sars cov 2 s protein  (Sino Biological)


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    Structured Review

    Sino Biological sars cov 2 s protein
    CTSL cleaves the <t>SARS-CoV-2</t> spike (S) protein, and this cleavage promotes cell–cell fusion. a Overview of the SARS-CoV-1 and SARS-CoV-2 S1/S2 cleavage sites. FP (fusion peptide), HR1 (heptad repeat 1), and HR2 (heptad repeat 2) are units of the S2 subunit that function in membrane fusion. b Analysis of CTSL-mediated S-protein cleavage. Purified SARS-CoV-1 or SARS-CoV-2 S protein was incubated in the presence or absence (assay buffer, pH = 5.5) of CTSL (2 or 10 μg/ml in assay buffer, pH = 5 .5) at 37 °C for 1 h. The reaction system of 2 μg/ml CTSL was further supplemented with CTSL inhibitors (20 μM E64d or 20 μM SID 26681509), as indicated. Proteins were subjected to SDS-PAGE and detected by silver staining. Representative data from three independent experiments are shown. c Syncytium-formation assay: Huh7 cells were untransfected (Null) or transfected with plasmid to express the SARS-CoV-2 S protein. Cells were incubated in the presence or absence (PBS, pH = 7.4) of trypsin (2 μg/ml in PBS, pH = 7.4) or in the presence or absence (PBS, pH = 5.8) of CTSL (2 or 4 μg/ml in PBS, pH = 5.8) for 20 min. Images were acquired after an additional 16 h incubation in the medium. (scale bars, 50 μm). The black arrowheads indicate syncytia. Representative data from seven independent experiments are shown. d Quantitative analysis of syncytia in panel c . n = 7. Statistical significance was assessed by one-way ANOVA with Tukey’s post hoc test. The data are expressed as the mean ± s.e.m. values. * P
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    Images

    1) Product Images from "Cathepsin L plays a key role in SARS-CoV-2 infection in humans and humanized mice and is a promising target for new drug development"

    Article Title: Cathepsin L plays a key role in SARS-CoV-2 infection in humans and humanized mice and is a promising target for new drug development

    Journal: Signal Transduction and Targeted Therapy

    doi: 10.1038/s41392-021-00558-8

    CTSL cleaves the SARS-CoV-2 spike (S) protein, and this cleavage promotes cell–cell fusion. a Overview of the SARS-CoV-1 and SARS-CoV-2 S1/S2 cleavage sites. FP (fusion peptide), HR1 (heptad repeat 1), and HR2 (heptad repeat 2) are units of the S2 subunit that function in membrane fusion. b Analysis of CTSL-mediated S-protein cleavage. Purified SARS-CoV-1 or SARS-CoV-2 S protein was incubated in the presence or absence (assay buffer, pH = 5.5) of CTSL (2 or 10 μg/ml in assay buffer, pH = 5 .5) at 37 °C for 1 h. The reaction system of 2 μg/ml CTSL was further supplemented with CTSL inhibitors (20 μM E64d or 20 μM SID 26681509), as indicated. Proteins were subjected to SDS-PAGE and detected by silver staining. Representative data from three independent experiments are shown. c Syncytium-formation assay: Huh7 cells were untransfected (Null) or transfected with plasmid to express the SARS-CoV-2 S protein. Cells were incubated in the presence or absence (PBS, pH = 7.4) of trypsin (2 μg/ml in PBS, pH = 7.4) or in the presence or absence (PBS, pH = 5.8) of CTSL (2 or 4 μg/ml in PBS, pH = 5.8) for 20 min. Images were acquired after an additional 16 h incubation in the medium. (scale bars, 50 μm). The black arrowheads indicate syncytia. Representative data from seven independent experiments are shown. d Quantitative analysis of syncytia in panel c . n = 7. Statistical significance was assessed by one-way ANOVA with Tukey’s post hoc test. The data are expressed as the mean ± s.e.m. values. * P
    Figure Legend Snippet: CTSL cleaves the SARS-CoV-2 spike (S) protein, and this cleavage promotes cell–cell fusion. a Overview of the SARS-CoV-1 and SARS-CoV-2 S1/S2 cleavage sites. FP (fusion peptide), HR1 (heptad repeat 1), and HR2 (heptad repeat 2) are units of the S2 subunit that function in membrane fusion. b Analysis of CTSL-mediated S-protein cleavage. Purified SARS-CoV-1 or SARS-CoV-2 S protein was incubated in the presence or absence (assay buffer, pH = 5.5) of CTSL (2 or 10 μg/ml in assay buffer, pH = 5 .5) at 37 °C for 1 h. The reaction system of 2 μg/ml CTSL was further supplemented with CTSL inhibitors (20 μM E64d or 20 μM SID 26681509), as indicated. Proteins were subjected to SDS-PAGE and detected by silver staining. Representative data from three independent experiments are shown. c Syncytium-formation assay: Huh7 cells were untransfected (Null) or transfected with plasmid to express the SARS-CoV-2 S protein. Cells were incubated in the presence or absence (PBS, pH = 7.4) of trypsin (2 μg/ml in PBS, pH = 7.4) or in the presence or absence (PBS, pH = 5.8) of CTSL (2 or 4 μg/ml in PBS, pH = 5.8) for 20 min. Images were acquired after an additional 16 h incubation in the medium. (scale bars, 50 μm). The black arrowheads indicate syncytia. Representative data from seven independent experiments are shown. d Quantitative analysis of syncytia in panel c . n = 7. Statistical significance was assessed by one-way ANOVA with Tukey’s post hoc test. The data are expressed as the mean ± s.e.m. values. * P

