Journal: Viruses

Article Title: Stabilization of the Metastable Pre-Fusion Conformation of the SARS-CoV-2 Spike Glycoprotein through N-Linked Glycosylation of the S2 Subunit

doi: 10.3390/v16020223

Figure Lengend Snippet: α-glycosidase inhibitors impaired SARS-CoV-2 S-mediated membrane fusion. ( A ) HEK293T cells transfected with plasmids expressing the SARS-CoV-2 S protein were treated with 100 µg/mL castanospermine or NB-DNJ for 40 h, after which the expression levels of S protein in cell lysates were detected using Western blot analysis with antibodies against the S2 subunit of the SARS-CoV-2 S protein. β-actin was used as a loading control. ( B ) HEK293T cells expressing the SARS-CoV-2 S protein and GFP were treated with α-glycosidase inhibitors for 40 h and then trypsinized at 40 h post-transfection and co-cultured with HEK293/hACE2 for another 4 h, then imaged using a fluorescence microscope. The area of syncytia in each image was measured using ImageJ. An unpaired two-tailed Student’s t -test was used for statistical analysis, ** p < 0.01; * p < 0.05. ( C ) Effect of α-glycosidase inhibitors on the proliferation of HEK293T cells. HEK293T cells were treated with 100 µg/mL castanospermine or NB-DNJ for 40 h, after which cell viability was determined using the CCK8 assay. ( D ) Western blot analysis of SARS-CoV-2 S-pseudotyped lentiviral particles produced in HEK293T cells in the absence or presence of α-glycosidase inhibitors. p24 was used as a loading control. ( E ) The transduction efficiency of SARS-CoV-2 S-pseudotyped lentiviral particles produced in HEK293T cells in the absence or presence of α-glycosidase inhibitors. An unpaired two-tailed Student’s t -test was used for statistical analysis, ** p < 0.01.

Article Snippet: Subsequently, the cells were washed and incubated with 1 μg/mL polyclonal goat anti-human ACE2 antibody (R&D Systems, Minneapolis, MN, USA) for 1 h. Next, the cells were incubated with FITC-conjugated rabbit anti-goat secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA) for 1 h. After washing, the cells were analyzed using a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).

Techniques: Membrane, Transfection, Expressing, Western Blot, Control, Cell Culture, Fluorescence, Microscopy, Two Tailed Test, CCK-8 Assay, Produced, Transduction

Journal: Viruses

Article Title: Stabilization of the Metastable Pre-Fusion Conformation of the SARS-CoV-2 Spike Glycoprotein through N-Linked Glycosylation of the S2 Subunit

doi: 10.3390/v16020223

Figure Lengend Snippet: The expression levels and receptor-binding capacity of the N-glycosylation-site mutants of the SARS-CoV-2 S protein. ( A ) Schematic representation of N-linked glycosylation sites in the SARS-CoV-2 S protein. ( B ) HEK293T cells were transfected with plasmids encoding wild-type S protein or with N-glycosylation-site mutants. After 40 h of transfection, the cells were lysed and analyzed via Western blot with antibodies against the SARS-CoV-2 S2 subunit. β-actin was used as a loading control. ( C ) Cell-surface expression of wild-type S protein and different N-glycosylation-site mutants, determined via flow cytometry. ( D ) HEK293T cells were transfected with plasmids encoding wild-type S protein or different N-glycosylation mutants. After 40 h of transfection, the cells were detached with 1 mM EDTA, incubated with soluble hACE2 for 1 h on ice, incubated with primary antibodies against hACE2, and then incubated with FITC-conjugated secondary antibodies. The cells were then subjected to flow cytometry.

Article Snippet: Subsequently, the cells were washed and incubated with 1 μg/mL polyclonal goat anti-human ACE2 antibody (R&D Systems, Minneapolis, MN, USA) for 1 h. Next, the cells were incubated with FITC-conjugated rabbit anti-goat secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA) for 1 h. After washing, the cells were analyzed using a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).

Techniques: Expressing, Binding Assay, Glycoproteomics, Transfection, Western Blot, Control, Flow Cytometry, Incubation

Journal: Viruses

Article Title: Stabilization of the Metastable Pre-Fusion Conformation of the SARS-CoV-2 Spike Glycoprotein through N-Linked Glycosylation of the S2 Subunit

doi: 10.3390/v16020223

Figure Lengend Snippet: Cell–cell fusion capacity of N-glycosylation mutants of the SARS-CoV-2 S protein. ( A ) HEK293T cells were co-transfected with plasmids encoding either wild-type S protein or different N-glycosylation-site-mutant S proteins and plasmids encoding GFP. After 40 h of transfection, the cells were trypsinized and co-cultured with HEK293/hACE2 cells for an additional 4 h and then imaged using a fluorescence microscope. ( B ) Quantification of cell–cell fusion. The total number of nuclei and the number of nuclei in fused cells for each image were counted. The fusion efficiency was calculated as the number of nuclei in syncytia/the total number of nuclei ×100.

Article Snippet: Subsequently, the cells were washed and incubated with 1 μg/mL polyclonal goat anti-human ACE2 antibody (R&D Systems, Minneapolis, MN, USA) for 1 h. Next, the cells were incubated with FITC-conjugated rabbit anti-goat secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA) for 1 h. After washing, the cells were analyzed using a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).

