Journal: Cell Discovery

Article Title: Heparan sulfate assists SARS-CoV-2 in cell entry and can be targeted by approved drugs in vitro

doi: 10.1038/s41421-020-00222-5

Figure Lengend Snippet: a The chemical structures of heparan sulfate and heparin. b The experimental scheme for inhibitor testing in HEK293T-ACE2-GFP cells. c Heparin mitigates the entry of SARS-Cov and SARS-CoV-2 PP. ACE2-GFP HEK293T cells were transduced with SARS-Cov and SARS-CoV-2 PP in the presence of heparin as indicated. The ratio of luciferase vs GFP was measured 24 h post-transduction. Error bars indicate SEM, n = 4. d Heparin interacts with Spike in a salt sensitive manner. Spike (600 ng) was incubated with either control or heparin-conjugated beads in a buffer containing 0 or 150 mM NaCl. Bound proteins were analyzed by SDS-PAGE and Coomassie blue staining. Note that a small amount of Spike interacts with heparin in the presence of 150 mM NaCl.

Article Snippet: HEK293T cells stably expressing human ACE2 tagged with GFP (ACE2-GFP) were generated by transfecting cells with pCMV-ACE2-GFP (Covex), and stable clones were hand-picked after neomycin (1 mg/mL) selection for 1 week.

Techniques: Transduction, Luciferase, Incubation, Control, SDS Page, Staining

Journal: Cell Discovery

Article Title: Heparan sulfate assists SARS-CoV-2 in cell entry and can be targeted by approved drugs in vitro

doi: 10.1038/s41421-020-00222-5

Figure Lengend Snippet: a The HSPG biosynthetic pathway. Genes chosen for knockdown or knockout (KO) are in red. b Knockdown of XYLT2 reduces SARS-Cov and SARS-CoV-2 PP entry. ACE2-GFP cells transfected with either control or XYLT2 siRNA were transduced with SARS-Cov (gray) or SARS-CoV-2 (orange) PP for 24 h and the ratio of luciferase/GFP was determined. A parallel experiment done without the virus provides another control for the effect of gene knockdown on cell viability (blue). Error bars indicate SEM, n = 4, ** P < 0.01, **** P < 0.0001 by unpaired Student t -test. c SCL35B2 is required for SARS-Cov and SARS-CoV-2 cell entry. As in b , except that control and SLC35B2 CRISPR KO cells were used. d , e SLC35B2 promotes the binding of SARS-CoV-2 PP to cells. d ACE2-GFP cells were spin-infected at 4 °C for 1 h. After washing, the virus bound to the cells was detected by immunoblotting. e The binding of SARS-CoV-2 PP to control and SLC35B2 -deficient cells was analyzed by immunoblotting. S and S2 indicate the full-length and furin-cleaved S2 fragment, respectively.

Article Snippet: HEK293T cells stably expressing human ACE2 tagged with GFP (ACE2-GFP) were generated by transfecting cells with pCMV-ACE2-GFP (Covex), and stable clones were hand-picked after neomycin (1 mg/mL) selection for 1 week.

Techniques: Knockdown, Knock-Out, Transfection, Control, Transduction, Luciferase, Virus, CRISPR, Binding Assay, Infection, Western Blot

Journal: Cell Discovery

Article Title: Heparan sulfate assists SARS-CoV-2 in cell entry and can be targeted by approved drugs in vitro

