Journal: Journal of Virology

Article Title: Concanavalin A targets phylogenetically conserved N-linked glycans on coronavirus spike proteins for broad-spectrum antiviral activity

doi: 10.1128/jvi.01679-25

Figure Lengend Snippet: Concanavalin A broadly inhibits SARS-CoV-2 spike-mediated membrane fusion and viral entry. ( A ) Schematics of the cell-cell fusion co-culture model used to quantify or qualify spike-mediated syncytium formation. Cells co-expressing SARS-CoV-2 spike and Cre were co-cultured with LoxP-luc or LoxP-mCherry expressing HEK293T cells for 16 h, before syncytia formation was captured using the fluorescence derived from mCherry reporter or quantified based on bioluminescence signal derived from the firefly luciferase. Different concentrations of Concanavalin A (ConA) were added at the time of co-culture. ( B ) Representative fluorescent images of mCherry reporter and nuclei counterstained with 100 ng/mL Hoescht33342 collected from SARS-CoV-2 WT and SARS-CoV-2 variants (Omicron BA.4, XBB.1.5, and EG.5.1) spike-expressing cells, co-cultured with LoxP-mCherry control HEK293T or HEK293T-ACE2 cells for 16 h in the absence or presence of 10 µg/mL ConA. The scale bar is representative of 100 μm. ( C ) Luciferase activity (RLU) detected from WT, Omicron BA.4, XBB.1.5, or EG.5.1 spike-expressing cells, co-cultured with LoxP-luciferase control HEK293T or HEK293T-ACE2 cells for 16 h in the absence or presence of 19.60, 65.29, and 196.07 nM (2, 6.6, and 20 µg/mL) ConA. Data are representative of three repeats, and data points are represented as mean ± SEM. Statistical significance was determined using one-way ANOVA with Sidak post-hoc multiple comparison. P values are indicated as ns, not significant, * P < 0.05, ** P < 0.005, *** P < 0.0005, **** P < 0.0001. ( D ) The IC 50 curve (black) and cytotoxicity (red) of ConA against Omicron XBB.1.5 and EG.5.1 spike variants pseudovirus entry, respectively, in Caco-2 cells. Data are representative of three repeats. ( E ) Normalized IC 50 curve of ConA-mediated inhibition of SARS-CoV-2 WT viral copies in the infected Caco-2 cells supernatant at MOI = 0.01 for 24 h. Viral copies were determined using quantitative PCR targeting the SARS-CoV-2 E genes. ( F ) Representative fluorescent images of SARS-CoV-2 nucleocapsid (N) and nuclei counterstained with 1 μg/mL DAPI, captured from Caco-2 cells infected with SARS-CoV-2 (MOI = 0.01) in the absence or presence of 32.65 and 98.04 nM ConA for 24 h. The scale bar is representative of 100 μm.

Article Snippet: For ConA magnetic pulldown of SARS-CoV-2, hCoV-NL63, and SADS-CoV spikes, HEK293T cells transiently transfected with respective spikes were lysed in the NP-40 lysis buffer supplemented with the 1× EDTA-free protease inhibitor cocktail (TargetMol, C0001).

Techniques: Membrane, Co-culture Assay, Expressing, Cell Culture, Fluorescence, Derivative Assay, Luciferase, Control, Activity Assay, Comparison, Inhibition, Infection, Real-time Polymerase Chain Reaction, Co-Culture, Cell-Cell Fusion Assay, Immunofluorescence

