Journal: Viruses

Article Title: Metalloprotease-Dependent S2′-Activation Promotes Cell–Cell Fusion and Syncytiation of SARS-CoV-2

doi: 10.3390/v14102094

Figure Lengend Snippet: Serine protease-independent SARS-CoV-2 cell–cell fusion and syncytiation. ( A ) Experimental schematic of a qualitative in vitro cell–cell fusion assay with fluorescent reporters. Target HEK293T cells were transiently co-transfected with human ACE2 and mCherry (HEK293T-ACE2-mCherry), with or without human TMPRSS2, and were co-cultured with effector HEK293T cells transiently co-transfected with the SARS-CoV-2 (SARS2) spike protein and GFP (HEK293T-SARS2-GFP). ( B ) Western blot analysis of ACE2 and TMPRSS2 in HEK293T-ACE2-mCherry cells and the SARS2 spike protein in HEK293T-SARS2-GFP cells. ACE2 and the SARS2 spike protein were immunodetected with an anti-1D4 rhodopsin antibody, directed against the C-terminal C9-tag; TMPRSS2, with an anti-TMPRSS2-specific antibody. Western blotting data are from one experiment representative of at least three independent experiments. Immunodetection of β-actin served as a loading control. ( C ) Representative fluorescent micrographs of HEK293T-ACE2-mCherry and HEK293T-SARS2-GFP co-cultures at 16-h post-co-culture, in the presence or absence of the serine protease inhibitor camostat mesylate (120 µM; 16-h). ACE2-FL: full-length ACE2; S0: full-length SARS-CoV-2 spike protein; S2: S2 fragment of the SARS-CoV-2 spike protein; S2′: S2′ fragment of the SARS-CoV-2 spike protein. Scale bar: 500 μm.

Article Snippet: The following plasmids were obtained from Addgene: a plasmid for human ACE2 (pcDNA3.1-hACE2: Addgene plasmid #1786) was a gift from Hyeryun Choe; a plasmid for the ancestral Wuhan-Hu-1 SARS-CoV-2 spike protein (pcDNA3.1-SARS2-Spike: Addgene plasmid #145032) was a gift from Fang Li; plasmids for the SARS-CoV-2 spike proteins of the Delta (pCAGGS-S-B.1.617.2: Addgene plasmid #177097) and Omicron (pCAGGS-S-B.1.1.529: Addgene #180774) variants were gifts from Daniel Conway; a plasmid for human TMPRSS2 (pCSDest-TMPRSS2: Addgene plasmid #53887) was a gift from Roger Reeves; and a plasmid for human ADAM17 (pRK5F-TACE: Addgene plasmid #31713) was a gift from Rik Derynck.

Techniques: In Vitro, Cell-Cell Fusion Assay, Transfection, Cell Culture, Western Blot, Immunodetection, Control, Co-Culture Assay, Protease Inhibitor

Journal: Viruses

Article Title: Metalloprotease-Dependent S2′-Activation Promotes Cell–Cell Fusion and Syncytiation of SARS-CoV-2

doi: 10.3390/v14102094

Figure Lengend Snippet: Metalloprotease-dependent syncytiation of the SARS-CoV-2 spike protein ( A ) Experimental schematic of the quantitative in vitro cell–cell fusion assay with split β-galactosidase reporter. ( B ) Effects of protease inhibitors (AEBSF, 1 mM; aprotinin, 10 μM; camostat mesylate, 120 μM; EDTA, 2.5 mM; EGTA, 2.5 mM; leupetin, 10μM; pepstatin A, 10 μM; 16-h) on fusion-associated β-galactosidase-activity of HEK293T-ACE2-⍺ and HEK293T-SARS2-ω co-cultures (one-way ANOVA; Dunnett post hoc). ( C ) Effects of hydroxamate-based metalloprotease inhibitors on fusion-associated β-galactosidase-activity of HEK293T-ACE2-⍺ and HEK293T-SARS2-ω co-cultures (two-way ANOVA; Tukey post hoc). ( D ) Representative fluorescent micrographs of HEK293T-ACE2 and HEK293T-SARS2-GFP co-cultures post-treatment with hydroxamate-based metalloprotease inhibitors. ( E ). Effects of hydroxamate-based metalloprotease inhibitors on fusion-associated β-galactosidase-activity of Huh-7-⍺ and HEK293T-SARS2-ω co-cultures (two-way ANOVA; Tukey post hoc). ( F ) Representative fluorescent micrographs of Huh-7 and HEK293T-SARS2-GFP co-cultures post-treatment with TAPI-1 and/or camostat mesylate. Syncytiation was visualised at 16-h post-co-culture on an EVOS FL Auto Imaging System (Thermo Fisher Scientific). Camostat: Camostat mesylate (120 μM; 16-h); BATIM: Batimastat (20 μM; 16-h); MARIM: Marimastat (20 μM; 16-h); TAPI: TAPI-1 (20 μM; 16-h); SARS2: SARS-CoV-2 spike protein. Probability value: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. Scale bar: 500 μm. Fluorescent micrographs are also available in .

