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Receptor and virus sequences

The acquisition of sequences of 46 bat ACE2 and hACE2 was described in our previous study29. The five bat DPP4 and hDPP4 sequences were directly retrieved from GenBank (human DPP4, NM_001935.3; Bat37, P. pipistrellus, KC249974.1) or extracted from whole genome sequence assemblies of the bat species retrieved from GenBank (Bat25, Sturnira hondurensis, GCA_014824575.2; Bat29, Mormoops blainvillei, GCA_004026545.1; Bat36, Aeorestes cinereus, GCA_011751065.1; Bat40, A. pallidus, GCA_007922775.1). The whole genome sequences of different coronaviruses were retrieved from GenBank. Accession numbers are as follows: MERS-CoV (NC_019843.3), Camel MERS-CoV KFU-HKU 19Dam (KJ650296.1), HKU4 (NC_009019.1), HKU5 (NC_009020.1), ErinaceusCoV/HKU31 strain F6 (MK907286.1), NeoCoV (KC869678.4), PDF-2180 (NC_034440.1), ErinaceusCoV/2012-174 (NC_039207.1), BtVs-BetaCoV/SC2013 (KJ473821.1), BatCoV/H.savii/Italy (MG596802.1), BatCoV HKU25 (KX442564.1), BatCoV ZC45 (MG772933.1), SARS-CoV-2 (NC_045512.2), NL63 (JX504050.1) and 229E (MT797634.1). All receptor and viral gene sequences used in this study were commercially synthesized by Genewiz, GenScript or GeneArt. The sources, accession numbers and sequences of the receptors and viruses are summarized in Supplementary Table 4.

Collection of SARS-CoV-2 antisera

All of the vaccinated sera were collected from volunteers at about 21 days after the third dose of the WHO-approved inactivated SARS-CoV-2 vaccine (CoronaVac, Sinovac). The median age of volunteers was 37 years. A total of 44% of participants were male and 56% were female. All of the volunteers were recruited by Sinovac. None of the participants had a history of previous SARS-CoV-2 infection, and none reported serious adverse events after vaccination. All of the volunteers were provided informed written consent forms, and the whole study was conducted according to the requirements of Good Clinical Practice of China. The procedures about human participants were approved by the Ethics Committee (seal) of Beijing Youan Hospital, Capital Medical University with an approval number of LL-2021-042-K.

Bioinformatic and computational analyses

Protein sequence alignment was performed using the MUSCLE algorithm by MEGA-X software (v.10.1.8) or ClustalW (https://www.genome.jp/tools-bin/clustalw). For phylogenetic analysis, nucleotide or protein sequences of the viruses were first aligned using the ClustalW and the MUSCLE algorithm, respectively. Phylogenetic trees were subsequently generated using the maximal-likelihood method in MEGA-X (1,000 bootstraps). The model and the other parameters used for phylogenetic analysis were applied following the recommendations after finding the best DNA/protein models using the software. The nucleotide similarity of coronaviruses was analysed using SimPlot (v.3.5.1) with a sliding windows size of 1,000 nucleotides and a step size of 100 nucleotides using gap-stripped alignments and the Kimura (two-parameter) distance model. Molecular dynamics prediction of the effect of residue mutations on protein–protein interactions was conducted by mCSM-PPI2 (http://biosig.unimelb.edu.au/mcsm_ppi2/)52.

