SARS-CoV-2 and Three Related Coronaviruses Utilize Multiple ACE2 Orthologs and Are Potently Blocked by an Improved ACE2-Ig

Ethics statement.All experiments involving SARS-CoV-2 live virus infections were approved (no. QS2020050050) by the ethics committee of Shenzhen Center for Disease Control and Prevention (SZCDC) and the ethics committee of Shenzhen Bay Laboratory. SARS-CoV-2 live virus was isolated from SARS-CoV-2 RNA RT-qPCR-positive oropharyngeal swab samples by SZCDC, and all SARS-CoV-2 live virus experiments were performed in the biosafety level 3 facility at SZCDC.

Cells.293T cells and Vero cells were kindly provided by Stem Cell Bank, Chinese Academy of Sciences, confirmed mycoplasma-free by the provider, and maintained in Dulbecco modified Eagle medium (DMEM; Life Technologies) at 37°C in a 5% CO₂-humidified incubator. Growth medium was supplemented with 2 mM GlutaMAX-I (Gibco, catalog no. 35050061), 100 μM nonessential amino acids (Gibco, catalog no. 11140050), 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco, catalog no. 15140122), and 10% FBS (Gibco, catalog no. 10099141C). 293T-based stable cells expressing human ACE2 were maintained under the same culture condition as 293T, except that 3 μg/ml of puromycin was added to the growth medium. 293F cells for recombinant protein production were generously provided by Yu J. Cao (School of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School) and maintained in SMM 293-TII serum-free medium (Sino Biological, catalog no. M293TII) at 37°C, 8% CO₂, in a shaker incubator at 125 rpm.

Plasmids.DNA fragments encoding spike proteins of SARS-CoV-2 WHU01 (GenBank accession no. MN988668.1), SARS-CoV BJ01 (GenBank AY278488.2), Pangolin-CoV (National Genomics Data Center GWHABKW00000000; https://bigd.big.ac.cn/search/?dbId=gwh&q=GWHABKW00000000&page=1) (11), and Bat-CoV RaTG13 (GenBank MN996532) (8), were synthesized by the Beijing Genomic Institute (BGI, China) and Sangon Biotech (Shanghai, China) and then cloned into pcDNA3.1(+) plasmid or pCAGGS plasmid between EcoRI and XhoI restriction sites. Plasmids encoding recombinant RBD and soluble ACE2 variants were generated by cloning each of the gene fragments into a pCAGGS-based mouse-IgG2a or human IgG1 Fc fusion protein expression plasmid between NotI and BspEI sites. The retroviral reporter plasmids encoding a Gaussia luciferase or a green fluorescent protein (GFP) reporter gene were constructed by cloning the reporter genes into pQCXIP plasmid (Clontech), respectively. DNA fragments encoding C-terminally S-tagged ACE2 orthologs were synthesized in pUC57 backbone plasmid by Sangon Biotech (Shanghai, China). These fragments were then cloned into pQCXIP plasmid (Clontech) between SbfI and NotI restriction sites.

IgG Fc fusion protein production and purification.293F cells at the density of 6 × 10⁵ cells/ml were seeded into 100 ml of SMM 293-TII serum-free medium (Sino Biological, catalog no. M293TII) 1 day before transfection. The cells were then transfected with 100 μg of plasmid in complex with 250 μg of PEI MAX 4000 (Polysciences, Inc., catalog no. 24765-1). Cell culture supernatants were collected at 48 to 72 h posttransfection. Recombinant Fc fusion proteins are purified using protein A-Sepharose CL-4B (GE Healthcare, catalog no. 17-0780-01), eluted with 0.1 M citric acid at pH 4.5, and neutralized with 1 M Tris-HCl at pH 9.0. Buffers were then exchanged to phosphate-buffered saline (PBS), and proteins were concentrated by 30-kDa cutoff Amicon Ultra-15 centrifugal filter units (Millipore, catalog no. UFC903096).