    Techniques Used: Purification, Incubation, SDS Page, Silver Staining, Tube Formation Assay, Transfection, Plasmid Preparation

    2) Product Images from "Development and structural basis of a two-MAb cocktail for treating SARS-CoV-2 infections"

    Article Title: Development and structural basis of a two-MAb cocktail for treating SARS-CoV-2 infections

    Journal: Nature Communications

    doi: 10.1038/s41467-020-20465-w

    Cryo-EM structures of the SARS-CoV-2 S trimer in complex with the 3C1 Fab. a , b S-3C1-F3b cryo-EM map ( a ) and pseudo atomic model ( b ). All the three RBDs are up and each of them binds with a 3C1 Fab. The heavy chain of the 3C1 Fab in medium blue and light chain in violet red. c , d S-3C1-F3a cryo-EM map ( c ) and pseudo atomic model ( d ). There are two up RBDs and one down RBD, with each bound with a 3C1 Fab. e Structural alignment of the three up RBDs of S-3C1-F3b (in color) and the only up RBD from S-open (gray), suggesting 3C1 induced outward tilt of the RBDs within the S trimer. f , g Conformational comparation between S-3C1-F1 and S-open ( f ), as well as between S-3C1-F3a and S-3C1-F2 ( g ). h RBD/3C1 interaction interface (take RBD-3/3C1 of S-3C1-F3b as an example), with major involved structural elements labeled. i ACE2 (coral, PDB: 6M0J) would clash with the heavy chain of 3C1 Fab (blue). They share overlapping epitopes on the RBM (dotted black circle); additionally, the framework of 3C1-VH would clash with ACE2 (dotted black frame), which could be enhanced by the presence of an N-linked glycan at site N322 of ACE2. j 3C1 showed two distinct orientations to bind RBD within S trimer, i.e., adopting orientation 1 to associate with up RBD while orientation 2 with down RBD. k Contact footprint variations of 3C1 on up RBD (left) compared with that on down RBD (right), with unique epitopes indicated by dotted black frame. l – m Potential simultaneous binding of RBD by 2H2 and 3C1 cocktail. In 3C1 orientation 1, 3C1 and 2H2 could have minor clash (indicated by black frame, l ); while in origination 2, there is no clash between 3C1 and 2H2 Fabs ( m ).
    Figure Legend Snippet: Cryo-EM structures of the SARS-CoV-2 S trimer in complex with the 3C1 Fab. a , b S-3C1-F3b cryo-EM map ( a ) and pseudo atomic model ( b ). All the three RBDs are up and each of them binds with a 3C1 Fab. The heavy chain of the 3C1 Fab in medium blue and light chain in violet red. c , d S-3C1-F3a cryo-EM map ( c ) and pseudo atomic model ( d ). There are two up RBDs and one down RBD, with each bound with a 3C1 Fab. e Structural alignment of the three up RBDs of S-3C1-F3b (in color) and the only up RBD from S-open (gray), suggesting 3C1 induced outward tilt of the RBDs within the S trimer. f , g Conformational comparation between S-3C1-F1 and S-open ( f ), as well as between S-3C1-F3a and S-3C1-F2 ( g ). h RBD/3C1 interaction interface (take RBD-3/3C1 of S-3C1-F3b as an example), with major involved structural elements labeled. i ACE2 (coral, PDB: 6M0J) would clash with the heavy chain of 3C1 Fab (blue). They share overlapping epitopes on the RBM (dotted black circle); additionally, the framework of 3C1-VH would clash with ACE2 (dotted black frame), which could be enhanced by the presence of an N-linked glycan at site N322 of ACE2. j 3C1 showed two distinct orientations to bind RBD within S trimer, i.e., adopting orientation 1 to associate with up RBD while orientation 2 with down RBD. k Contact footprint variations of 3C1 on up RBD (left) compared with that on down RBD (right), with unique epitopes indicated by dotted black frame. l – m Potential simultaneous binding of RBD by 2H2 and 3C1 cocktail. In 3C1 orientation 1, 3C1 and 2H2 could have minor clash (indicated by black frame, l ); while in origination 2, there is no clash between 3C1 and 2H2 Fabs ( m ).

    Techniques Used: Labeling, Binding Assay

    A proposed model of stepwise binding of 2H2/3C1 Fabs to the RBD of SARS-CoV-2 S trimer. a 2H2 and 3C1 Fabs appear to follow similar pathway to induce generally comparable conformational transitions of the S trimer to neutralize the virus. RBD-1, RBD-2, and RBD-3 are colored in light green, light blue, and gold, respectively; 2H2 and 3C1 Fab in violent red and medium blue, respectively. Red ellipsoid and black ellipsoid indicate Fab bound to up RBD and down RBD, respectively. The maps of S-2H2 and S-3C1 complexes shown here were generated by lowpass filtering of the corresponding models to 10 Å resolution. b Population distribution for the S-2H2 and S-3C1 dataset.
    Figure Legend Snippet: A proposed model of stepwise binding of 2H2/3C1 Fabs to the RBD of SARS-CoV-2 S trimer. a 2H2 and 3C1 Fabs appear to follow similar pathway to induce generally comparable conformational transitions of the S trimer to neutralize the virus. RBD-1, RBD-2, and RBD-3 are colored in light green, light blue, and gold, respectively; 2H2 and 3C1 Fab in violent red and medium blue, respectively. Red ellipsoid and black ellipsoid indicate Fab bound to up RBD and down RBD, respectively. The maps of S-2H2 and S-3C1 complexes shown here were generated by lowpass filtering of the corresponding models to 10 Å resolution. b Population distribution for the S-2H2 and S-3C1 dataset.