Plasmids

Human codon-optimized sequences of various receptors and their mutants were cloned into a lentiviral transfer vector (pLVX-EF1a-Puro, Genewiz) with a C-terminal 3×Flag tag (DYKDHD-G-DYKDHD-I-DYKDDDDK). The DNA sequences of human codon-optimized NeoCoV S (GenBank: AGY29650.2), PDF-2180 S (GenBank: YP_009361857.1), HKU4 S (GenBank: AWH65899.1), HKU5 S (GenBank: YP_001039962.1), HKU31 S (GenBank: QGA70692.1), SARS-CoV-2 S (GenBank: YP_009724390.1) and MERS-CoV S (GenBank: YP_009047204.1) were cloned into the pCAGGS vector or pcDNA3.1(−) vector with a deletion of the last 13–15 residues (or 18 amino-acids in the SARS-CoV-2 S construct) or replacement by an HA tag (YPYDVPDYA) for higher VSV pseudotyping efficiency53. Plasmids expressing coronavirus RBD–IgG-hFc fusion proteins were generated by inserting the coding sequences of NeoCoV RBD (380–585), PDF-2180 RBD (381–586), HKU4 (382–593), HKU5 RBD (385–586), HKU31 RBD (366–575), SARS-CoV-2 RBD (331–524) and MERS-CoV RBD (377–588) into the pCAGGS vector with an N-terminal CD5 secretion leading sequence (MPMGSLQPLATLYLLGMLVASVL) and C-terminal hFc tag for easy purification and detection. Plasmids expressing soluble bat and human ACE2 proteins (corresponding to residues 18–740 in hACE2) were constructed by inserting the ectodomain-coding sequences (containing a collectrin-like dimerization domain) into the pCAGGS vector with an N-terminal CD5 leading sequence and a C-terminal twin-strep tag and 3×Flag tandem sequences (WSHPQFEKGGGSGGGSGGSAWSHPQFEK-GGGRS-DYKDHDGDYKDHDIDYKDDDDK). Virus S proteins or receptor mutants or chimeras were generated by overlapping PCR. For DSP-based cell–cell fusion assays, the dual reporter split proteins were expressed by pLVX-EF1a-Puro-based plasmids carrying the rLucN(1–155)-sfGFP1–7(1–157) and sfGFP8–11(158–231)-rLucC(156–311) coding sequences (summarized in Supplementary Table 4), which were constructed in the laboratory based on a previous study54,55. Plasmids expressing codon-optimized anti-ACE2 antibodies (H11B11; GenBank MZ514137 and MZ514138)31, B644, S2P645 and S2H1440 were constructed by inserting the heavy-chain and light-chain coding sequences into the pCAGGS vector with N-terminal CD5 leader sequences, respectively. For anti-MERS-CoV nanobody–hFc fusion proteins, nanobody coding sequences were synthesized and cloned into the pCAGGS vector with N-terminal CD5 leader sequences and C-terminal hFc tags38,39 (Supplementary Table 4). MERS-CoV-specific nanobodies and H11B11 coding sequences were synthesized by Sangon Biotech or Tsingke Biotechnology. For S trimer cryo-EM analysis, PDF-2180 S full-length gene (GenBank: YP_009361857.1) was synthesized by GeneArt, codon-optimized for expression in mammalian cells, cloned into pcDNA3.1(−) between XbaI and BamHI in frame with a Kozak’s sequence to direct translation and with the endogenous signal peptide. PDF-2180 S ectodomain gene was derived from the S full-length construct and comprised residues 1 to 1,286 followed by a foldon trimerization domain and a C-terminal His tag to assist purification. The full-length gene for expression of A. pallidus ACE2 (QJF77789) used in pseudotyped virus assays was synthesized by GeneScript and cloned in pCDNA3(+). The full-length gene for expressing hACE2 was described in one of our previous studies41.

Cell lines

HEK293T (CRL-3216), Vero E6 cells (CRL-1586), A549 (CCL-185), BHK-21 (CCL-10), Caco-2 (HTB-37) and the bat epithelial cell line Tb 1 Lu (CCL-88) were purchased from American Type Culture Collection (ATCC). The human hepatocellular carcinoma cell line Huh-7 (SCSP-526) were obtained from the Cell Bank of Type Culture Collection, Chinese Academy of Sciences. All these cells were cultured in Dulbecco’s modified Eagle medium, (DMEM, Monad) supplemented with 10% fetal bovine serum (FBS), 2.0 mM of l-glutamine, 110 mg l−1 of sodium pyruvate and 4.5 g l−1 of d-glucose. An I1-hybridoma (CRL-2700) cell line secreting a neutralizing mouse monoclonal antibody against the VSV glycoprotein (VSVG) was cultured in minimum essential medium with Earles’s balanced salts solution, 2.0 mM l-glutamine (Gibco) and 10% FBS. All cells were cultured at 37 °C in 5% CO2 with regular passaging every 2–3 days. HEK Expi 293F cells (A14527, Thermo Fisher Scientific) expressing protein for cryo-EM analysis was cultured in OPM-293 CD03 serum-free medium (OPM, Shanghai OPM Biosciences).