Techniques: Glycoproteomics, Transfection, Mutagenesis, Cell Culture, Fluorescence, Microscopy

Journal: Viruses

Article Title: Stabilization of the Metastable Pre-Fusion Conformation of the SARS-CoV-2 Spike Glycoprotein through N-Linked Glycosylation of the S2 Subunit

doi: 10.3390/v16020223

Figure Lengend Snippet: Removal of N-glycans from the SARS-CoV-2 S protein destabilized the S protein. ( A ) HEK293T cells were co-transfected with plasmids encoding EGFP and plasmids encoding wild-type SARS-CoV-2 S protein, the N1074Q mutant, or the T1076A mutant. After 40 h of transfection, the cells were visualized using a fluorescence microscope. ( B ) Endogenous ACE2 knockout in HEK293T cells was confirmed through DNA sequencing. A single-nucleotide insertion in the ACE2 gene caused a frameshift and led to the premature termination of ACE2 translation. ( C ) The endogenous-ACE2-knockout HEK293T cells were co-transfected with plasmids encoding EGFP and plasmids encoding wild-type SARS-CoV-2 S protein, the N1074Q mutant, or the T1076A mutant. After 40 h of transfection, the cells were visualized using a fluorescence microscope. ( D , E ) HEK293T cells were transfected with plasmids encoding wild-type SARS-CoV-2 S protein, the N1074Q mutant, or the T1076A mutant. After 40 h of transfection, the S protein in cell lysates ( D ) and supernatants ( E ) was analyzed via Western blot with antibodies against the S2 subunit and the RBD subunit of the SARS-CoV-2 S protein, respectively.

Article Snippet: Subsequently, the cells were washed and incubated with 1 μg/mL polyclonal goat anti-human ACE2 antibody (R&D Systems, Minneapolis, MN, USA) for 1 h. Next, the cells were incubated with FITC-conjugated rabbit anti-goat secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA) for 1 h. After washing, the cells were analyzed using a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).

Techniques: Transfection, Mutagenesis, Fluorescence, Microscopy, Knock-Out, DNA Sequencing, Western Blot

Journal: Viruses

Article Title: Stabilization of the Metastable Pre-Fusion Conformation of the SARS-CoV-2 Spike Glycoprotein through N-Linked Glycosylation of the S2 Subunit

doi: 10.3390/v16020223

Figure Lengend Snippet: Removal of the furin cleavage site compensated for the instability of the N-glycosylation mutant N1074Q. ( A ) HEK293T cells were transfected with plasmids encoding wild-type SARS-CoV-2 S protein, wild-type S protein with a mutant furin cleavage site (WT-delFurin), mutant N1074Q, or N1074Q with a mutant furin cleavage site (N1074Q-delFurin). After 40 h of transfection, the cells were lysed and analyzed via Western blot with antibodies against the SARS-CoV-2 S2 subunit. β-actin was used as a loading control. ( B ) HEK293T cells were co-transfected with plasmids encoding GFP and plasmids encoding wild-type SARS-CoV-2 S protein, the WT-delFurin mutant, the N1074Q mutant, or the N1074Q-delFurin mutant. After 40 h of transfection, the cells were visualized using a fluorescence microscope. ( C ) A cell–cell fusion assay was performed to assess the fusogenicity of wild-type SARS-CoV-2 S protein, the WT-delFurin mutant, the N1074Q mutant, and the N1074Q-delFurin mutant. HEK293T cells were co-transfected with plasmids encoding wild-type or mutant S proteins and plasmids encoding GFP. After 30 h of transfection, the cells were trypsinized and co-cultured with HEK293/hACE2 cells for an additional 4 h and then imaged using a fluorescence microscope.

Article Snippet: Subsequently, the cells were washed and incubated with 1 μg/mL polyclonal goat anti-human ACE2 antibody (R&D Systems, Minneapolis, MN, USA) for 1 h. Next, the cells were incubated with FITC-conjugated rabbit anti-goat secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA) for 1 h. After washing, the cells were analyzed using a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).

Techniques: Glycoproteomics, Mutagenesis, Transfection, Western Blot, Control, Fluorescence, Microscopy, Cell-Cell Fusion Assay, Cell Culture

Journal: Viruses

Article Title: Stabilization of the Metastable Pre-Fusion Conformation of the SARS-CoV-2 Spike Glycoprotein through N-Linked Glycosylation of the S2 Subunit

doi: 10.3390/v16020223

Figure Lengend Snippet: Removal of N-glycans from the S2 subunit of SARS-CoV S protein destabilized the S protein. ( A ) Amino-acid sequence alignment of S2 subunits from SARS-CoV and SARS-CoV-2. Red asterisks indicate N-linked glycosylation sites. ( B ) HEK293T cells were transfected with an empty vector or with plasmids encoding the wild-type SARS-CoV S protein or one of the various glycosylation mutants. After 40 h of transfection, the cells were lysed and analyzed with polyclonal rabbit anti-SARS S1 antibodies T62. β-actin was used as a loading control. ( C ) HEK293T cells were co-transfected with plasmids encoding EGFP and plasmids encoding wild-type SARS-CoV S protein or one of the various glycosylation mutants. After 40 h of transfection, the cells were visualized using a fluorescence microscope. ( D ) HEK293T/ACE2-KO cells were co-transfected with plasmids encoding EGFP and plasmids encoding wild-type SARS-CoV S protein, the N699Q mutant, or the N1080Q mutant. After 40 h of transfection, the cells were visualized using a fluorescence microscope.

Article Snippet: Subsequently, the cells were washed and incubated with 1 μg/mL polyclonal goat anti-human ACE2 antibody (R&D Systems, Minneapolis, MN, USA) for 1 h. Next, the cells were incubated with FITC-conjugated rabbit anti-goat secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA) for 1 h. After washing, the cells were analyzed using a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).

Techniques: Sequencing, Glycoproteomics, Transfection, Plasmid Preparation, Control, Fluorescence, Microscopy, Mutagenesis