    Techniques Used: Binding Assay, Generated

    Cryo-EM structures of the SARS-CoV-2 S trimer in complex with 2H2 Fab. a , b Side and top views of the S-2H2-F3a cryo-EM map ( a ) and pseudo atomic model ( b ). RBD-1 and RBD-2 are in up configuration, while RBD-3 is down, with each of the RBDs bound with a 2H2 Fab. Protomer 1, 2, and 3 are shown in light green, powder blue, and gold, respectively. This color scheme is followed throughout. Heavy chain and light chain of 2H2 Fab in royal blue and violet red, respectively. c , d Side and top views of the S-2H2-F2 cryo-EM map ( c ) and pseudo atomic model ( d ), with two up RBDs (RBD-1 and RBD-2) each bound with a 2H2 Fab. e , f 2H2 Fab-induced conformational changes of the S trimer. Shown is the structural comparation of RBDs between S-2H2-F1 (in color) and S-open (dim gray) ( e ), and between S-2H2-F3a (in color) and S-2H2-F2 (dim gray) ( f ). g 2H2 Fab mainly binds to the RBM (light sea green surface) of RBD, with major involved structural elements labeled. RBD core is rendered as light green surface. h 2H2 Fab (left) and ACE2 (right, gold, PDB: 6M0J) share overlapping epitopes on RBM (second row) and would clash upon binding to the S trimer. i , j The involved regions/residues forming potential contacts between the light chain (in violent red, i ) or heavy chain (in royal blue, j ) of 2H2 and the RBD-1 of S-2H2-F3a. Asterisks highlight residues also involved in the interactions with ACE2. Note that considering the local resolution limitation in the RBD-2H2 portion of the map due to intrinsic dynamic nature in these regions, we analyzed the potential interactions that fulfill criteria of both
    Figure Legend Snippet: Cryo-EM structures of the SARS-CoV-2 S trimer in complex with 2H2 Fab. a , b Side and top views of the S-2H2-F3a cryo-EM map ( a ) and pseudo atomic model ( b ). RBD-1 and RBD-2 are in up configuration, while RBD-3 is down, with each of the RBDs bound with a 2H2 Fab. Protomer 1, 2, and 3 are shown in light green, powder blue, and gold, respectively. This color scheme is followed throughout. Heavy chain and light chain of 2H2 Fab in royal blue and violet red, respectively. c , d Side and top views of the S-2H2-F2 cryo-EM map ( c ) and pseudo atomic model ( d ), with two up RBDs (RBD-1 and RBD-2) each bound with a 2H2 Fab. e , f 2H2 Fab-induced conformational changes of the S trimer. Shown is the structural comparation of RBDs between S-2H2-F1 (in color) and S-open (dim gray) ( e ), and between S-2H2-F3a (in color) and S-2H2-F2 (dim gray) ( f ). g 2H2 Fab mainly binds to the RBM (light sea green surface) of RBD, with major involved structural elements labeled. RBD core is rendered as light green surface. h 2H2 Fab (left) and ACE2 (right, gold, PDB: 6M0J) share overlapping epitopes on RBM (second row) and would clash upon binding to the S trimer. i , j The involved regions/residues forming potential contacts between the light chain (in violent red, i ) or heavy chain (in royal blue, j ) of 2H2 and the RBD-1 of S-2H2-F3a. Asterisks highlight residues also involved in the interactions with ACE2. Note that considering the local resolution limitation in the RBD-2H2 portion of the map due to intrinsic dynamic nature in these regions, we analyzed the potential interactions that fulfill criteria of both

    Techniques Used: Labeling, Binding Assay

    3) Product Images from "Development of cell-based pseudovirus entry assay to identify potential viral entry inhibitors and neutralizing antibodies against SARS-CoV-2"

    Article Title: Development of cell-based pseudovirus entry assay to identify potential viral entry inhibitors and neutralizing antibodies against SARS-CoV-2