Article Snippet: The following plasmids were obtained from Addgene: a plasmid for human ACE2 (pcDNA3.1-hACE2: Addgene plasmid #1786) was a gift from Hyeryun Choe; a plasmid for the ancestral Wuhan-Hu-1 SARS-CoV-2 spike protein (pcDNA3.1-SARS2-Spike: Addgene plasmid #145032) was a gift from Fang Li; plasmids for the SARS-CoV-2 spike proteins of the Delta (pCAGGS-S-B.1.617.2: Addgene plasmid #177097) and Omicron (pCAGGS-S-B.1.1.529: Addgene #180774) variants were gifts from Daniel Conway; a plasmid for human TMPRSS2 (pCSDest-TMPRSS2: Addgene plasmid #53887) was a gift from Roger Reeves; and a plasmid for human ADAM17 (pRK5F-TACE: Addgene plasmid #31713) was a gift from Rik Derynck.

Techniques: In Vitro, Cell-Cell Fusion Assay, Activity Assay, Co-Culture Assay, Imaging

Journal: Viruses

Article Title: Metalloprotease-Dependent S2′-Activation Promotes Cell–Cell Fusion and Syncytiation of SARS-CoV-2

doi: 10.3390/v14102094

Figure Lengend Snippet: ADAM17 promotes S2′-activation of the SARS-CoV-2 spike protein. ( A , B ) Effects of phorbol ester-induced endogenous ADAM17 activity or exogenous ADAM17 overexpression on fusion-associated β-galactosidase-activity of HEK293T-ACE2-⍺ cells co-cultured with HEK293T-SARS2-ω cells, with or without hydroxamate-based metalloprotease (two-way ANOVA; Tukey post hoc). ( C , D ) Western blot analysis of the SARS-CoV-2 spike protein (SARS2) under conditions of phorbol ester-induced ADAM17 activity or exogenous ADAM17 overexpression, with or without TAPI-1. The SARS-CoV-2 spike protein (SARS2) was immunodetected with an anti-1D4 rhodopsin antibody, directed against the C-terminal C9-tag. Western blotting data are from one experiment representative of at least three independent experiments. Immunodetection of β-actin served as a loading control. PMA: Phorbol 12-myristate 13-acetate (200 ng/mL; 2-h) BATIM: Batimastat (20 μM; 16-h); MARIM: Marimastat (20 μM; 16-h); TAPI: TAPI-1 (20 μM; 16-h); S0: full-length SARS-CoV-2 spike protein; S2: S2 fragment of the SARS-CoV-2 spike protein; S2′: S2′ fragment of the SARS-CoV-2 spike protein. Probability value: ***, p < 0.001; ****, p < 0.0001.

Article Snippet: The following plasmids were obtained from Addgene: a plasmid for human ACE2 (pcDNA3.1-hACE2: Addgene plasmid #1786) was a gift from Hyeryun Choe; a plasmid for the ancestral Wuhan-Hu-1 SARS-CoV-2 spike protein (pcDNA3.1-SARS2-Spike: Addgene plasmid #145032) was a gift from Fang Li; plasmids for the SARS-CoV-2 spike proteins of the Delta (pCAGGS-S-B.1.617.2: Addgene plasmid #177097) and Omicron (pCAGGS-S-B.1.1.529: Addgene #180774) variants were gifts from Daniel Conway; a plasmid for human TMPRSS2 (pCSDest-TMPRSS2: Addgene plasmid #53887) was a gift from Roger Reeves; and a plasmid for human ADAM17 (pRK5F-TACE: Addgene plasmid #31713) was a gift from Rik Derynck.

