Methods

Constructs and plasmids
Codon-optimized cDNA (sequence shown in Supplementary Table 2) encoding SARS-CoV-2 S glycoprotein (QHU36824.1) with C-terminal 19 amino acids deletion was synthesized and cloned into eukaryotic cell expression vector pCMV14-3×Flag between the Hind III and BamH I sites. Plasmids encoding SARS-CoV S glycoprotein, MERS-CoV S glycoprotein and MHV S glycoprotein (sequences shown in Supplementary Table 2) were constructed by inserting DNA fragment encoding codon-optimized SARS-CoV S protein (AAP13441.1) lacking the last 19 amino acids (aa), MERS-CoV S protein (AFS88936.1) lacking the last 16 aa but with a FLAG tag at the C terminus, or full-length MHV S protein (AAU06356.1) into pcDNA3.1between BamH I and Not I sites, respectively. The VSV-G encoding plasmid and lentiviral packaging plasmid psPAX2 were obtained from Addgene (Cambridge, MA). The pLenti-GFP lentiviral reporter plasmid that expresses GFP and luciferase was generously gifted by Fang Li, Duke University. All primers used in this study are listed in Supplementary Table 1.

Cell lines
Human embryonic kidney cell line 293 (#CRL-1573) and 293T expressing the SV40 T-antigen (#CRL-3216), human airway epithelial cell line Calu3 (#HTB-55), human alveolar epithelial cell line A549 (#CCL-185), human fibroblasts derived from lung tissue MRC5 (#CCL-171), African green monkey kidney cell line Vero E6 (#CRL-1586) and Vero 81 (#CCL-81), and human cervical carcinoma cell line HeLa(#CCL-2) were obtained from ATCC (Manassas, VA, USA). Human carcinoma cell line derived from hepatocyte Huh7 was kindly provided by Dr. Wei Yang (Chinese Academy of Medical Sciences, Beijing, China). Bat embryo fibroblast cells RS were isolated mid-gestation fetuses from Rhinolophus sinicus bat. HEK 239 cells stably expressing recombinant human ACE2 (293/hACE2), baby hamster kidney fibroblasts stably expressing recombinant human APN (BHK/hAPN), HeLa cells stably expressing recombinant human DPP4 (HeLa/hDPP4) were established in our lab by overexpression of these receptors. All above cells were maintained in Dulbecco’s MEM containing 10% fetal bovine serum and 100 unit penicillin, 100 μg streptomycin, and 0.25 μg Fungizone (1% PSF, Gibco) per milliliter. Rhesus monkeys kidney cell line LLC-MK2 (#CCL-7) from ATCC was maintained in Opti-MEM containing 10% FBS and 1%PSF.

Antibodies and inhibitors
Broad-spectrum cysteine protease inhibitor E64D (#HY-100229), Cathepsin L-specific inhibitor SID 26681509 (#HY-103353), Cathepsin B-specific inhibitor CA-074 (#HY-103350), PIKfyve inhibitors apilimod (#HY-14644) and YM201636 (#HY-13228), were purchased from Med Chem Express (MCE, New Jersey, USA). Calcium channel blocker tetrandrine (#S2403) was purchased from Selleck Chemicals (Texas, USA). Endosome acidification inhibitors NH4Cl (#A9434) and bafilomycin A (#19-148) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Rabbit polyclonal against SARS S1 antibodies (#40150-T62), mouse monoclonal against MERS-CoV S2 antibody (#40070-MM11), mouse monoclonal against SARS S1 antibody (#40150-MM02), rabbit polyclonal against HIV-1 Gag-p24 antibody (11695-RP01) were purchased from Sino Biological Inc. (Beijing, China). Goat polyclonal against human ACE2 antibody (#AF933) was purchased from R&D Systems (Minnesota, USA). Mouse monoclonal anti-FLAG M2 antibody was purchased from Sigma-Aldrich. Donkey Anti-Rabbit IgG (#711-035-152), Goat Anti-Mouse IgG (#115-035-146), and Rabbit Anti-Goat IgG (#305-035-003) were purchased from Jackson ImmunoResearch (West Grove, PA, USA). Alexa Fluor 488-conjugated goat anti-rabbit IgG, Alexa Fluor 488-conjugated goat anti-mouse IgG were purchased from ZSGB-Bio LLC (Beijing, China).