    Journal: Genes & Diseases

    doi: 10.1016/j.gendis.2020.07.006

    Detection of SARS-CoV-2 spike (S) protein expression and localization. (A) Schematic illustration of the SARS-CoV-2 full-length spike (S-FL) and mutant S variants. The RBD (receptor binding domain) is in subunit S1; the FP (fusion peptide), HR1 (heptad repeat 1), HR2 (heptad repeat 2), TM (transmembrane domain), and CT (cytoplasmic tail) are in subunit S2. The endoplasmic reticulum retrieval signals (“KxHxx” motif) in the CT domain of S-FL were destroyed in S-Mut protein. The C-terminal 19 amino acids were lacking in S-C19del. (B) Detection of SARS-CoV-2 S expression in HKE293T cells by Western blot using the anti-RBD monoclonal antibody. Cells were transfected with pS-FL, pS-Mut, and pS-C19del plasmids or with an empty vector. (C) Detection of SARS-CoV-2 S subcellular localization in HKE293T cells by confocal microscopy. Cells were grown on glass coverslips for 24 h preceding transfection of plasmids encoding S protein variants. The cells were harvested and labeled with the corresponding antibodies. Calreticulin, ER marker. Nuclei were counterstained with DAPI. Bar = 20 μm.
    Figure Legend Snippet: Detection of SARS-CoV-2 spike (S) protein expression and localization. (A) Schematic illustration of the SARS-CoV-2 full-length spike (S-FL) and mutant S variants. The RBD (receptor binding domain) is in subunit S1; the FP (fusion peptide), HR1 (heptad repeat 1), HR2 (heptad repeat 2), TM (transmembrane domain), and CT (cytoplasmic tail) are in subunit S2. The endoplasmic reticulum retrieval signals (“KxHxx” motif) in the CT domain of S-FL were destroyed in S-Mut protein. The C-terminal 19 amino acids were lacking in S-C19del. (B) Detection of SARS-CoV-2 S expression in HKE293T cells by Western blot using the anti-RBD monoclonal antibody. Cells were transfected with pS-FL, pS-Mut, and pS-C19del plasmids or with an empty vector. (C) Detection of SARS-CoV-2 S subcellular localization in HKE293T cells by confocal microscopy. Cells were grown on glass coverslips for 24 h preceding transfection of plasmids encoding S protein variants. The cells were harvested and labeled with the corresponding antibodies. Calreticulin, ER marker. Nuclei were counterstained with DAPI. Bar = 20 μm.

    Techniques Used: Expressing, Mutagenesis, Binding Assay, Western Blot, Transfection, Plasmid Preparation, Confocal Microscopy, Labeling, Marker

    Detection of SARS-CoV-2 S pseudotyped virus infectivity. (A) Schematic representation of the pseudovirus production and neutralization assay and the applications of the pseudovirus. (B) HEK293T and 293T-ACE2 cells were infected with lentiviruses pseudotyped with vesicular stomatitis virus G (VSV-G) and SARS-CoV-2 S protein variants. The y -axis shows the relative luminescence units (RLU) detected at 48 h post-pseudovirus inoculation. (C) Optimization of the incubation time for pseudovirus luciferase assay. Luciferase activities were measured 24–72 h post-virus infection. For this purpose, 72 h was chosen as the optimized incubation time. The data are presented as the means ± standard deviations (SDs) of three independent biological replicates.
    Figure Legend Snippet: Detection of SARS-CoV-2 S pseudotyped virus infectivity. (A) Schematic representation of the pseudovirus production and neutralization assay and the applications of the pseudovirus. (B) HEK293T and 293T-ACE2 cells were infected with lentiviruses pseudotyped with vesicular stomatitis virus G (VSV-G) and SARS-CoV-2 S protein variants. The y -axis shows the relative luminescence units (RLU) detected at 48 h post-pseudovirus inoculation. (C) Optimization of the incubation time for pseudovirus luciferase assay. Luciferase activities were measured 24–72 h post-virus infection. For this purpose, 72 h was chosen as the optimized incubation time. The data are presented as the means ± standard deviations (SDs) of three independent biological replicates.

    Techniques Used: Infection, Neutralization, Incubation, Luciferase

    4) Product Images from "SARS-CoV-2 Infection Depends on Cellular Heparan Sulfate and ACE2"

    Article Title: SARS-CoV-2 Infection Depends on Cellular Heparan Sulfate and ACE2

    Journal: Cell

    doi: 10.1016/j.cell.2020.09.033

    SARS-CoV-2 Spike Ectodomain Protein Binding to Cells Is Differentially Affected by HS from Different Organs and Potently Inhibited by Heparinoids (A) LC-MS/MS disaccharide analysis of HS isolated from human kidney, liver, tonsil, and lung tissue. (B) Inhibition of binding of recombinant SARS-CoV-2 S RBD protein to H1299 cells, using tissue HS. Analysis by flow cytometry. (C) Inhibition of recombinant trimeric SARS-CoV-2 protein (20 μg/mL) binding to H1299 cells, using CHO HS, heparin, MST heparin, and split-glycol heparin. Analysis by flow cytometry. (D) Similar analysis of A549 cells. Curve fitting was performed using non-linear regression and the inhibitor versus response least-squares fit algorithm. IC 50 values are listed in Table 1 . Graphs show representative experiments performed in technical duplicates or triplicates. (ns: p > 0.05, ∗ : p ≤ 0.05, ∗∗ : p ≤ 0.01, ∗∗∗ : p ≤ 0.001, ∗∗∗∗ : p ≤ 0.0001).
    Figure Legend Snippet: SARS-CoV-2 Spike Ectodomain Protein Binding to Cells Is Differentially Affected by HS from Different Organs and Potently Inhibited by Heparinoids (A) LC-MS/MS disaccharide analysis of HS isolated from human kidney, liver, tonsil, and lung tissue. (B) Inhibition of binding of recombinant SARS-CoV-2 S RBD protein to H1299 cells, using tissue HS. Analysis by flow cytometry. (C) Inhibition of recombinant trimeric SARS-CoV-2 protein (20 μg/mL) binding to H1299 cells, using CHO HS, heparin, MST heparin, and split-glycol heparin. Analysis by flow cytometry. (D) Similar analysis of A549 cells. Curve fitting was performed using non-linear regression and the inhibitor versus response least-squares fit algorithm. IC 50 values are listed in Table 1 . Graphs show representative experiments performed in technical duplicates or triplicates. (ns: p > 0.05, ∗ : p ≤ 0.05, ∗∗ : p ≤ 0.01, ∗∗∗ : p ≤ 0.001, ∗∗∗∗ : p ≤ 0.0001).

