACE2 swap mutants with Bat37ACE2 counterparts. d,e, Expression levels of hACE2 mutants were analysed using western blotting (d) and immunofluorescence (e). h, human. f,g, The receptor function of hACE2 mutants was evaluated by NeoCoV RBD binding (f) and NeoCoV S pseudotyped virus entry in HEK293T-hACE2 (h-WT) cells (g). The fold changes of pseudotyped virus entry relative to wild-type hACE2 are shown. For e and f, scale bars, 100 μm. h, Mutations in the interaction between NeoCoV RBD and Bat37ACE2 were taken as the input of mCSM-PPI2 to predict the change of free binding energy (ΔΔG, kcal mol⁻¹). Mutations with lower ΔΔG are marked in blue and those with higher ΔΔG are marked in red. i, Computational modelling of the NeoCoV RBD–hACE2 complex obtained by superposing the hACE2 structure (PDB: 6M0J, blue) on the Bat37ACE2-bound NeoCoV RBD (red) structure described here. Magnified view of the T510F NeoCoV RBD mutation. j–l, The effect of NeoCoV and PDF-2180 RBM mutations on hACE2 recognition assessed by RBD–hFc binding (j), spike expression (lysate) and virion incorporation (supernatant) (k), and pseudotyped virus entry efficiency (l) in HEK293T-hACE2 and HEK293T-Bat37ACE2 cells. For j, scale bars, 100 μm. m, hACE2-dependent entry of NeoCoV-T510F S pseudotyped virus in Caco-2 cells in the presence of 50 μg ml⁻¹ anti-ACE2 (H11B11) or anti-VSVG (I1) antibodies. Mock, no antibody. For d–g and j–l, data are representative of two infection assays with independent transfections. For m, data are representative of two infection assays. Data are mean ± s.d. for g (n = 4 biologically independent cells) and l and m (n = 3 biologically independent cells). Statistical analysis was performed using two-tailed unpaired Student’s t-tests. Source data

Fig. 5. The architecture and antigenicity of the PDF-2180 spike glycoprotein.

a,b, Ribbon diagram of the cryo-EM structure of PDF-2180 S ectodomain trimer viewed along two orthogonal orientations (side (a) and top (b)). c,d, Sequence conservation between PDF-2180 S and the spikes of different isolates of MERS-CoV plotted on the PDF-2180 S structure viewed from the side (c) and top (d). Conservation analysis was performed using Consurf. e, Ribbon diagram of the PDF-2180 (left) and MERS-CoV (right) S₁ subunits. f, Superimposition of the PDF-2180 and MERS-CoV S₁ subunits. g, Ribbon diagram of the superposed PDF-2180 and MERS-CoV S₂ subunits highlighting the similarity of the fusion machinery. Inset: magnified view of the fusion peptide region. h, Neutralizing activity of CoronaVac vaccine-elicited (three doses) human sera against SARS-CoV-2, NeoCoV and PDF-2180 S pseudotyped viruses. i, Neutralizing activity of MERS-CoV RBD-targeting nanobodies against MERS-CoV, NeoCoV and PDF-2180 S pseudotyped viruses^,. HEK293T-hACE2 cells for SARS-CoV-2; HEK293T-hDPP4 cells for MERS-CoV; HEK293T-Bat37ACE2 cells for NeoCoV and PDF-2180. For h and i, data are mean ± s.d. n = 10 sera or antibodies. Each point represents the mean neutralization value of three biologically independent infection replicates. Statistical analysis was performed using two-tailed paired Student’s t-tests. The inhibitory efficiency of specific samples is summarized in Extended Data Fig. 10b,c. j–o, PDF-2180 (j,k), NeoCoV (l,m) and NeoCoV-T510F S (n,o) pseudotyped viruses entry in the presence of the indicated dilutions of B6 (red) and S2P6 (blue) antibodies, and a SARS-CoV-2-RBD-specific IgG (S2H14, black) was used as negative control. HEK293T cells transiently transfected with Bat40ACE2 (j,l,n) or hACE2 (k,m,o) were used as target cells. For j–o, the average of technical duplicates (one representative experiment out of two independent experiments, that is, a biological duplicate) is shown. For h–o, data are representative of two independent neutralization assays. Source data

Extended Data Fig. 1. Pseudotyped virus entry efficiency of six merbecoviruses in HEK293T cells stably expressing the indicated human ACE2, DPP4, or APN.