Stable cell lines

Stable cell lines overexpressing different receptors were generated by lentivirus transduction and antibiotic selection. Specifically, the lentiviruses carrying the target genes were produced by co-transfection of lentiviral transfer (pLVX-EF1a-Puro,) and packaging plasmids pMD2G (Addgene, 12259) and psPAX2 (Addgene, 12260) into HEK293T cells using Lip2000 transfection reagent (Biosharp, BL623B). The lentivirus-containing supernatant was collected and pooled at 24 and 48 h after transfection. Cells were transduced by the lentivirus after 16 h in the presence of 8 μg ml−1 polybrene. Stable cells were selected and maintained in the growth medium with puromycin (1 μg ml−1). Cells selected for at least 10 days were considered to be stable cell lines and were used in various experiments.

Protein expression and purification

Antibodies, nanobody–hFc, soluble ACE2 ectodomain and RBD–hFc fusion proteins were expressed in HEK293T cells by transfecting the corresponding plasmids with GeneTwin reagent (Biomed, TG101-01) according to the manufacturer’s instructions. Then, 4 h after transfection, the culture medium of the transfected cells was replenished by SMM 293-TII Expression Medium (Sino Biological, M293TII). Protein-containing supernatants were collected every 2–3 days. Antibodies, nanobody–hFc and recombinant RBD–hFc proteins were captured by Pierce Protein A/G Plus Agarose (Thermo Scientific, 20424), washed with wash buffer (100 mM Tris/HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA), eluted with pH 3.0 glycine buffer (100 mM in H2O) and then immediately balanced by 1/10 volume of UltraPure 1 M Tris-HCI, pH 8.0 (15568025, Thermo Fisher Scientific). The twin-strep-tag-labelled proteins were captured by Strep-Tactin XT 4Flow high-capacity resin (IBA, 2-5030-002), washed with washing buffer and eluted with buffer BXT (100 mM Tris/HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 50 mM biotin). Eluted proteins were concentrated and buffer-changed to PBS through ultrafiltration. Protein concentrations were determined using the Omni-Easy Instant BCA Protein Assay Kit (Epizyme, ZJ102). Purified proteins were aliquoted and stored at −80 °C. For cryo-EM analysis, NeoCoV RBD(380–588), PDF-2018 RBD(381–589) and Bat37ACE2(18–740) were synthesized and subcloned into the pCAGGS vector with a C-terminal twin-strep tag. In brief, these proteins were expressed by transient transfection of HEK Expi 293F cells (Gibco, Thermo Fisher Scientific, A14527) using polyethylenimine MAX (MW 40,000; Polysciences) or 293-free Transfection Reagent (Thermo Fisher Scientific). After 4 days, the supernatant was collected and cells were kept in culture for an additional 4 days, yielding two collections per transfection. The RBD and ACE2 protein samples were further purified by size-exclusion chromatography using a Superdex 75 10/300 Increase column (GE Healthcare) or a Superdex 200 10/300 Increase column (GE Healthcare) in 20 mM HEPES, 100 mM NaCl, pH  7.5. For the RBD–receptor complex (NeoCoV RBD–Bat37ACE2/PDF-2180 RBD–Bat37ACE2), NeoCoV RBD or PDF-2180 RBD was mixed with Bat37ACE2 at the ratio of 1.2:1, and incubated for 30 min on ice. The mixture was then processed for gel-filtration chromatography. The fractions containing the complex were collected and concentrated to 2 mg ml−1.

For PDF-2180 S ectodomain expression, HEK293F cells were grown in suspension, with rotation at 130 rpm, in FreeStyle 293 Expression Medium (Life Technologies) at 37 °C in a humidified 8% CO2 incubator. The wild-type PDF-2180 S ectodomain construct was transfected into 500 ml cultures with cells grown to a density of 1 million cells per ml using 293-free Transfection Reagent (Thermo Fisher Scientific). After 4 days, the supernatant was collected and cells were kept in culture for an additional 4 days, yielding two collections per transfection. The supernatants were clarified by centrifugation at 800g for 10 min, supplemented with 350 mM NaCl and 25 mM Tris-HCl pH 8.0, further centrifuged at 14,000g for 30 min and passed through a 1 ml His trap HP column (Cytiva) that had been equilibrated with binding buffer (25 mM Tris pH 7.4 and 350 mM NaCl). The PDF-2180 S ectodomain was eluted using a linear gradient of 500 mM imidazole. Purified protein was concentrated, buffer-exchanged into Tris-saline buffer (25 mM Tris pH 8, 150 mM NaCl) and quantified using absorption at 280 nm. Purified S glycoprotein was concentrated, and flash-frozen before negative staining and cryo-EM analysis.