    Techniques Used: Protein Binding, Liquid Chromatography with Mass Spectroscopy, Isolation, Inhibition, Binding Assay, Recombinant, Flow Cytometry

    Binding of RBD Protein to Hep3B Mutants, Related to Figure 3 Binding of SARS-CoV-2 S RBD protein (20 μg/mL) to Hep3B mutants. Binding was measured by flow cytometry. Statistical analysis by unpaired t test. (ns: p > 0.05, ∗ : p ≤ 0.05, ∗∗ : p ≤ 0.01, ∗∗∗ : p ≤ 0.001, ∗∗∗∗ : p ≤ 0.0001).
    Figure Legend Snippet: Binding of RBD Protein to Hep3B Mutants, Related to Figure 3 Binding of SARS-CoV-2 S RBD protein (20 μg/mL) to Hep3B mutants. Binding was measured by flow cytometry. Statistical analysis by unpaired t test. (ns: p > 0.05, ∗ : p ≤ 0.05, ∗∗ : p ≤ 0.01, ∗∗∗ : p ≤ 0.001, ∗∗∗∗ : p ≤ 0.0001).

    Techniques Used: Binding Assay, Flow Cytometry

    SARS-CoV-2 Spike Ectodomain Binding to Cells Is Dependent on Cellular HS (A) Titration of recombinant SARS-CoV-2 spike protein binding to human H1299 cells with and without treatment with a mix of heparin lyases I, II, and III (HSase). (B) Recombinant SARS-CoV-2 spike protein binding (20 μg/mL) to H1299, A549, and Hep3B cells with and without HSase treatment. (C) SARS-CoV-2 S RBD protein binding (20 μg/mL) to H1299, A549, and Hep3B cells with and without HSase treatment. (D) SARS-CoV-2 spike protein binding (20 μg/mL) to H1299 and A375 cells with and without HSase treatment. (E) Anti-HS (F58-10E4) staining of H1299, A549, Hep3B, and A375 cells with and without HSase treatment. (F) Binding of recombinant SARS-CoV-2 spike protein (20 μg/mL) to Hep3B mutants altered in HS biosynthesis enzymes. Specific enzymes that were lacking in the mutants are listed along the x axis. All values were obtained by flow cytometry. Graphs shows representative experiments performed in technical triplicate. The experiments were repeated at least three times. Statistical analysis by unpaired t test (ns: p > 0.05, ∗ : p ≤ 0.05, ∗∗ : p ≤ 0.01, ∗∗∗ : p ≤ 0.001, ∗∗∗∗ : p ≤ 0.0001). See also Figure S4 .
    Figure Legend Snippet: SARS-CoV-2 Spike Ectodomain Binding to Cells Is Dependent on Cellular HS (A) Titration of recombinant SARS-CoV-2 spike protein binding to human H1299 cells with and without treatment with a mix of heparin lyases I, II, and III (HSase). (B) Recombinant SARS-CoV-2 spike protein binding (20 μg/mL) to H1299, A549, and Hep3B cells with and without HSase treatment. (C) SARS-CoV-2 S RBD protein binding (20 μg/mL) to H1299, A549, and Hep3B cells with and without HSase treatment. (D) SARS-CoV-2 spike protein binding (20 μg/mL) to H1299 and A375 cells with and without HSase treatment. (E) Anti-HS (F58-10E4) staining of H1299, A549, Hep3B, and A375 cells with and without HSase treatment. (F) Binding of recombinant SARS-CoV-2 spike protein (20 μg/mL) to Hep3B mutants altered in HS biosynthesis enzymes. Specific enzymes that were lacking in the mutants are listed along the x axis. All values were obtained by flow cytometry. Graphs shows representative experiments performed in technical triplicate. The experiments were repeated at least three times. Statistical analysis by unpaired t test (ns: p > 0.05, ∗ : p ≤ 0.05, ∗∗ : p ≤ 0.01, ∗∗∗ : p ≤ 0.001, ∗∗∗∗ : p ≤ 0.0001). See also Figure S4 .

    Techniques Used: Binding Assay, Titration, Recombinant, Protein Binding, Staining, Flow Cytometry