a, The expression levels of coronaviruses S proteins in transiently transfected HEK293T cells were analysed by Western blot detecting the C-terminal fused HA tags. GAPDH was used as a loading control. Experiments were performed in triplicates. b, Pseudotyped S virus entry efficiency of six merbecoviruses in HEK293T cells stably expressing hACE2, hDPP4, or hAPN. The dashed lines indicate the baseline of background signals (mean values of vector-only groups with MERS-CoV excluded). Blot representative of experiments of three independent transfections for generating each pseudoviruses. Data are presented as mean ± SEM (n = 4 biologically independent infected cells), representative of three independent infection assays with similar results. Two-tailed unpaired Student’s t-test; ****P < 0.001. RLU: relative light units. Source data

Extended Data Fig. 2. Bat ACE2 orthologs tested in this study and their ability to support the entry of NeoCoV and PDF-2180.

a, Phylogenetic tree and assessment of the ability of a panel of ACE2 orthologs from 46 bat species to support NeoCoV and PDF-2180 S-mediated entry (relative to Bat40 ACE2). ACE2 orthologs deficient in supporting the entry of both viruses are highlighted in red. The GenBank accession numbers and protein sequences were summarized in Supplementary Table 4. b, Immunofluorescence assay detecting the 46 bat ACE2 orthologs C-terminal 3×Flag tag in the HEK293T stable cells. Mock indicates cells were transduced with lentiviruses produced by empty vector only. Data representative of three independent immunostaining assays with similar results.

Extended Data Fig. 3. Entry of indicated pseudoviruses in HEK293T cells stably expressing different bat ACE2 orthologs.

a-e, Entry efficiency of NeoCoV (a) PDF-2180 (b-c), HKU4 (d), and HKU5 (e) S pseudotyped viruses as indicated by GFP (a-b) or luciferase (c-e) intensity. Data representative of two independent infection assays. Data are presented as mean ± SD (biological triplicates for a-c and biological duplicates for d-e). f-g, TPCK-treated trypsin treatment (100 μg/ml) significantly enhanced NeoCoV (f) and PDF-2180 (g) S pseudotyped virus entry in HEK293T cells expressing different ACE2 orthologs. Data are presented as mean ± SD (biological triplicates of infected cells), representative of two independent experiments. Two-tailed unpaired Student’s t-test; ****P < 0.001. h, The expression level of ACE2 was evaluated by immunofluorescence detecting the C-terminal fused 3×Flag tag. Experiments performed twice with similar results and representative data are shown. i, Entry of SARS-CoV-2 and HKU31 S pseudotyped viruses into cells expressing hACE2 or hedgehog hgACE2. Representative data are presented as mean ± SD (biological triplicates of infected cells) examined over two independent experiments. Source data

Extended Data Fig. 4. ACE2 and DPP4 receptor usage of different merbecoviruses.

a, Western blot analysis of the expression levels of ACE2 and DPP4 orthologs in HEK293T cells. Blot representative of immunoblotting based on biological independent duplicates of transfected cells. b, Evaluation of bat ACE2 expression level by immunofluorescence assay detecting the C-terminal 3×Flag tag. Data representative of two independent immunofluorescence assays based on two different transfection experiments. c-d, Pseudotyped virus entry (c) and RBD binding (d) of different CoVs on HEK293T cells expressing different ACE2 or DPP4 orthologs. The experiment was repeated independently twice with similar results, and representative data are shown.

Extended Data Fig. 5. ACE2 and Domain B dependent entry of NeoCoV, PDF-2180 S pseudotyped viruses in different cell types.

a-c, BHK-21, HEK293T, Vero E6, A549, Huh-7, and Tb 1 Lu cells were transfected with either Bat40ACE2 or Bat40DPP4. Expression, pseudotyped virus entry (GFP) (a), and luciferase intensity (b-c) were detected at 16 h post-infection. Data are presented as mean ± SD (biological triplicates). Data representative of two independent transfection and infection assays. d, Expression levels of the indicated chimeric viral S glycoproteins (anti-HA) in HEK293T whole cell lysates. GAPDH was used as a loading control. e, Chimeric viral S glycoproteins (anti-HA) and VSV-M level in pseudotyped viruses precipitated from virus-containing supernatant. Blots representative of two independent transfection assays for pseudovirus production. Source data

Extended Data Fig. 6. Cryo-EM data processing and validation of the NeoCoV RBD-Bat37ACE2 complex and PDF-2180 RBD-Bat37ACE2 complex cryo-EM datasets.