doi: 10.1038/s41421-020-00222-5

Figure Lengend Snippet: a , b Sunitinib and BNTX inhibit Spike-dependent entry of PP. HEK293-ACE2-GFP cells were transduced with SARS-Cov and SARS-CoV-2 PP in the presence the indicated drugs. The luciferase expression was measured 48 h post-transduction. Error bars indicate SEM, n = 4. As a control for cytotoxicity, cells treated with the drugs without virus were analyzed in parallel by an ATP-based cytotoxicity assay. c BNTX protects Vero E6 cells from SARS-CoV-2-induced CPE. Viability of Vero E6 cells was measured after treatment with the indicated drugs in the presence (black) or absence (red) of the SARS-CoV-2 virus. Error bars indicate SEM, n = 2. d , e Sunitinib stimulates actin filament formation at the cell periphery. d Confocal snapshots of a U2OS cell stably expressing Tractin-EGFP before or after Sunitinib (5 μM) treatment. The red arrows indicate new actin filaments formed in a direction perpendicular to the existing filaments. The inset shows an enlarged view of the box, which highlights newly formed filopodia. e Live-cell imaging of actin filament formation after Sunitinib treatment (5 μM, 60 min). The insets show an enlarged view of the boxed area. The red arrowhead in the inset and the blue arrowhead indicate two examples of actin filament assembly. Scale bar, 5 μm. f , g BNTX disrupts actin filaments at the cell periphery. f Confocal snapshots of a Tractin-EGFP cell before and after BNTX (10 μM) treatment. Arrows indicate the peripheral actin network disrupted by BNTX. g Live-cell imaging shows the shrinking of peripheral membranes after BNTX treatment. Yellow arrowheads mark a retracting actin bundle. The blue arrowhead indicates the cell edge. h Latrunculin A inhibits SARS-CoV-2 entry. ACE2-GFP cells incubated with SARS-CoV-2 PP in the presence of Latrunculin A were analyzed for luciferase expression (entry). A parallel treatment in the absence of the virus showed the toxicity of Latrunculin A (orange). Error bars indicate SEM. n = 2, *** P < 0.001, **** P < 0.0001.

Article Snippet: HEK293T cells stably expressing human ACE2 tagged with GFP (ACE2-GFP) were generated by transfecting cells with pCMV-ACE2-GFP (Covex), and stable clones were hand-picked after neomycin (1 mg/mL) selection for 1 week.

Techniques: Transduction, Luciferase, Expressing, Control, Virus, Cytotoxicity Assay, Stable Transfection, Live Cell Imaging, Incubation

Journal: Cell Discovery

Article Title: Heparan sulfate assists SARS-CoV-2 in cell entry and can be targeted by approved drugs in vitro

doi: 10.1038/s41421-020-00222-5

Figure Lengend Snippet: a Mitoxantrone inhibits Spike-mediated entry of PP. ACE2-GFP HEK293 cells were transduced with SARS-Cov and SARS-CoV-2 PP for 48 h in the presence of Mitoxantrone as indicated. Error bars indicate SEM, n = 4. Cells treated without the virus were used to control drug cytotoxicity. b The subcellular distribution of Mitoxantrone. The scheme illustrates the experimental procedure. HEK293T cells were treated with 5 μM Mitoxantrone for 30 min at 37 °C before fractionation. The membrane pellet (P100) and the cytosol supernatant (S100) fractions were analyzed by immunoblotting and by spectrometry at the indicated wavelengths. Error bars indicate SEM, n = 3. c The membrane association of Mitoxantrone requires SLC35B2 ( 35B2 ). Cells of the indicated genotypes were treated with 5 μM Mitoxantrone for 30 min at 37 °C and then fractionated as in b . The absorbance of the P100 fractions was measured and the control sample at 680 nm was normalized to 1. Bottom panels show the cells stained with DAPI (blue) and GFP + (green) to label DNA and HS (Green), respectively. Error bars indicate SEM, n = 3. d Mitoxantrone has a higher affinity for heparin and HS than chondroitin sulfate (CS). A 650 of Mitoxantrone (5 μM) mixed with the indicated oligosaccharides was determined. The decrease in absorbance after the addition of the oligosaccharides was plotted. Error bars indicate SEM, n = 2. e The chemical structures of Mitoxantrone (MTAN) and Banoxantrone (BANO). f Banoxantrone (BANO) has reduced antiviral activity. ACE2-GFP HEK293T cells were incubated with SARS-CoV-2 in the presence of the indicated drugs for 24 h before measuring the luciferase activity. Error bars indicate SEM, n = 4. g Mitoxantrone inhibits SARS-CoV-2 cell entry. ACE2-GFP HEK293T cells were pretreated with DMSO as a control or Mitoxantrone for 30 min before incubation with SARS-CoV-2 PP. Cells were fixed 3 h later and stained with anti-Spike antibodies (red) and DAPI (blue).

Article Snippet: HEK293T cells stably expressing human ACE2 tagged with GFP (ACE2-GFP) were generated by transfecting cells with pCMV-ACE2-GFP (Covex), and stable clones were hand-picked after neomycin (1 mg/mL) selection for 1 week.

Techniques: Transduction, Virus, Control, Fractionation, Membrane, Western Blot, Staining, Activity Assay, Incubation, Luciferase