Cells and viruses
HEK293FT cells expressing the SARS-CoV-2 S protein and DSP8-11 (293FT/SARS-CoV-2 S/DSP8-11) were maintained in D-MEM supplemented with 10% foetal calf serum (FCS), 100 mg/mL penicillin, 100 mg/mL streptomycin, 1 mg/mL puromycin, and 10 mg/mL blasticidin. HEK293FT cells expressing ACE2, TMPRSS2, and DSP1-7 (293FT/ACE2/TMPRSS2/DSP1-7) were maintained in D-MEM supplemented with 10% FCS, 100 mg/mL penicillin, 100 mg/mL streptomycin, 1 mg/mL puromycin, 10 mg/mL blasticidin, and 300 mg/mL hygromycin. HEK293FT cells expressing DSP8-11 (293FT/DSP8-11) were maintained in D-MEM supplemented with 10% FCS, 100 µg/mL penicillin, 100 µg/mL streptomycin, and 1 mg/mL puromycin. VeroE6TMPRSS2 cells were obtained from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan)51. VeroE6TMPRSS2 cells were maintained in D-MEM supplemented with 10% FCS, 100 µg/mL of penicillin, 100 µg/mL of streptomycin, and 1 mg/mL of geneticin antibiotic. SARS-CoV-2 NCGM-05-2N strain (SARS-CoV-205-2N) was isolated from nasopharyngeal swabs of a patient with COVID-19 who was admitted to the National Center for Global Health and Medicine, Tokyo, Japan.
Chemicals and antibodies
Myriocin, SKI-II, and FTY720 were obtained from Abcam (Cambridge, UK). Fumonicin B1, CBE, amitriptyline, and ceranib-2 were obtained from Sigma-Aldrich (St. Louis, MO, USA). 4-HPR and HPA12 were obtained from Tokyo Chemical Industry (Tokyo, Japan). GT11 was obtained from Avanti Polar Lipids (Alabaster, AL, USA). DL-threo-PPMP, DL-erythro-PPMP, and GW4869 were obtained from Cayman Chemical (Ann Arbor, MI, USA). All compounds were dissolved in DMSO and diluted to a final concentration of 0.2% DMSO in cell culture medium.
Mouse IgG1 monoclonal anti-ACE2 antibody (catalogue no. 66699-1-Ig) was obtained from Proteintech Group Inc. (Rosemont, IL, USA). Mouse IgG1 isotype control was obtained from BioLegend (San Diego, CA, USA). Goat PE-conjugated anti-mouse IgG polyclonal antibody (catalogue no. 12-4010-82) was obtained from Thermo Fisher Scientific (Waltham, MA, USA). N-palmitoyl-d31-D-erythro-sphingosine (D31-Cer) and N-palmitoyl-d31-D-erythro-sphingosylphosphorylcholine (D31-SM) were obtained from Avanti Polar Lipids, and N-omega-d3-hexadecanoyl-glucopsychosine (D3-GlcCer) was obtained from Cayman Chemical.
Rabbit polyclonal anti-ACE2 antibody (catalogue no. ab15348), rabbit IgG monoclonal anti-transferrin receptor antibody (catalogue no. ab214039), and rabbit IgG monoclonal anti-flotillin 1 antibody (catalogue no. ab133497) were obtained from Abcam. Goat horseradish peroxidase-conjugated anti-rabbit IgG antibody (catalogue no. 7074S) was obtained from Cell Signaling Technology (Beverly, MA, USA).
Cytotoxicity assay
293FT/ACE2/TMPRSS2/DSP1-7 and VeroE6TMPRSS2 cells were seeded in 96-well plates at 1 × 104 cells/well and 5 × 103 cells/well, respectively. On the following day, the cells were cultured with the specific compounds for 2 days (293FT/ACE2/TMPRSS2/DSP1-7 cells) or 3 days (VeroE6TMPRSS2 cells), and then the 50% cytotoxic concentration (CC50) values were determined using the WST-8 assay employing Cell Counting Kit-8 (Dojindo, Kumamoto, Japan).