Techniques: Activation Assay, Activity Assay, Over Expression, Cell Culture, Western Blot, Immunodetection, Control

Journal: Viruses

Article Title: Metalloprotease-Dependent S2′-Activation Promotes Cell–Cell Fusion and Syncytiation of SARS-CoV-2

doi: 10.3390/v14102094

Figure Lengend Snippet: SARS-CoV-2 variants of concern are susceptible to metalloprotease inhibition. ( A ) Western blot analysis of the SARS-CoV-2 spike protein (SARS2) of the ancestral Wuhan-Hu-1 strain or the Omicron (B.1.1.5129) or Delta (B.1.617.2) variants in transiently transfected HEK293T cells. The SARS-CoV-2 spike protein of the ancestral Wuhan-Hu-1 strain was immunodetected with an anti-1D4 rhodopsin antibody, directed against the C-terminal C9-tag, and the spike proteins of the SARS-CoV-2 Omicron (B.1.1.5129) or Delta (B.1.617.2) variants were immunodetected with an anti-FLAG antibody, directed against the C-terminal FLAG tag. Western blotting data are from one experiment representative of at least three independent experiments. Immunodetection of β-actin served as a loading control. ( B ) Comparison of the fusion-associated β-galactosidase-activity of HEK293T-ACE2-⍺ and HEK293T-SARS2-ω co-cultures, wherein HEK293T-SARS2-ω express either the ancestral Wuhan-Hu-1 strain, the Omicron (B.1.5129), Delta (B.1.617.2) SARS-CoV-2 spike protein (one-way ANOVA; Dunnett post hoc). ( C , D ) Effects of hydroxamate-based metalloprotease inhibitors on fusion-associated β-galactosidase-activity of HEK293T-ACE2-⍺ and HEK293T-SARS2-ω co-cultures, wherein HEK293T-SARS2-ω express either the Delta (B.1.617.2) or Omicron (B.1.5129) (two-way ANOVA; Tukey post hoc). BATIM: Batimastat (20 μM; 16-h); MARIM: Marimastat (20 μM; 16-h); TAPI: TAPI-1 (20 μM; 16-h); SARS2: SARS-CoV-2 spike protein; S0: full-length SARS-CoV-2 spike protein; S2: S2 fragment of the SARS-CoV-2 spike protein. Probability value: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Article Snippet: The following plasmids were obtained from Addgene: a plasmid for human ACE2 (pcDNA3.1-hACE2: Addgene plasmid #1786) was a gift from Hyeryun Choe; a plasmid for the ancestral Wuhan-Hu-1 SARS-CoV-2 spike protein (pcDNA3.1-SARS2-Spike: Addgene plasmid #145032) was a gift from Fang Li; plasmids for the SARS-CoV-2 spike proteins of the Delta (pCAGGS-S-B.1.617.2: Addgene plasmid #177097) and Omicron (pCAGGS-S-B.1.1.529: Addgene #180774) variants were gifts from Daniel Conway; a plasmid for human TMPRSS2 (pCSDest-TMPRSS2: Addgene plasmid #53887) was a gift from Roger Reeves; and a plasmid for human ADAM17 (pRK5F-TACE: Addgene plasmid #31713) was a gift from Rik Derynck.

Techniques: Inhibition, Western Blot, Transfection, FLAG-tag, Immunodetection, Control, Comparison, Activity Assay

Journal: Viruses

Article Title: Metalloprotease-Dependent S2′-Activation Promotes Cell–Cell Fusion and Syncytiation of SARS-CoV-2

doi: 10.3390/v14102094

Figure Lengend Snippet: TMPRSS2-dependent SARS-CoV-2 cell–cell fusion and syncytiation is resistant to metalloprotease inhibition. ( A ) Effect of TMPRSS2 overexpression on fusion-associated β-galactosidase activity of HEK293T-ACE2-⍺ and HEK293T-SARS2-ω co-cultures in the presence or absence of the serine protease inhibitor camostat mesylate, or ( B ) hydroxamate-based metalloprotease inhibitors (two-way ANOVA; Tukey post hoc). ( C ) Effect of TAPI-1 and/or camostat mesylate on fusion-associated β-galactosidase activity of HEK293T-ACE2-⍺, with or without TMPRSS2, and HEK293T-SARS2-ω co-cultures (two-way ANOVA; Tukey post hoc). ( D ) Effects of TAPI-1 and/or camostat mesylate on fusion-associated β-galactosidase activity of Caco-2-⍺ and HEK293T-SARS2-ω co-cultures (two-way ANOVA; Tukey post hoc). ( E ) Representative fluorescent micrographs of Caco-2 and HEK293T-SARS2-GFP co-cultures post-treatment with TAPI-1 and/or camostat mesylate. ( F ) Effects of TAPI-1 and/or camostat mesylate on fusion-associated β-galactosidase activity of Calu-3-⍺ and HEK293T-SARS2-ω co-cultures (two-way ANOVA; Tukey post hoc). ( G ) Representative fluorescent micrographs Calu-3 and HEK293T-SARS2-GFP co-cultures post-treatment with TAPI-1 and/or camostat mesylate. Syncytiation was visualised at 16-h post-co-culture on an EVOS FL Auto Imaging System (Thermo Fisher Scientific). Camostat mesylate (120 μM; 16-h); BATIM: Batimastat (20 μM; 16-h); MARIM: Marimastat (20 μM; 16-h); TAPI: TAPI-1 (20 μM; 16-h); SARS2: SARS-CoV-2 spike protein. Probability value: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. Scale bar: 500 μm. Fluorescent micrographs are also available in .