Journal: Journal of Virology

Article Title: Concanavalin A targets phylogenetically conserved N-linked glycans on coronavirus spike proteins for broad-spectrum antiviral activity

doi: 10.1128/jvi.01679-25

Figure Lengend Snippet: Concanavalin A directly interacts with spike via N-linked glycans. ( A ) A schematic of ConA heat inactivation and its effect on spike binding. ( B ) Indirect ELISA of His-tag containing soluble spike ectodomain binding to the immobilized native or heat-inactivated ConA. ( C ) Luciferase activity (RLU) detected from cell lysates of Cre and SARS-CoV-2 spike-expressing cells, co-cultured with LoxP-luciferase control HEK293T or HEK293T-ACE2 for 16 h in the absence or presence of 196.07 nM (20 µg/mL) native or heat-inactivated ConA. Data are representative of three individual repeats. ( D ) Luciferase activity (RLU) detected from cell lysates of Cre and SARS-CoV-2 spike-expressing cells, co-cultured with LoxP-luciferase control HEK293T or HEK293T-ACE2 for 16 h in the absence or presence of 196.07 nM native ConA and various types of monosaccharides; 25 mM D-glucose, D-mannose, and D-galactose were supplemented at the beginning of the co-culture assay. Data are representative of three individual repeats. Data are representative of three repeats, and data points are represented as mean ± SEM. Statistical significance was determined using one-way ANOVA with Sidak post-hoc multiple comparison test. P values are indicated as ns, not significant, * P < 0.05, *** P < 0.0005. ( E ) Competition ELISA of 500 ng/mL soluble spike ectodomain binding to the immobilized native ConA in the presence of increasing concentrations (0.002–200 mM) of D-mannose or Methyl alpha-D-mannopyranoside (MADP). ( F ) Luciferase activity (RLU) was detected from cell lysates of Cre and SARS-CoV-2 spike-expressing cells, co-cultured with LoxP-luciferase control HEK293T or HEK293T-ACE2 for 16 h in the absence or presence of 196.07 nM native ConA and various concentrations of methyl alpha-D-mannopyranoside (MADP); 0.1–10 mM MADP was supplemented at the beginning of the co-culture assay. Data are representative of three individual repeats. ( G ) Schematics of ConA ELISA with purified prefusion spike trimers treated with Endo H- and PNGase F. ( H ) ConA ELISA showing the binding of control, Endo H-, or PNGase F-treated prefusion spike HexaPro ectodomain (S-6P-ECD) to the ELISA plate coated with ConA; relative binding of ConA to untreated and treated spikes at 1 μg/mL was shown in the bar chart. ( I ) Schematics of ConA-mediated pulldown of spike or deglycosylated spike from cell lysates. ( J ) Immunoblots showing the full-length SARS-CoV-2 spike and S2, collected from the ConA magnetic beads pulldown from control, Endo H- or PNGase F-treated cell lysate samples. Total cell lysates were used as input controls. Blots are representative of three individual repeats. ( K ) Immunoblots showing the full-length SADS-CoV spike and S2, collected from the ConA magnetic beads pulldown from control, Endo H- or PNGase F-treated cell lysate samples. Total cell lysates were used as input controls. Blots are representative of two individual repeats. ( L ) Immunoblots showing the full-length hCoV-NL63 spike collected from the ConA magnetic beads pulldown from hCoV-NL63 viral supernatants. Viral supernatants were used as input controls, and protein A/G magnetic beads were used as negative controls. Blots are representative of three individual repeats.

Article Snippet: For ConA magnetic pulldown of SARS-CoV-2, hCoV-NL63, and SADS-CoV spikes, HEK293T cells transiently transfected with respective spikes were lysed in the NP-40 lysis buffer supplemented with the 1× EDTA-free protease inhibitor cocktail (TargetMol, C0001).