DSP-based cell-cell fusion assay
The cell-cell fusion assay was conducted as previously described by Yamamoto et al.25, with slight modifications. Target cells (293FT/ACE2/TMPRSS2/DSP1-7) and effector cells (293FT/SARS-CoV-2 S/DSP8-11) were seeded in 24-well plates at 4 × 104 cells/well. On the following day, the medium of the target cells was exchanged with D-MEM containing 10% FCS and various concentrations of each of the compounds to be tested, and the cells were incubated for an additional 2 days. Cells were washed with phosphate-buffered saline (PBS) and were detached using Cellstripper containing EDTA but no trypsin (Corning, Christiansburg, VA, USA). Cells were resuspended in serum-free D-MEM and centrifuged at 500 × g for 2 min. After aspirating the suspension, cells were resuspended in serum-free D-MEM containing 1% Nutridoma SP (Roche, Basel, Switzerland) and 6 mM EnduRen (Promega, Madison, WI, USA), a substrate for RL. The effector and target cells were mixed in the wells of a 96-well plate, and after incubating at 37 °C for 4 h the RL activity was measured using a SpectraMax i3x Microplate Reader (Molecular Devices, San Jose, CA, USA).
Antiviral assay
VeroE6TMPRSS2 cells were seeded in 96-well plates (5 × 103 cells/well). On the following day, the cells were cultured with each of the tested compounds for 3 days before adding SARS-CoV-205-2N. The cells were inoculated at a multiplicity of infection of 0.01. After culturing the cells with the specific compounds and SARS-CoV-205-2N for 3 days, the level of cytopathic effect observed in SARS-CoV-2-exposed cells was determined using the WST-8 assay.
Lipid extraction and quantification of sphingolipids by LC-MS/MS
Lipid extraction and quantification of sphingolipids by LC-MS/MS were performed as described previously52-54. 293FT/ACE2/TMPRSS2/DSP1-7 and VeroE6TMPRSS2 cells were seeded in 6-well plates at 2 × 105 cells/well and 7 × 104 cells/well, respectively. On the following day, the cells were cultured with each of the tested compounds for 2 days (293FT/ACE2/TMPRSS2/DSP1-7 cells) or 3 days (VeroE6TMPRSS2 cells), after which the cells were washed once with cold PBS, collected in 100 mL of cold PBS, and then homogenised by sonication. Part of the sample (5 mL) was used in the bicinchoninic acid protein assay to determine the amount of protein. Total lipids were extracted by adding 375 mL of chloroform:methanol (1:2, v/v) containing 40 pmol of each component, specifically D31-Cer, D31-SM, and D3-GlcCer. Deuterated D31-Cer, D31-SM, and D3-GlcCer were used as internal standards for Cer and DHCer, SM and DHSM, and GlcCer and DHGlcCer, respectively. After sonicating the single-phase mixture, 100 mL of chloroform:methanol:5N NaOH (1:2:0.8, v/v/v) was added and the solution was incubated for 1 h at 37 °C, followed by neutralisation with acetic acid. Subsequently, 158 mL of chloroform and 158 mL of water were added and the mixture was then vigorously shaken using a vortex, before centrifuging for 1 min at 13,000 × g at 4 °C. The lower phase was withdrawn and dried and then resuspended in acetonitrile:methanol (1:1, v/v), sonicated for 10 s, centrifuged at 14,000 × g for 5 min, and the supernatant was transferred to vials. The concentrations of Cer, DHCer, GlcCer, DHGlcCer, SM, and DHSM were analysed using the QTRAP4500 instrument (SCIEX, Framingham, MA, USA). Sphingolipids containing C16:0, C18:0, C20:0, C22:0, C24:0, and C24:1 fatty acids were detected using a multiple reaction monitoring method, as described in Supplementary Table 1.