Article Snippet: The following plasmids were obtained from Addgene: a plasmid for human ACE2 (pcDNA3.1-hACE2: Addgene plasmid #1786) was a gift from Hyeryun Choe; a plasmid for the ancestral Wuhan-Hu-1 SARS-CoV-2 spike protein (pcDNA3.1-SARS2-Spike: Addgene plasmid #145032) was a gift from Fang Li; plasmids for the SARS-CoV-2 spike proteins of the Delta (pCAGGS-S-B.1.617.2: Addgene plasmid #177097) and Omicron (pCAGGS-S-B.1.1.529: Addgene #180774) variants were gifts from Daniel Conway; a plasmid for human TMPRSS2 (pCSDest-TMPRSS2: Addgene plasmid #53887) was a gift from Roger Reeves; and a plasmid for human ADAM17 (pRK5F-TACE: Addgene plasmid #31713) was a gift from Rik Derynck.

Techniques: Inhibition, Over Expression, Activity Assay, Protease Inhibitor, Co-Culture Assay, Imaging

Journal: Viruses

Article Title: Metalloprotease-Dependent S2′-Activation Promotes Cell–Cell Fusion and Syncytiation of SARS-CoV-2

doi: 10.3390/v14102094

Figure Lengend Snippet: The SARS-CoV-2 spike protein induces ACE2 ectodomain shedding. ( A ). Western blot analysis of the SARS-CoV-2 spike protein and ( B ) ACE2 in monocultures (MC) or co-cultures (CC) of HEK293T cells overexpressing ACE2, with a C-terminal eGFP-tag (HEK293T-ACE2e), and SARS-CoV-2 spike protein (HEK293T-SARS2), in the presence of absence of TAPI-1. ( C ) Total soluble ACE2 and ( D ) relative ACE2 C-peptidase activity in the conditioned media of cultures in ( B ) (unpaired Student’s t -test). ( E ) Western blot analysis of the soluble S1 fragment precipitated from artificial SARS-CoV-2 conditioned media (αSCM) of HEK293T-SARS2 cells; parental basal conditioned media (pBCM) from untransfected cells served as a negative control. ( F ) Western blot analysis of ACE2 in HEK293T-ACE2e incubated with αSCM (2-h). ( G ) Total ACE2 determined by ELISA and ( H ) relative ACE2 C-peptidase activity in the conditioned media of HEK293T-ACE2-GFP incubated with αSCM (2-h) (unpaired Student’s t -test). ( I ) Western blot analysis of ACE2 in HEK293T-ACE2-GFP incubated with αSCM, in the presence of absence of TAPI-1. The SARS-CoV-2 spike protein (SARS2) was immunodetected with an anti-1D4 rhodopsin antibody, directed against the C-terminal C9-tag; ACE2, with an anti-eGFP antibody, directed against the C-terminal eGFP-tag. Western blotting data are from one experiment representative of at least three independent experiments. Immunodetection of β-actin served as a loading control. TAPI: TAPI-1 (20 μM; 16-h) SARS2: SARS-CoV-2 spike protein; S0: full-length SARS-CoV-2 spike protein; S2: S2 fragment of the SARS-CoV-2 spike protein. ACE2-FL: full-length ACE2; ACE2-CTF: ACE2 C-terminal fragment. Probability value: *, p < 0.05; **, p < 0.01.

Article Snippet: The following plasmids were obtained from Addgene: a plasmid for human ACE2 (pcDNA3.1-hACE2: Addgene plasmid #1786) was a gift from Hyeryun Choe; a plasmid for the ancestral Wuhan-Hu-1 SARS-CoV-2 spike protein (pcDNA3.1-SARS2-Spike: Addgene plasmid #145032) was a gift from Fang Li; plasmids for the SARS-CoV-2 spike proteins of the Delta (pCAGGS-S-B.1.617.2: Addgene plasmid #177097) and Omicron (pCAGGS-S-B.1.1.529: Addgene #180774) variants were gifts from Daniel Conway; a plasmid for human TMPRSS2 (pCSDest-TMPRSS2: Addgene plasmid #53887) was a gift from Roger Reeves; and a plasmid for human ADAM17 (pRK5F-TACE: Addgene plasmid #31713) was a gift from Rik Derynck.