Cryo-EM sample preparation and data collection

For cryo-EM sample preparation, the NeoCoV RBD–Bat37ACE2 or PDF-2018 RBD–Bat37ACE2 complexes were diluted to 0.5 mg ml−1. Holy-carbon gold grids (Cflat R1.2/1.3 mesh 300) were freshly glow-discharged with a Solarus 950 plasma cleaner (Gatan) for 30 s. A 3 μl aliquot of the mixture complex was transferred onto the grids, blotted with filter paper at 16 °C and 100% humidity, and plunged into the ethane using the Vitrobot Mark IV (FEI). For these complexes, micrographs were collected at 300 kV using the Titan Krios microscope (Thermo Fisher Scientific), equipped with a K2 detector (Gatan), using SerialEM automated data collection software (v.3.8). Videos (32 frames, each 0.2 s, total dose 60 e Å−2) were recorded at a final pixel size of 0.82 Å with a defocus of between −1.2 and −2.0 μm. For PDF-2180 S trimer sample preparation, 3 μl of PDF-2180 S at 1 mg ml−1 was applied to a 2/2 C-flat grid (Protochips) that had been glow discharged for 30 s at 20 mA. The grids were plunge-frozen into liquid ethane using an FEI Mark IV Vitrobot with a 6.5–7.5 s blot time at 100% humidity and 20 °C. Data were acquired using the FEI Titan Krios transmission electron microscope operated at 300 kV and equipped with a Gatan K2 Summit direct detector and Gatan Quantum GIF energy filter, operated in zero-loss mode with a slit width of 20 eV. Automated data collection was carried out using Leginon (v.3.1)56 at a nominal magnification of ×130,000 with a super-resolution pixel size of 0.525 Å and a defocus range between −0.8 μm and −1.5 μm. The dose rate was adjusted to 8 counts per physical pixel per s, and each video was dose-fractionated in 50 frames of 200 ms.

Image processing

For the NeoCoV RBD–Bat37ACE2 complex, a total of 4,234 micrographs were recorded. For the PDF-2018 RBD–Bat37ACE2 complex, a total of 3,298 micrographs were recorded. Both datasets were similarly processed. Raw data were processed using MotionCor2 (v.1.3.0). The raw data were aligned and averaged into motion-corrected summed images, after which, defocus values for each micrograph were determined using Gctf. Next, particles were picked and extracted for two-dimensional alignment. Well-defined partial particles were selected for initial model reconstruction in Relion57. The initial model was used as a reference for 3D classification. After refinement and post-processing, the overall resolution of the PDF-2018 RBD–Bat37ACE2 complex was up to 3.8 Å based on the gold-standard Fourier shell correlation (FSC; threshold = 0.143)58. For the NeoCoV RBD–Bat37ACE2 complex, the C2 symmetry was expanded before 3D refinement. Finally, the resolution of the NeoCoV RBD–Bat37ACE2 complex reached 3.5 Å. The quality of the local resolution was evaluated using ResMap (v.1.95)59. For the PDF-2180 spike trimer, a total of 1,746 micrographs were collected. Video frame alignment, estimation of the microscope contrast-transfer function parameters, particle picking and extraction were performed using Warp (v.1.0.9)60. Particles were extracted with a box size of 800 binned to 400 px2. Reference-free 2D classification was performed using Relion (v.3.0) to select well-defined particles images before 3D classification without symmetry applied using a MERS-CoV cryo-EM map61 as an initial model in Relion. 3D refinements and CTF refinement (to refine per-particle defocus values) were performed in Relion (v.3.0)62. Particle images were processed using the Bayesian polishing procedure implemented in Relion (v.3.0)63 before performing another round of 3D refinement and per-particle defocus refinement. Subsequently, 3D classification without alignment was performed using a mask focused on the N-terminal domain to improve its resolution, and the selected particles were processed for 3D refinement imposing C3 symmetry using non-uniform refinement in cryoSPARC (v.3.3.1)64, which yielded a final reconstruction of the PDF-2180 S at a resolution of 2.5 Å. Local-resolution estimation, filtering and sharpening were performed using CryoSPARC (v.3.3.1)65. Reported resolutions are based on the gold-standard FSC of 0.143 criterion, and FSC curves were corrected for the effects of soft masking by high-resolution noise substitution66,67.