    SARS-CoV-2 Pseudovirus Infection Depends on Heparan Sulfate (A) Left, SARS-CoV-2 spike protein (20 μg/mL) binding to Vero cells measured by flow cytometry with and without HSase. Right, heparin and split-glycol heparin inhibit SARS-CoV-2 spike protein (20 μg/mL) binding to Vero cells by flow cytometry. Statistical analysis by unpaired t test. (B) Western blot analysis of ACE2 expression in Vero E6 cells compared to A549, H1299, and A375 cells. A representative blot of three extracts is shown for each strain. (C) Infection of Vero E6 cells with SARS-CoV-2 spike protein expressing pseudotyped virus expressing GFP. Infection was done with and without HSase treatment of the cells. Insert shows GFP expression in the infected cells by imaging. Counting was performed by flow cytometry with gating for GFP-positive cells as indicated by “infected.” (D) Quantitative analysis of GFP-positive cells. (E) Infection of Vero E6 cells with SARS-CoV-2 S protein pseudotyped virus expressing luciferase, as measured by the addition of Bright-Glo and detection of luminescence. The figure shows infection experiments done at low and high titer. (F) HSase treatment diminishes infection by SARS-CoV-2 S protein pseudotyped virus (luciferase) at low and high titer. (G) Heparin (0.5 μg/mL) blocks infection with SARS-CoV-2 S protein pseudotyped virus (luciferase). (H) Effect of HSase treatment of Vero E6 cells on the infection of both SARS-CoV-1 S and SARS-CoV-2 S protein pseudotyped virus expressing luciferase. (I) Infection of Hep3B with and without HSase and in Hep3B cells containing mutations in EXT1 , NDST1 , and HS6ST1 / HS6ST2 . Cells were infected with SARS-CoV-2 S protein pseudotyped virus expressing luciferase. All experiments were repeated at least three times. Graphs shows representative experiments performed in technical triplicates. Statistical analysis by unpaired t test. (ns: p > 0.05, ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001). See also Figure S6 .
    Figure Legend Snippet: SARS-CoV-2 Pseudovirus Infection Depends on Heparan Sulfate (A) Left, SARS-CoV-2 spike protein (20 μg/mL) binding to Vero cells measured by flow cytometry with and without HSase. Right, heparin and split-glycol heparin inhibit SARS-CoV-2 spike protein (20 μg/mL) binding to Vero cells by flow cytometry. Statistical analysis by unpaired t test. (B) Western blot analysis of ACE2 expression in Vero E6 cells compared to A549, H1299, and A375 cells. A representative blot of three extracts is shown for each strain. (C) Infection of Vero E6 cells with SARS-CoV-2 spike protein expressing pseudotyped virus expressing GFP. Infection was done with and without HSase treatment of the cells. Insert shows GFP expression in the infected cells by imaging. Counting was performed by flow cytometry with gating for GFP-positive cells as indicated by “infected.” (D) Quantitative analysis of GFP-positive cells. (E) Infection of Vero E6 cells with SARS-CoV-2 S protein pseudotyped virus expressing luciferase, as measured by the addition of Bright-Glo and detection of luminescence. The figure shows infection experiments done at low and high titer. (F) HSase treatment diminishes infection by SARS-CoV-2 S protein pseudotyped virus (luciferase) at low and high titer. (G) Heparin (0.5 μg/mL) blocks infection with SARS-CoV-2 S protein pseudotyped virus (luciferase). (H) Effect of HSase treatment of Vero E6 cells on the infection of both SARS-CoV-1 S and SARS-CoV-2 S protein pseudotyped virus expressing luciferase. (I) Infection of Hep3B with and without HSase and in Hep3B cells containing mutations in EXT1 , NDST1 , and HS6ST1 / HS6ST2 . Cells were infected with SARS-CoV-2 S protein pseudotyped virus expressing luciferase. All experiments were repeated at least three times. Graphs shows representative experiments performed in technical triplicates. Statistical analysis by unpaired t test. (ns: p > 0.05, ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001). See also Figure S6 .

    Techniques Used: Infection, Binding Assay, Flow Cytometry, Western Blot, Expressing, Imaging, Luciferase

    Molecular Modeling of the SARS-Cov-2 Spike RBD Interaction with Heparin (A) A molecular model of SARS CoV-2 S protein trimer (PDB: 6VSB and 6M0J ) rendered with Pymol. ACE2 is shown in blue and the RBD open conformation in green. A set of positively charged residues lies distal to the ACE2 binding site. (B) Electrostatic surface rendering of the SARS-CoV-2 RBD (PDB: 6M17 ) docked with dp4 heparin oligosaccharides. Blue and red surfaces indicate electropositive and electronegative surfaces, respectively. Oligosaccharides are represented using standard CPK format. (C) Mesh surface rendering of the RBD (green) docked with dp4 heparin oligosaccharides (red). (D) Number of contacts between the RBD amino acids and a set of docked heparin dp4 oligosaccharides from (A and B). (E) Calculated energy contributions of each amino acid residue in the RBD that can interact with heparin. (F) Amino acid sequence alignment of the SARS-CoV-1 and SARS-Cov-2 RBD. Red boxes indicate amino acid residues contributing to the electropositive patch in (A and B). Identical residues are shaded dark gray. Conservative substitutions have backgrounds in blue. Non-conserved residues have a white background (G) Structural alignment of SARS-CoV-1 (cyan; PDB: 3BGF ) and SARS-CoV-2 (red; PDB: 6M17 ) RBD. (H) Electrostatic surface rendering of the SARS-CoV-1 and SAR-CoV-2 RBDs. See also Figure S1 .
    Figure Legend Snippet: Molecular Modeling of the SARS-Cov-2 Spike RBD Interaction with Heparin (A) A molecular model of SARS CoV-2 S protein trimer (PDB: 6VSB and 6M0J ) rendered with Pymol. ACE2 is shown in blue and the RBD open conformation in green. A set of positively charged residues lies distal to the ACE2 binding site. (B) Electrostatic surface rendering of the SARS-CoV-2 RBD (PDB: 6M17 ) docked with dp4 heparin oligosaccharides. Blue and red surfaces indicate electropositive and electronegative surfaces, respectively. Oligosaccharides are represented using standard CPK format. (C) Mesh surface rendering of the RBD (green) docked with dp4 heparin oligosaccharides (red). (D) Number of contacts between the RBD amino acids and a set of docked heparin dp4 oligosaccharides from (A and B). (E) Calculated energy contributions of each amino acid residue in the RBD that can interact with heparin. (F) Amino acid sequence alignment of the SARS-CoV-1 and SARS-Cov-2 RBD. Red boxes indicate amino acid residues contributing to the electropositive patch in (A and B). Identical residues are shaded dark gray. Conservative substitutions have backgrounds in blue. Non-conserved residues have a white background (G) Structural alignment of SARS-CoV-1 (cyan; PDB: 3BGF ) and SARS-CoV-2 (red; PDB: 6M17 ) RBD. (H) Electrostatic surface rendering of the SARS-CoV-1 and SAR-CoV-2 RBDs. See also Figure S1 .