Flow cytometry for detecting interactions of RBD-Ig proteins with cell surface ACE2 orthologs.293T cells were seeded at 20% density in 48-well plates at 12 to 15 h before transfection. Cells in each well were then transfected with 0.5 μl of Lipofectamine 2000 (Life Technologies, catalog no. 11668019) in complex with 200 ng of plasmid encoding one of the 16 ACE2 orthologs or a D30E mutant of the human ACE2. Culture medium was changed at 6 h after transfection. Cells were then detached with 5 mM EDTA (Life Technologies, catalog no. 15575020) at 36 h posttransfection. The cells were then stained with 5 μg/ml RBD-Ig proteins at 37°C for 10 min, washed three times, and then stained with 2 μg/ml Alexa488-conjugated goat anti-mouse IgG secondary antibody (Invitrogen, catalog no. A-11001) at room temperature for 20 min. After another three washes, cells were analyzed by Attune NxT flow cytometer (Thermo Fisher), and signals of 10,000 FSC/SSC-gated cells were collected for each sample.

Western Blot to detect S-tagged ACE2 (ACE2-S-tag) or C9-tagged spike (Spike-C9-tag) expression in 293T cells.293T cells were seeded at 20% density in 6-well plates at 12 to 15 h before transfection. Cells in each well were then transfected with 2 μg of plasmid in complex with 5 μl of Lipofectamine 2000 (Life Technologies, catalog no. 11668019). At 36 h after transfection, the cells were lysed, and 10 μg of total protein was used for Western blotting. ACE2-S-tag expression was detected by using 6.2, a mouse anti-S-tag monoclonal antibody (Invitrogen, catalog no. MA1-981), and a horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG Fc secondary antibody (Invitrogen, catalog no. 31437). Beta-actin was used as an internal control. Spike-C9-tag expression was then detected by using 1D4, a mouse anti-C9-tag monoclonal antibody (Invitrogen, catalog no. MA1-722), and the HRP-conjugated goat anti-mouse IgG Fc secondary antibody (Invitrogen, catalog no. 31437).

Production of reporter retroviruses pseudotyped with coronavirus spike proteins or VSV-G.MLV retroviral vector-based coronavirus-spike and VSV-G pseudotypes were produced using a previously described protocol (23) with some modifications. 293T cells were seeded at 30% density in 150 mm dish at 12 to 15 h before transfection. Cells were then transfected with 67.5 μg of PEI MAX 4000 (Polysciences, Inc., catalog no. 24765-1) in complex with 11.25 μg of plasmid encoding a coronavirus spike protein or VSV-G, 11.25 μg of plasmid encoding murine leukemia virus) Gag and Pol proteins, and 11.25 μg of a pQCXIP-based GFP or luciferase reporter plasmid. Eight hours after transfection, cell culture medium was refreshed and changed to growth medium containing 2% fetal bovine serum (FBS; Gibco, catalog no. 10099141C) and 25 mM HEPES (Gibco, catalog no. 15630080). Cell culture supernatants were collected at 36 to 48 h posttransfection, spun down at 3,000 × g for 10 min, and filtered through 0.45-μm filter units to remove cell debris. Coronavirus spike-pseudotyped viruses were then concentrated 10 times at 2,000 × g using 100-kDa cutoff Amicon Ultra-15 centrifugal filter units (Millipore, catalog no. UFC910024).

Pseudovirus infection of 293T cells expressing ACE2 orthologs.293T cells were seeded at 20% density in polylysine precoated 48-well plates 12 to 15 h before transfection. Cells in each well were then transfected with 0.5 μl of Lipofectamine 2000 (Life Technologies, catalog no. 11668019) in complex with 200 ng of a vector control plasmid or a plasmid encoding one of the 16 ACE2 orthologs. Cell culture medium was refreshed at 6 h posttransfection. After another 18 h, the cells in each well were infected with 50 μl of SARS-CoV-2 wild-type pseudovirus (10× concentrated), 20 μl of SARS-CoV-2 ΔFurin pseudovirus (10× concentrated), 50 μl of Bat-CoV RaTG13 pseudovirus (10× concentrated), 10 μl of SARS-CoV pseudovirus (10× concentrated), or 10 μl of VSV-G pseudovirus diluted in 200 μl of culture medium containing 2% FBS (Gibco, catalog no. 10099141C). The culture medium was refreshed at 2 h after pseudovirus infection, and the medium was refreshed every 12 h. For the luciferase reporter virus-infected cells, the cell culture supernatants were collected and subjected to a Gaussia luciferase assay at 48 h postinfection. The GFP reporter virus-infected cells were stained with Hoechst 33342 (Invitrogen, catalog no. H3570) and subjected to fluorescence microscopy (IX73 microscope; Olympus) at 48 h postinfection.