Model building and refinement

The NeoCoV RBD–Bat37ACE2 complex structures were manually built into the refined maps using Coot (v.0.9.4)68. The atomic models were further refined by positional and B-factor refinement in real space using Phenix (v.1.19)59. For building the PDF-2018 RBD–Bat37ACE2 complex model, the refinement NeoCoV RBD–Bat37ACE2 complex structures were manually docked into the refined maps using UCSF Chimera (v.1.15) and further corrected manually by real-space refinement in Coot. The atomic models were further refined by using Phenix (v.1.19) and Rosetta (v.1.2.5)69,70. For the PDF-2180 S model building, UCSF ChimeraX (v.1.1)71 and Coot were used to fit a MERS-CoV spike atomic model (PDB: 5W9J) into the PDF-2180 cryo-EM map. The model was subsequently manually rebuilt using Coot. N-linked glycans were hand built into the density where visible, and the models were rebuilt and refined using Rosetta (v.1.2.5)69,70. All of the models were validated and analysed using MolProbity72 and Privateer73. The figures were generated using ChimeraX. The datasets and refinement statistics are shown in Supplementary Table 1.

Immunofluorescence assay

Expression levels of receptors were evaluated by immunofluorescence assay detecting the C-terminal 3×Flag tags. Cells expressing receptors were seeded in the 96-well plate (poly-lysine-pretreated plates for HEK293 cell lines) at a cell density of about 1–5 × 104 cells per well and cultured for 24 h. Cells were fixed with 100% methanol at room temperature for 10 min and then incubated with a mouse monoclonal antibody (M2) targeting the Flag-tag (Sigma-Aldrich, F1804) diluted in 1% BSA/PBS at 37 °C for 1 h. After one wash with PBS, cells were incubated with 2 μg ml−1 of the Alexa Fluor 594-conjugated goat anti-mouse IgG (Thermo Fisher Scientific, A32742) diluted in 1% BSA/PBS at 37 °C for 1 h. The nuclei were stained with Hoechst 33342 (1:5,000 dilution in PBS). Images were captured with a fluorescence microscope (Mshot, MI52-N).