    Techniques Used: Binding Assay, Sequencing

    5) Product Images from "Functional evaluation of proteolytic activation for the SARS-CoV-2 variant B.1.1.7: role of the P681H mutation"

    Article Title: Functional evaluation of proteolytic activation for the SARS-CoV-2 variant B.1.1.7: role of the P681H mutation

    Journal: bioRxiv

    doi: 10.1101/2021.04.06.438731

    Cell-cell assays of S-expressing VeroE6 and Vero-TMPRSS2 cells. Cells were transfected with the SARS-CoV-2 WT, B.1.1.7 or P681H S gene and syncytia formation was evaluated through IFA after 28 hours. Syncytia was observed in all cells, regardless the expressed S type. More extensive syncytia formation was observed in Vero-TMPRSS2 cells and was consistent among the three S proteins.
    Figure Legend Snippet: Cell-cell assays of S-expressing VeroE6 and Vero-TMPRSS2 cells. Cells were transfected with the SARS-CoV-2 WT, B.1.1.7 or P681H S gene and syncytia formation was evaluated through IFA after 28 hours. Syncytia was observed in all cells, regardless the expressed S type. More extensive syncytia formation was observed in Vero-TMPRSS2 cells and was consistent among the three S proteins.

    Techniques Used: Expressing, Transfection, Immunofluorescence

    Pseudoparticle infectivity assays in VeroE6 and Vero-TMPRSS2 cells. VeroE6 and Vero-TMPRSS2 cells were infected with MLVpps harboring the VSV-G, SARS-CoV-2 S WT, SARS-CoV-2 S B.1.1.7 variant, SARS-CoV-2 S WT with P681H mutation. Data represents the average luciferase activity of cells of three biological replicates. No significant differences in luciferase transduction were observed between the infected cells.
    Figure Legend Snippet: Pseudoparticle infectivity assays in VeroE6 and Vero-TMPRSS2 cells. VeroE6 and Vero-TMPRSS2 cells were infected with MLVpps harboring the VSV-G, SARS-CoV-2 S WT, SARS-CoV-2 S B.1.1.7 variant, SARS-CoV-2 S WT with P681H mutation. Data represents the average luciferase activity of cells of three biological replicates. No significant differences in luciferase transduction were observed between the infected cells.

    Techniques Used: Infection, Variant Assay, Mutagenesis, Luciferase, Activity Assay, Transduction

    Furin cleavage score analysis of CoV S1/S2 cleavage sites. CoV S sequences were analyzed using the ProP 1.0 and PiTou 3.0 furin prediction algorithm, generating a score with bold numbers indicating predicted furin cleavage. (| ) denotes the position of the predicted S1/S2 cleavage site. Basic resides, arginine (R) and lysine (K), are highlighted in blue, with histidine (H) in purple. Sequences corresponding to the S1/S2 region of SARS-CoV-2 (QHD43416.1), SARS-CoV (AAT74874.1), MERS-CoV (AFS88936.1), HCoV-HKU1 (AAT98580.1), HCoV-OC43 (KY369907.1) were obtained from GenBank. Sequences corresponding to the S1/S2 region of SARS-CoV-2 B.1.1.7 (EPI_ISL_1374509) was obtained from GISAID.
    Figure Legend Snippet: Furin cleavage score analysis of CoV S1/S2 cleavage sites. CoV S sequences were analyzed using the ProP 1.0 and PiTou 3.0 furin prediction algorithm, generating a score with bold numbers indicating predicted furin cleavage. (| ) denotes the position of the predicted S1/S2 cleavage site. Basic resides, arginine (R) and lysine (K), are highlighted in blue, with histidine (H) in purple. Sequences corresponding to the S1/S2 region of SARS-CoV-2 (QHD43416.1), SARS-CoV (AAT74874.1), MERS-CoV (AFS88936.1), HCoV-HKU1 (AAT98580.1), HCoV-OC43 (KY369907.1) were obtained from GenBank. Sequences corresponding to the S1/S2 region of SARS-CoV-2 B.1.1.7 (EPI_ISL_1374509) was obtained from GISAID.

    Techniques Used:

    Fluorogenic peptide cleavage assays of the SARS-CoV-2 S1/S2 cleavage site. Peptides mimicking the S1/S2 site of the SARS-CoV-2 WT and B.1.1.7 variants were evaluated for in vitro cleavage by trypsin and furin proteases at pH 7.4 (trypsin), and pH 6.5, 6.0 and 7.5 (furin) conditions. A significant decrease in the cleavage of the B.1.1.7 S1/S2 peptide by furin was observed at pH 6.5 and 7.0 compared to WT. In contrast, a non-significant increase in the furin cleavage of the B.1.1.7 peptide was observed at pH 7.5.
    Figure Legend Snippet: Fluorogenic peptide cleavage assays of the SARS-CoV-2 S1/S2 cleavage site. Peptides mimicking the S1/S2 site of the SARS-CoV-2 WT and B.1.1.7 variants were evaluated for in vitro cleavage by trypsin and furin proteases at pH 7.4 (trypsin), and pH 6.5, 6.0 and 7.5 (furin) conditions. A significant decrease in the cleavage of the B.1.1.7 S1/S2 peptide by furin was observed at pH 6.5 and 7.0 compared to WT. In contrast, a non-significant increase in the furin cleavage of the B.1.1.7 peptide was observed at pH 7.5.