Techniques: Inactivation, Binding Assay, Indirect ELISA, Luciferase, Activity Assay, Expressing, Cell Culture, Control, Co-culture Assay, Comparison, Enzyme-linked Immunosorbent Assay, Purification, Western Blot, Magnetic Beads, Cell-Cell Fusion Assay

Journal: Journal of Virology

Article Title: Concanavalin A targets phylogenetically conserved N-linked glycans on coronavirus spike proteins for broad-spectrum antiviral activity

doi: 10.1128/jvi.01679-25

Figure Lengend Snippet: Concanavalin A binding to conserved mannose glycans interferes with coronavirus spike proteolytic processing. ( A ) Indirect ELISA showing immobilized ConA binding to 1 ng/mL to 10 μg/mL half-logarithmic dilutions of prefusion spike HexaPro ectodomain (S-6P-ECD), but not the same concentrations of the receptor binding domain (RBD). Data are representative of two replicates. ( B ) Indirect ELISA showing immobilized S-6P-ECD binding to half-logarithmic dilutions of 3.33 ng to 33.33 μg/mL soluble ACE2-Fc in the absence or presence of 10 μg/mL ConA (left) or half-logarithmic dilutions of 3.33 ng to 33.33 μg/mL ConA to 10 μg/mL ACE2-Fc (right). Data are representative of two replicates. ( C ) A table summarizing the reported percentage of oligomannose and complex sugar occupancies on the SARS-CoV-2 WT spike protein. ( D ) ConA-mediated inhibition of spike-mediated cell-cell fusion, derived from cells co-expressing 8 individual glycosylation mutant spikes (N61Q, N122Q, N234Q, N603Q, N709Q, N717Q, N801Q, and N1074Q) and Cre, co-cultured with LoxP-luciferase HEK293T-ACE2 cells for 16 h. Percentage inhibition was normalized against the bioluminescence readings from each spike mutant in the absence of ConA. The concentration of ConA used was 196.07 nM (20 µg/mL). Data are representative of four repeats, and data points are represented as mean ± SEM. ( E ) The IC 50 of ConA on pseudoviruses bearing the WT BA.4, N717Q, and N801Q spike mutants in HEK293T-ACE2 cells. The inhibitory activity of ConA at 32.64 nM (3.33 µg/mL) on pseudovirus entry mediated by the WT, N717Q, and N801Q spike mutants was shown as a bar chart. Data are representative of four repeats, and data points are represented as mean ± SEM. Statistical significance was determined using one-way ANOVA with Sidak post-hoc multiple comparison test. P -values are indicated as ns, not significant, * P < 0.05, ** P < 0.005, **** P < 0.0001. ( F ) Amino acid sequence alignment of spikes N717 and N801 regions with reference to the SARS-CoV-2 spike. ( G ) Structural representation of SARS-CoV-2 spike N717 and N801 residues modeled on the prefusion spike (pdb: 6VXX). ( H ) Immunoblots of full-length spike, S2, and cleaved S2′ collected from SARS-CoV-2 WT spike-expressing HEK293T cells co-cultured with control HEK293T or HEK293T-ACE2 cells in the absence or presence of 196.07 nM ConA. Blots are representative of two repeats. ( I ) Immunoblots of full-length spike, S2, and cleaved S2′ collected from SARS-CoV-2 Omicron XBB.1.5 spike-expressing HEK293T cells co-cultured with control HEK293T or HEK293T-ACE2 cells in the absence or presence of 196.07 nM ConA. Blots are representative of two repeats. ( J ) Immunoblots of full-length spike, S2, and cleaved S2′ collected from SARS-CoV-2 Omicron EG.5.1 spike-expressing HEK293T cells co-cultured with control HEK293T or HEK293T-ACE2 cells in the absence or presence of 196.07 nM ConA. Blots are representative of two repeats.

Article Snippet: For ConA magnetic pulldown of SARS-CoV-2, hCoV-NL63, and SADS-CoV spikes, HEK293T cells transiently transfected with respective spikes were lysed in the NP-40 lysis buffer supplemented with the 1× EDTA-free protease inhibitor cocktail (TargetMol, C0001).

Techniques: Binding Assay, Indirect ELISA, Inhibition, Derivative Assay, Expressing, Glycoproteomics, Mutagenesis, Cell Culture, Luciferase, Concentration Assay, Activity Assay, Comparison, Sequencing, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Cell-Cell Fusion Assay