Gaussia luciferase luminescence flash assay.To measure Gaussia luciferase expression, 20 μl of cell culture supernatant of each sample and 100 μl of assay buffer containing 4 μM coelenterazine native (Biosynth Carbosynth, catalog no. C-7001) were added to one well of a 96-well black opaque assay plate (Corning, catalog no. 3915) and measured with the Tristar5 multifunctional microplate reader (Berthold Technologies) for 0.1 s/well.

Coronavirus pseudovirus neutralization assay.Coronavirus spike protein-pseudotyped luciferase reporter viruses were prediluted in DMEM (2% FBS, heat inactivated) containing titrated amounts of RBD-Ig or ACE2-Ig variant proteins. An Fc fusion protein of an anti-influenza hemagglutinin (HA) antibody, F10-scFv (25), was used as a control protein here. Virus-inhibitor mixtures were then added to ACE2-expressing 293T or HeLa cells in polylysine (Sigma, catalog no. P4832-50ml) precoated 96-well plates and incubated overnight at 37°C. The cells were then washed with serum-free medium and incubated in 150 μl of DMEM (2% FBS) at 37°C. Cell culture supernatants were collected for Gaussia luciferase assay at 48 h postinfection.

SARS-CoV-2 live virus infection of ACE2 ortholog-expressing 293T cells.293T cells expressing one of the 16 ACE2 orthologs were inoculated with SARS-CoV-2 live virus at 800 50% tissue culture infective dose(s) (TCID₅₀) and incubated for 1 h at 37°C. The cells were then washed with serum-free medium and incubated in 150 μl of DMEM (2% FBS) at 37°C for an additional 24 h. The cells were then fixed with 4% paraformaldehyde in PBS, permeabilized with 0.5% Triton X-100, and sequentially stained with 1:200-diluted rabbit anti-SARS-CoV-2 nucleocapsid polyclonal antibody (Sino Biological, catalog no. 40588-T62) at 37°C for 30 min, 4 μg/ml of Alexa Fluor 568 goat anti-rabbit IgG (Invitrogen, catalog no. A-11011) at 37°C for 20 min, and 0.5 μg/ml of DAPI (4′,6′-diamidino-2-phenylindole; Sigma-Aldrich, catalog no. D9542-5mg) at room temperature for 10 min. Stained cells were then examined under fluorescence microscope (IX73 microscope; Olympus).

SARS-CoV-2 live virus neutralization by ACE2-Ig variants.SARS-CoV-2 live virus at 800 TCID₅₀ were prediluted in DMEM (2% FBS, heat inactivated) containing titrated amounts of ACE2-Ig variant proteins and incubated at 37°C for 1 h. Virus-inhibitor mixtures were then added to ACE2-expressing 293T or HeLa cells in polylysine-precoated 48-well plates and incubated for 1 h at 37°C. The cells were then washed with serum-free medium and incubated in 400 μl of DMEM (2% FBS) at 37°C for 24 to 40 h. Cells were then fixed for immunofluorescence staining of viral nucleocapsid proteins as described above.

Data collection and analysis.All the experiments were repeated two to four times. All of the infection assays for Fig. 3 were independently performed by two different people, and all the data are reproducible in different hands. The key neutralization assays of Fig. 4 to 6 were independently performed by two or three different people, and all of the data are reproducible in different hands. Attune NxT Software (Thermo Fisher) was used to collect and analyze flow cytometry data. Image Lab Software (Bio-Rad) was used to collect SDS-PAGE and Western blot image data. Cell Sens Software (Olympus) was used to collect fluorescence microscopy data. ICE Software (Berthold Technologies) was used to collect luciferase assay data. GraphPad Prism 6.0 software was used for figure preparation and statistical analyses.

Statistical analysis.Data expressed as mean values ± the standard deviations (SD). Statistical analyses were performed using two-sided two-sample Student t test using GraphPad Prism 6.0 software when applicable. Differences were considered significant at P < 0.01.

Data availability.The study did not generate unique data sets or code. Our research resources, including methods, plasmids, and protocols, are available upon reasonable request to qualified academic investigators for noncommercial research purposes. All reagents developed, such as vector plasmids, as well as detailed methods, will be made available upon written request.