Pseudotype virus production and titration

Coronavirus S pseudotyped viruses (CoV-PSVs) were generated according to a previously described protocol based on a replication-deficient VSV-based rhabdoviral pseudotyping system (VSV-dG)74. The VSV-G glycoprotein-deficient VSV co-expressing GFP and firefly luciferase (VSV-dG-GFP-fLuc) was rescued using a reverse genetics system in the laboratory and helper plasmids from Karafast. For CoV-PSV production, HEK293T or Vero E6 cells were transfected with the plasmids overexpressing the coronavirus spike proteins using the Lip2000 transfection reagent (Biosharp, BL623B). After 24–36 h, the transfected cells were transduced with VSV-dG-GFP-fLuc diluted in DMEM with 8 μg ml−1 polybrene for 4 h at 37 °C with a 50% tissue culture infectious dose (TCID50) of 1 × 106 TCID50 per ml. After 2–5 h infection, virus inoculum was removed, and the cells were washed twice with DMEM or PBS before addition of DMEM supplemented with anti-VSV-G-antibody-containing supernatant (from IL-mouse hybridoma) with 10–50-fold dilution to minimize background from the parental viruses. CoV-PSV-containing supernatants were collected at 24 h after the medium change, clarified at 4,000 rpm for 5 min to remove cellular debris, aliquoted and frozen at −80 °C. The TCID50 of pseudotyped viruses was determined using a threefold-serial-dilution-based infection assay on HEK293T-bat40ACE2 cells for NeoCoV and PDF-2180 S pseudotypes, HEK293T-hDPP4 cells for MERS-CoV and HKU4 S pseudotypes, and BHK21-hACE2 cells for SARS-CoV-2 S pseudotypes. The TCID50 was calculated according to the Reed–Muench method75,76. Relative light unit values of ≥1,000 were considered to be positive. Viral titres (genome equivalents) of HKU5 and HKU31 without an ideal infection system were determined by quantitative PCR with reverse transcription using the HiScript II Q RT SuperMix (Vazyme, R223-01). RNA copies in the virus-containing supernatants were detected using primers for the VSV-N gene sequences (VSV-N-F, 5′-ACGGCGTACTTCCAGATGG-3′; VSV-N-R, 5′-CTCGGTTCAAGATCCAGGT-3′). For PDF-2180-, NeoCoV- and NeoCoV-T510F-neutralization assays, the S pseudotyped viruses were generated by transfecting the HEK293T cells using Lipofectamine 2000 (Life Technologies) according to the manufacturer’s instructions. After 5 h at 37 °C, DMEM supplemented with 20% FBS and 2% penicillin–streptomycin was added, and cells were incubated at 37 °C. The next day, cells were washed three times with DMEM and were transduced with VSVΔG-luc. After 2 h, virus inoculum was removed, and the cells were washed five times with DMEM before addition of DMEM supplemented with anti-VSV-G antibody (IL-mouse hybridoma supernatant diluted 1:25 (v/v)) to minimize parental background. After 18–24 h, the supernatants containing pseudotyped VSVs were collected, centrifuged at 2,000g for 5 min to remove cellular debris, filtered with a 0.45 µm membrane, concentrated ten times using a 30 kDa cut-off membrane (Amicon), aliquoted and frozen at −80 °C.

Pseudotyped virus entry assay

Cells obtained by plasmid transfection or lentiviral transduction were trypsinized and incubated with different pseudotyped viruses (1 × 105 TCID50 per 100 μl) in a 96-well plate (5 × 104 per well) to allow attachment and entry simultaneously. For viruses without known susceptible cells, infections were performed using the same genome copies of a reference virus with a calculated TCID50 titre (commonly 1 × 105 TCID50 per 100 μl). For TPCK-treated trypsin (Sigma-Aldrich, T8802) treatment, pseudotyped viruses in serum-free DMEM were incubated with 100 μg ml−1 TPCK-treated trypsin for 10 min at 25 °C. The reactions were stopped by adding 100 μg ml−1 soybean trypsin inhibitor (Sigma-Aldrich, T6414) in DMEM + 10% FBS. After 16–20 h, GFP images were acquired using a fluorescence microscope (Mshot, MI52-N), and intracellular luciferase activity was determined using the Bright-Glo Luciferase Assay Kit (Promega, E2620) and measured using the SpectraMax iD3 Multi-well Luminometer (Molecular Devices) or a GloMax 20/20 Luminometer (Promega).

Pseudotyped virus neutralization assays

For viral RBD or soluble ACE2 neutralization assays, serial dilutions of proteins were prepared in DMEM. Pseudotyped viruses (1 × 105 TCID50 per well) were mixed with 25 µl of each dilution and the mixtures were incubated for 30–45 min at 37 °C before addition to receptor-expressing cells seeded the day before at 2 × 104 cells per well in a 96-well plate. After 16–20 h, the luciferase activity was measured in the same way as described for the pseudotype virus entry assay.