Analysis of cell-surface expression of ACE2
293FT/ACE2/TMPRSS2/DSP1-7 cells in 24-well plates were treated with the indicated concentrations of the compounds for 2 days. Cells were harvested by Cellstripper (Corning) and fixed with 4% formaldehyde before incubation with either anti-ACE2 antibody (Proteintech) or mouse IgG1 isotype control (BioLegend) antibody at 4 °C. Cells were then stained with PE-conjugated secondary antibody (Thermo Fisher Scientific) and analysed using the FACSCanto II instrument (BD Biosciences, San Jose, CA, USA). Data were analysed with FlowJo software (Tree Star Inc., San Carlos, CA, USA).
S protein stimulation of 293FT/SARS-CoV-2 S/DSP8-11 cells and isolation of lipid raft domains
Target cells (293FT/ACE2/TMPRSS2/DSP1-7), S protein expressing cells (293FT/SARS-CoV-2 S/DSP8-11), and cells not expressing S protein (293FT/DSP8-11) were seeded at 7 × 105 cells per 10-cm-diameter dish. On the following day, the medium of the target cells was exchanged with D-MEM containing 10% FCS and 5 mM 4-HPR, and the cells were incubated for an additional 2 days. Cells were then washed with PBS and detached by Cellstripper (Corning) before resuspending in serum free D-MEM and centrifuging at 500 × g for 2 min. After aspirating the suspension, the cells were resuspended in serum-free D-MEM containing 1% Nutridoma SP (Roche). The target cells were mixed with 293FT/SARS-CoV-2 S/DSP8-11 (presence of SARS-CoV-2 S protein stimulation) or 293FT/DSP8-11 (absence of SARS-CoV-2 S protein stimulation) cells, incubated for 30 min, and then washed with cold PBS. Subsequently, sucrose gradient analysis for ACE2 was performed as described previously55 with slight modifications. The pellets were homogenised in 2 mL of TNE buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 1 mM EDTA) with 0.1% TritonX-100. The sucrose content of the homogenate was then adjusted to 40% by adding 80% sucrose. A linear sucrose gradient (5–30%) in 7 mL of TNE buffer without TritonX-100 was layered over the lysate. The gradients were centrifuged for 17 h at 200,000 × g at 4 °C using a P40ST rotor (Hitachi Koki Co., Ltd, Tokyo, Japan). Eleven fractions were collected from the top of the gradient, followed by immunoblot analysis using anti-ACE2, anti-transferrin receptor (non-raft marker), and anti-flotillin 1 (raft marker) antibodies (all from Abcam).
Analysis of cellular membrane fluidity
Analysis of membrane fluidity was performed as described previously56 with slight modifications. Briefly, 293FT/ACE2/TMPRSS2/DSP1-7 cells in 24-well plates were treated with the indicated concentrations of the compounds for 2 days. Cells were detached by Cellstripper (Corning), resuspended in PBS, and centrifuged at 500 × g for 2 min. The suspension was aspirated and the cells were labelled with a solution of 15 mM pyrenedecanoic acid and 0.08% F-127 in perfusion buffer, in the dark for 20 min with rocking at 25 °C. Excess probe was removed and the cells were washed with PBS. The cells were resuspended in 450 mL PBS; 200 mL was aliquoted into a 96-well plate and fluorescence was measured using an excitation wavelength of 350 nm and two emission wavelengths: 400 nm for the monomer and 470 nm for the excimer, using a SpectraMax i3x Microplate reader (Molecular Devices). The ratio of excimer to monomer fluorescence is related to cellular membrane fluidity, with a higher ratio reflecting greater fluidity.
Statistical analysis
All statistical analyses were performed with GraphPad PRISM 6 (GraphPad Software, San Diego, CA, USA).
Data availability
Data that support the findings of this study are available from the corresponding author upon reasonable requests. Source data are provided within this paper.