Production of SARS-CoV-2 S pseudovirions and virus entry
Pseudovirions were produced by co-transfection 293T cells with psPAX2, pLenti-GFP, and plasmids encoding either SARS-CoV-2 S, SARS-CoV S, VSV-G, or empty vector by using polyetherimide (PEI). The supernatants were harvested at 40, 64 h post transfection, passed through 0.45 μm filter, and centrifuged at 800 × g for 5 min to remove cell debris. To transduce cells with pseudovirions, cells were seeded into 24-well plates and inoculated with 500 μl media containing pseudovirions. After overnight incubation, cells were fed with fresh media. About 40 h post inoculation, cells were lysed with 120 μl medium containing 50% Steady-glo (promega) at room temperature for 5 min. The transduction efficiency was measured by quantification of the luciferase activity using a Modulus II microplate reader (Turner Biosystems, Sunnyvale, CA, USA). All experiments were done in triplicates, and repeated at least twice or more.

Detection of S protein of SARS-CoV-2 by western blot
The spike glycoprotein of SARS-CoV-2 in cells and on pseudovirions were detected by using western blot. Briefly, cells transfected with plasmids encoding spike glycoprotein of SARS-CoV-2, SARS-CoV, MERS-CoV, and MHV were lysed at 40 h post transfection by RIPA buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% NP40, 1×protease inhibitor cocktail). To pellet down pseudovirions, the viral supernatants were centrifuged at 25,000 rpm for 2 h in a Beckman SW41 rotor at 4 °C through a 20% sucrose cushion, and virus pellets were resuspended into 30 μl RIPA buffer. The samples were boiled for 10 min and separated in a 10% SDS-PAGE gel and transferred to nitrocellulose filter membranes. After blocked by 5% milk, the membranes were blotted with primary antibodies, followed by incubated with horseradish peroxidase (HRP) conjugated secondary antibodies (1:5000) and visualized with Chemiluminescent Reagent (Bio-Rad). MHV S proteins were detected using polyclonal goat anti-MHV S antibody AO4 (1:2000); SARS-CoV S proteins were blotted with either polyclonal anti-SARS S1 antibodies T62 (1:2000) (Sinobiological Inc, Beijing, China) or mouse monoclonal against SARS S1 antibody MM02 (1:1000) (Sinobiological Inc, Beijing, China), MERS-CoV and SARS-CoV-2 S proteins were detected using mouse monoclonal anti-MERS S (1:1000) (Sinobiological Inc, Beijing, China) and anti-FLAG M2 antibody (1:1000) (Sigma, St. Louis, MO, USA), respectively.

Effects of agents and inhibitors on pseudovirion entry
HEK 293/hACE2 cells were pretreated with either lysosomotropic agents (endosome acidification inhibitor NH4Cl and bafilomycin A; PIKfyve inhibitor apilimod and YM201636; calcium channel blocker tetrandrine) or cathepsin inhibitors (cathepsin L inhibitor SID26881509, cathepsin B inhibitor CA-074, and cathepsin inhibitor E64D) for 1 h at 37 °C, then spin-inoculated with pseudovirions in the presence of inhibitor at 1200 × g for 30 min. After overnight incubation, cells were fed with fresh medium without inhibitor. Cells were lysed at 48 h post inoculation and their luciferase activity was measured.

Flow cytometric analysis of S protein expression
Briefly, 293T cells were transfected with 2 μg of plasmids encoding either SARS-CoV-2 S, SARS-CoV S or MERS-CoV S protein using PEI. Forty hours later, cells were detached by using PBS with 1 mM EDTA. After washing, cells were incubated with polyclonal rabbit anti-SARS S1 antibodies T62 (1:200 dilution) (Sinobiological Inc., Beijing, China) for 1 h on ice, followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:200) (ZSGB-Bio LLC, Beijing, China) for 1 h. Cells were then analyzed by flow cytometry.