For pseudotyped virus neutralization with B6, SP6 and S2H14 IgGs, HEK293T cells were transfected with plasmids encoding for full-length hACE2 or Bat40 (A. pallidus) ACE2. In brief, HEK293T cells at 90% confluency were transfected using Lipofectamine 2000 (Life Technologies) and trypsinized at 5 h after transfection and seeded into 96-well plates at 50,000 cells per well overnight at 37 °C. For neutralizations, twofold serial dilutions of B6, S2P6 or S2H14 IgGs were prepared in DMEM. Then, 5 µl of the corresponding pseudotyped viruses was mixed with 20 µl of DMEM and 25 µl of each IgG dilution, and the mixtures were incubated for 45 min at 37 °C. Transfected HEK293T cells were washed three times with DMEM before adding 40 μl of the mixture containing virus and IgG. Then, 1 h later, 40 μl DMEM was added to the cells. After 17–20 h, 70 μl of One-Glo-EX substrate (Promega) was added to each well and incubated on a plate shaker in the dark. After 5–15 min incubation, the plates were read using the Biotek Neo2 plate reader. Measurements were taken in duplicate with biological replicates. Relative light units were plotted and normalized in Prism (GraphPad, v.8). Cells alone without pseudotyped virus was defined as 0% infection, and cells with virus only was defined as 100% infection.

Western blot

Cells washed with PBS were lysed with 2% TritonX-100/PBS containing 1 mM freshly prepared PMSF (Beyotime, ST506) on ice for 10 min. Cell lysates were clarified by centrifugation at 12,000g at 4 °C for 5 min, mixed with 1:5 (v/v) 5× SDS-loading buffer, and incubated at 95 °C for 5 min. To detect the HA tag or VSV-M on pseudotyped viruses, 1 ml of virus-containing supernatant was incubated with 8% PEG at 4 °C overnight, precipitated by centrifugation at 7,000g for 10 min at 4 °C and resuspended with 50 μl 1×SDS loading buffer. After SDS–PAGE and PVDF membrane transfer, the blots were blocked with 5% milk in PBS containing 0.1% Tween-20 (PBST) or TBST (20 mM Tris-HCl pH 8.0, 150 mM NaCl) supplemented with 0.05% Tween-20 at room temperature for 1 h. Primary antibodies against Flag (Sigma-Aldrich, F1804), HA (BioLegend, 901515), VSV-M [23H12] (Kerafast, EB0011) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (AntGene, ANT325) were added at a 1:10,000 dilution in PBST with 1% milk, or 1:250 dilution in TBST with 1% milk in the case of the stem-helix targeting monoclonal antibody B6, and incubated on a shaker at 4 °C. After three washes in PBST or TBST, the blots were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody AffiniPure goat anti-mouse IgG (H+L) (Jackson Immuno Research, 115-035-003), AffiniPure goat anti-rabbit IgG (H+L) (Jackson Immuno Research, 111-035-003, 1:10,000 dilution) or Alexa Fluor 680-conjugated goat anti-human secondary antibody (1:50,000 dilution, Jackson ImmunoResearch, 109-625-098) for 1 hour. The blots were subsequently washed three times before visualization using the LI-COR Odyssey CLx or the Omni-ECL Femto Light Chemiluminescence Kit (EpiZyme, SQ201) and a ChemiDoc MP Imaging System (Bio-Rad). Information about antibodies is provided in Supplementary Table 3. Uncropped and unprocessed full scans of gel source data are provided in Supplementary Figs. 1 and 2.

Coronavirus RBD–hFc live-cell binding assays

Recombinant coronavirus RBD–hFc proteins were diluted in DMEM at the indicated concentrations and then incubated with the cells for 1 h at 37 °C. After incubation, cells were washed once with DMEM and then incubated with 2 μg ml−1 of Alexa Fluor 488-conjugated goat anti-human IgG (Thermo Fisher Scientific; A11013) diluted in Hanks’ balanced salt solution (HBSS) with 1% BSA for 1 h at 37 °C. Cells were washed twice with PBS and incubated with Hoechst 33342 (1:5,000 dilution in HBSS) for nucleus staining. Images were captured using a fluorescence microscope (MI52-N). For flow cytometry analysis, cells were detached by 5 mM of EDTA/PBS and analysed using the CytoFLEX Flow Cytometer (Beckman). The dead cells, as indicated by SSC/FSC, were excluded by gating. A total of 10,000 events in a gated live-cell population were analysed for all of the samples. The RBD–hFc-bound cells were gated as indicated by the fluorescence intensity compared with HEK293T control cells without receptor expression. The flow cytometry data were analysed using FlowJo (v.10). The gating strategy for flow cytometry analysis (Fig. 2b) is exemplified in Supplementary Fig. 3.