Techniques: Western Blot, Activity Assay, Negative Control, Incubation, Enzyme-linked Immunosorbent Assay, Immunodetection, Control

Journal: Viruses

Article Title: Metalloprotease-Dependent S2′-Activation Promotes Cell–Cell Fusion and Syncytiation of SARS-CoV-2

doi: 10.3390/v14102094

Figure Lengend Snippet: Metalloprotease-dependent ectodomain shedding of ACE2 contributes to SARS-CoV-2 cell–cell fusion and syncytiation. ( A ) Western blot analysis of wild type ACE2 and the ACE2 L584A mutant transiently overexpressed in HEK293T cells, in the presence or absence of phorbol ester stimulation or transient exogenous ADAM17 co-transfection. ( B ) Median fluorescence intensity (MFI) in arbitrary fluorescent units (AFU) of ACE2 on the surface of HEK293T cells transiently transfected with ACE2 or the ACE2 L584A mutant (unpaired Student’s t -test). ( C ) Total ACE2 determined by ELISA and ( D ) relative ACE2 C-peptidase activity in the conditioned media of HEK293T cells transiently transfected with human with ACE2 or the ACE2 L584A mutant, as described in ( A ) (two-way ANOVA; Tukey post hoc). ( E ) Effects of the ACE2 L584A mutation on fusion-associated β-galactosidase activity in co-cultures of target HEK293T-ACE2-⍺ cells or HEK293T-ACE2-L584A-⍺ and effector HEK293T-SARS2-ω cells (unpaired Student’s t -test). ( F ) Effect of the ACE2 L584A mutation on fusion-associated β-galactosidase activity in co-cultures of target HEK293T-ACE2-⍺ cells or HEK293T-ACE2-L584A-⍺ and effector HEK293T-SARS2-ω cells with or without hydroxamate-based metalloprotease inhibitors (two-way ANOVA; Tukey post hoc). ( G ) Western blot analysis of wild type ACE2 and the ACE2 L584A mutant, in the presence or absence of serine protease inhibition and/or transient TMPRSS2 co-transfection. ( H ) Total ACE2 determined by ELISA and ( I ) relative ACE2 C-peptidase activity in the conditioned media of HEK293T cells transiently transfected with human with ACE2 or the ACE2 L584A mutant, as described in ( G ) (two-way ANOVA; Tukey post hoc). ( J ) Effect of the ACE2 L584A mutation on fusion-associated β-galactosidase activity in co-cultures of target HEK293T-ACE2-⍺ cells or HEK293T-ACE2-L584A-⍺ and effector HEK293T-SARS2-ω cells, with or without the transient co-transfection of TMPRSS2 (two-way ANOVA; Tukey post hoc). ACE2 and the ACE2 L584A mutant were immunodetected with an anti-1D4 rhodopsin antibody, directed against the C-terminal C9-tag. Western blotting data are from one experiment representative of at least three independent experiments. Immunodetection of β-actin served as a loading control. PMA: Phorbol 12-myristate 13-acetate (200 ng/mL; 2-h); Camostat mesylate (120 μM; 16-h); BATIM: Batimastat (20 μM; 16-h); MARIM: Marimastat (20 μM; 16-h); TAPI: TAPI-1 (20 μM; 16-h); ACE2-FL: full-length ACE2; ACE2-CTF: ACE2 C-terminal fragment. Probability value: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Article Snippet: The following plasmids were obtained from Addgene: a plasmid for human ACE2 (pcDNA3.1-hACE2: Addgene plasmid #1786) was a gift from Hyeryun Choe; a plasmid for the ancestral Wuhan-Hu-1 SARS-CoV-2 spike protein (pcDNA3.1-SARS2-Spike: Addgene plasmid #145032) was a gift from Fang Li; plasmids for the SARS-CoV-2 spike proteins of the Delta (pCAGGS-S-B.1.617.2: Addgene plasmid #177097) and Omicron (pCAGGS-S-B.1.1.529: Addgene #180774) variants were gifts from Daniel Conway; a plasmid for human TMPRSS2 (pCSDest-TMPRSS2: Addgene plasmid #53887) was a gift from Roger Reeves; and a plasmid for human ADAM17 (pRK5F-TACE: Addgene plasmid #31713) was a gift from Rik Derynck.

Techniques: Western Blot, Mutagenesis, Cotransfection, Fluorescence, Transfection, Enzyme-linked Immunosorbent Assay, Activity Assay, Inhibition, Immunodetection, Control