Electron micrograph and flowchart for cryo-EM data processing, resolution estimation of the EM maps, density maps, and atomics models of NeoCoV RBD-Bat37ACE2 complex (a) and PDF-2180 RBD-Bat37ACE2 complex (b).

Extended Data Fig. 7. Structural and sequence analysis of different merbecoviruses.

a, Superimposition of overall structures of NeoCoV RBD-Bat37ACE2 (red) and PDF-2180 RBD-Bat37ACE2 complexes (blue). b, Structures of RBDs from different merbecoviruses. MERS-CoV (PDB:4KQZ), HKU4 (PDB:4QZV), HKU5 (PDB:5XGR). c, RBD sequence alignments were generated with ClustalW and rendered with ESPript. Identical residues are highlighted with a red background and similar residues are coloured red in blue boxes.

Extended Data Fig. 8. Characterization of NeoCoV and PDF-2180 RBM mutations enhancing human ACE2 binding and pseudovirus entry.

a, Analysis of binding of various concentrations of the NeoCoV and PDF-2180 RBD-hFc to HEK293T cells stably expressing hACE2. The SARS-CoV-2 RBD-hFc was used as a positive control. Mock indicates no protein added during protein incubation. Data representative of two live cell binding assays using independent preparations of RBD-hFc proteins. b, Analysis of binding kinetics of the interaction between NeoCoV RBD WT or T510F with hACE2 using BLI. Reported K_D values correspond to avidities due to the utilization of dimeric ACE2 constructs. Representative of two independent experiments. Unfitted curves can be found in Supplementary Figure 4b. c, Identification of PDF-2180 S mutations enhancing hACE2 binding. Sequence alignment of the NeoCoV and PDF-2180 RBMs and definition of the PDF-2180 mutants generated. d, Binding of PDF-2180 RBD-hFc mutants to hACE2- or Bat37ACE2-expressing HEK293T cells. Data presented were performed in two independent assays with similar results. e, Western blot analysis of the expression levels of PDF-2180 S protein mutants in HEK293T cells. f, Western blot analysis of the packaging efficiency of PDF-2180 S mutants in VSV pseudotyped viruses. g, Entry efficiency of PDF-2180 S mutant pseudotyped viruses into hACE2- or Bat37ACE2- expressing HEK293T cells. Experiments presented were independently performed twice with similar results for d-g. Representative data of g are presented as mean ± SD (n = 3 biological triplicates). Two-tailed unpaired Student’s t-test; *P < 0.05,**P < 0.01; ***P < 0.005, and ****P < 0.001 Source data

Extended Data Fig. 9. Cryo-EM data processing and validation of the PDF-2180 S cryo-EM dataset.

a, Representative electron micrograph (top) and class averages (bottom) of PDF-2180 S embedded in vitreous ice. Scale bar of the micrograph: 1000 Å. Scale bar of the class averages: 100 Å. b, Flowchart for cryo-EM data processing of PDF-2180 S trimer. c, Gold-standard Fourier shell correlation curve for the PDF-2180 S trimer. The 0.143 cut-off is indicated by a horizontal dashed grey line. d, Local resolution map for the PDF-2180 spike trimer.

Extended Data Fig. 10. S₁/S₂ cleavage and antibody neutralization efficiency of PDF-2180, NeoCoV, NeoCoV-T510F mutant, or MERS-CoV S pseudotyped viruses.

a, Western blot of VSV pseudotyped particles harbouring PDF-2180, NeoCoV, NeoCoV-T510F mutant or MERS-CoV S glycoproteins (detected using the B6 antibody) used for the B6 and S2P6 neutralization assays. Experiments were performed in duplicates, and representative data were shown. Mw: molecular weight ladder. Full-length S and S₂ subunit bands are indicated on the right-hand side of the blot. b-c, Inhibitory activity of 10 MERS-CoV RBD specific nanobodies at 10 μg/ml (b) and 10 SARS-CoV-2 specific anti-sera or two unvaccinated control sera at 50-fold dilution (c). Data are presented as mean ± SD (biological triplicates of two independent experiments). d-e, Dose-dependent inhibition of the entry of the indicated pseudotyped viruses by representative MERS-CoV RBD specific nanobodies (d) and SARS-CoV-2 specific anti-sera (e). Data are presented as mean (n = 2 biological duplicates), representative of two independent experiments. Source data