MTT assay
MTT assay was used to assess cell viability. Briefly, cells were seeded into a 96-well plate at a cell density of 3000 per well and allowed to adhere for 24 h, followed by treatment with serially diluted inhibitors for 12 h. Two microliters of 5 mg/ml MTT was added to the medium 24 h later and incubated for 4 h at 37 °C. After removing the culture medium, 100 µl of DMSO was added. The absorbance was measured at 570 nm using a microplate spectrophotometer (Multiskan FC, Thermo Scientific). All experiments were performed in triplicate, and the cell viability of cells was calculated as the ratio of each experimental condition to the control.

Cell–cell fusion assay
HEK-293T cells were co-transfected with plasmids encoding CoV S glycoprotein and eGFP. For trypsin-dependent cell–cell fusion, cells were detached with trypsin (0.25%) at 40 h post transfection and overlaid on an 80% confluent monolayer of 293/hACE2 cells at a ratio of approximately one S-expressing cell to three receptor-expressing cells. For trypsin-independent cell–cell fusion, cells were detached with 1 mM EDTA and overlaid on 293/hACE2 cells. After 4 h incubation, images of syncytia were captured with a Nikon TE2000 epifluorescence microscope running MetaMorph software (Molecular Devices). All experiments were done in triplicate and repeated at least three times.

Expression and purification of soluble human ACE2
DNA fragments encoding residues 19-615 of human hACE2 with N-terminal FLAG and 6×his tags were cloned between Sal I and Hind III of modified pFASTBac1 vector with gp67 signal peptide. The soluble receptors were expressed in High Five insect cells using the bac-to-bac system (Invitrogen) and purified using nickel affinity and ion-exchange chromatography.

Soluble hACE2-binding assay
HEK-293T cells were transfected with plasmids encoding SARS-CoV-2 S, SARS-CoV S, or MERS-CoV S protein. Cells were detached with 1 mM EDTA 40 h post transfection, washed twice with 3% FBS in 1×PBS, and incubated with 5 μg/ml soluble hACE2 for 1 h on ice. After washing three times with 3% FBS in 1×PBS, cells were incubated with polyclonal goat anti-human ACE2 antibody (1:200) (R&D Systems, MN, USA) for 1 h, followed by FITC-conjugated rabbit anti-goat secondary antibody (1:500) (Jackson ImmunoResearch, PA, USA). After washing, cells were then analyzed by flow cytometry.

Soluble hACE2 inhibition assay
Briefly, lentiviruses pseudotyped with SARS-CoV-2 S, SARS-CoV S or VSV-G were pre-incubated with serially diluted soluble hACE2 for 1 h on ice. The mixture were then added on 293/hACE2 cells, followed by centrifugation inoculation for 1 h at room temperature. Cells were fed with fresh medium 6 h later and lysed at 48 h post inoculation. Pseudoviral transduction was measured according to luciferase activities.

Pseudovirus neutralization assay
SARS-CoV S, SARS-CoV-2 S, and VSV-G pseudovirions were pre-incubated with serially diluted either polyclonal rabbit anti-SARS S1 antibodies T62 or patient sera for 1 h on ice, then virus-antibody mixture was added onto 293/hACE2 cells in a 96-well plate. After 6 h incubation, the inoculum was replaced with fresh medium. Cells were lysed 40 h later and pseudovirus transduction was measured as previously described. Prior to experiments, patient sera were incubated at 56 °C for 30 min to inactivate complement.

Serum collection
Five patients were hospitalized with pneumonia in Hengshui Third People’s Hospital, Hengshui, Heibei province, China, and their respiratory specimens were collected for coronavirus detection and were positive for SARS-CoV-2. After all patients recovered, their sera were collected right before discharge with informed consent. Serum from a recovered SARS patient was also collected at two years after recovery with informed consent.

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