BLI binding assays

Protein binding affinities were determined using BLI assays performed on the Octet RED96 instrument (Molecular Devices). In brief, 20 μg ml−1 RBD–hFc recombinant proteins were loaded onto protein A (ProA) biosensors (ForteBio, 18–5010) for 30 s. The loaded biosensors were then dipped into the kinetic buffer (PBST) for 90 s to wash out unbound RBD–hFc proteins. Subsequently, biosensors were dipped into the kinetic buffer containing soluble ACE2 ectodomain proteins with concentrations ranging from 0 to 500 nM for 120 s to record association kinetic and then dipped into kinetics buffer for 300 s to record dissociation kinetics. Kinetic buffer without ACE2 was used to define the background. The corresponding affinities were calculated with the Octet Data Analysis software (v.12.2.0.20) using curve-fitting kinetic analysis or steady-state analysis with global fitting. KD,app values were reported because of the use of dimeric ACE2.

ELISA

To evaluate binding between viral RBD and ACE2 in vitro, 96-well immuno-plates were coated with ACE2 ectodomains at the indicated concentrations in BSA/PBS (100 μl per well) overnight at 4 °C. After three washes with PBST, wells were blocked by 3% skimmed milk/PBS at 37 °C for 2 h. Next, different concentrations of RBD–hFc proteins (1–9 μg ml−1) diluted in 3% milk/PBST were added to the wells and incubated for 1 h at 37 °C. After extensive washes, wells were incubated with 1:2,000 diluted HRP-conjugated goat anti-human Fc antibody (Sigma-Aldrich, T8802) in 3% skimmed milk/PBS for 1 h at 37 °C. Finally, the substrate solution (Solarbio, PR1210) was added to the plates, and the absorbance at 450 nm after reaction termination was measured using the SpectraMax iD3 Multi-well Luminometer (Molecular Devices).

Cell–cell fusion assays

A cell–cell fusion assay based on DSPs was conducted on HEK293T cells stably expressing different receptors54. Group A cells were transfected with spike and rLucN(1–155)-sfGFP1–7(1–157) expressing plasmids. Group B cells were transfected with spike and sfGFP8–11(158–231)-rLuc(156–311) expressing plasmids. Then, 12 h after transfection, cells from both groups were trypsinized and mixed into a 96-well plate at 8 × 104 cells per well. Next, 24 h after transfection, cells were washed once with DMEM and then incubated with DMEM with or without 10–50 μg ml−1 TPCK-treated trypsin for 10 min at room temperature. Then, 6 h later, nuclei were stained with Hoechst 33342 (1:5,000 dilution in HBSS) for 30 min at 37 °C. Fluorescent images were subsequently captured using a fluorescence microscope (MI52-N; Mshot). For measurements of live-cell luciferase activity, 20 μM of EnduRen live-cell substrate (Promega, E6481) was added to the cells in DMEM and incubated for at least 1 h before detection using the Varioskan LUX Multi-well Luminometer (Thermo Fisher Scientific).

Biosafety

The infection-related experiments were conducted in the State Key Laboratory of Virology, Wuhan University, strictly following the bio-safety regulations. Indeed, a VSV-based pseudotype virus system was used for all entry and neutralization assays, including new mutations in NeoCoV or PDF-2180 spikes that expand tropism for human cells. These pseudotyped viruses are non-replicating and non-pathogenic to humans and are commonly used by researchers to study the mechanisms of coronavirus entry and host range determination. The gain-of-function mutation, NeoCoV-T510F, was generated as we found that an Phe residue was present in PDF-2180 S at the equivalent position and its effect on binding affinity is consistent with our prediction models. We cautiously avoided extensively testing and showing the gain of function mutations on NeoCoV to expand its tropism on human cells.

Statistical analysis

Most infection and live-cell binding-related experiments were repeated between 2 and 5 times with around 3–4 biological repeats. In vitro protein-binding-related experiments were conducted with 2–3 technical repeats. Similar results were obtained in all of the experiments, and representative data are shown. Data are presented as mean ± s.d. or mean ± s.e.m. as specified in the figure legends. Most statistical analyses were conducted using GraphPad Prism (v.8) using unpaired two-tailed Student’s t-tests, unless otherwise specified. P < 0.05 was considered to be significant; *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.



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