    Techniques Used: In Vitro

    6) Product Images from "Cathepsin L plays a key role in SARS-CoV-2 infection in humans and humanized mice and is a promising target for new drug development"

    Article Title: Cathepsin L plays a key role in SARS-CoV-2 infection in humans and humanized mice and is a promising target for new drug development

    Journal: Signal Transduction and Targeted Therapy

    doi: 10.1038/s41392-021-00558-8

    CTSL cleaves the SARS-CoV-2 spike (S) protein, and this cleavage promotes cell–cell fusion. a Overview of the SARS-CoV-1 and SARS-CoV-2 S1/S2 cleavage sites. FP (fusion peptide), HR1 (heptad repeat 1), and HR2 (heptad repeat 2) are units of the S2 subunit that function in membrane fusion. b Analysis of CTSL-mediated S-protein cleavage. Purified SARS-CoV-1 or SARS-CoV-2 S protein was incubated in the presence or absence (assay buffer, pH = 5.5) of CTSL (2 or 10 μg/ml in assay buffer, pH = 5 .5) at 37 °C for 1 h. The reaction system of 2 μg/ml CTSL was further supplemented with CTSL inhibitors (20 μM E64d or 20 μM SID 26681509), as indicated. Proteins were subjected to SDS-PAGE and detected by silver staining. Representative data from three independent experiments are shown. c Syncytium-formation assay: Huh7 cells were untransfected (Null) or transfected with plasmid to express the SARS-CoV-2 S protein. Cells were incubated in the presence or absence (PBS, pH = 7.4) of trypsin (2 μg/ml in PBS, pH = 7.4) or in the presence or absence (PBS, pH = 5.8) of CTSL (2 or 4 μg/ml in PBS, pH = 5.8) for 20 min. Images were acquired after an additional 16 h incubation in the medium. (scale bars, 50 μm). The black arrowheads indicate syncytia. Representative data from seven independent experiments are shown. d Quantitative analysis of syncytia in panel c . n = 7. Statistical significance was assessed by one-way ANOVA with Tukey’s post hoc test. The data are expressed as the mean ± s.e.m. values. * P
    Figure Legend Snippet: CTSL cleaves the SARS-CoV-2 spike (S) protein, and this cleavage promotes cell–cell fusion. a Overview of the SARS-CoV-1 and SARS-CoV-2 S1/S2 cleavage sites. FP (fusion peptide), HR1 (heptad repeat 1), and HR2 (heptad repeat 2) are units of the S2 subunit that function in membrane fusion. b Analysis of CTSL-mediated S-protein cleavage. Purified SARS-CoV-1 or SARS-CoV-2 S protein was incubated in the presence or absence (assay buffer, pH = 5.5) of CTSL (2 or 10 μg/ml in assay buffer, pH = 5 .5) at 37 °C for 1 h. The reaction system of 2 μg/ml CTSL was further supplemented with CTSL inhibitors (20 μM E64d or 20 μM SID 26681509), as indicated. Proteins were subjected to SDS-PAGE and detected by silver staining. Representative data from three independent experiments are shown. c Syncytium-formation assay: Huh7 cells were untransfected (Null) or transfected with plasmid to express the SARS-CoV-2 S protein. Cells were incubated in the presence or absence (PBS, pH = 7.4) of trypsin (2 μg/ml in PBS, pH = 7.4) or in the presence or absence (PBS, pH = 5.8) of CTSL (2 or 4 μg/ml in PBS, pH = 5.8) for 20 min. Images were acquired after an additional 16 h incubation in the medium. (scale bars, 50 μm). The black arrowheads indicate syncytia. Representative data from seven independent experiments are shown. d Quantitative analysis of syncytia in panel c . n = 7. Statistical significance was assessed by one-way ANOVA with Tukey’s post hoc test. The data are expressed as the mean ± s.e.m. values. * P

    Techniques Used: Purification, Incubation, SDS Page, Silver Staining, Tube Formation Assay, Transfection, Plasmid Preparation

    7) Product Images from "D614G mutation of SARS-CoV-2 spike protein enhances viral infectivity"

    Article Title: D614G mutation of SARS-CoV-2 spike protein enhances viral infectivity

    Journal: bioRxiv

    doi: 10.1101/2020.06.20.161323

    The S-G614 protein pseudotyped virus showed increased infectivity. a HEK 293T and 293T-ACE2 (human angiotensin-converting enzyme 2) cells were infected with lentiviruses pseudotyped with VSV-G and SARS-CoV-2 S protein variants. Virus titers were quantified by RT-qPCR and adjusted to 3.8 × 10 4 copies in 50 μL to normalize input virus doses. The relative luminescence units (RLU) detected 72 h post-infection (hpi). b Inhibition of pseudoviral entry by ACE2-Ig. Pseudoviruses were pre-incubated with ACE2-Ig and added to 293T-ACE2 cells, then RLU was measured at 72 hpi. c Viral entry efficiency meditated by S variants. The RLU was measured at 24-72 hpi. d-e D614G mutation facilitates elastase-2 induced pseudoviral entry. 293T-ACE2 cells were treated with elastase for 5 min and then infected with pseudotyped viruses containing the S-D614 or S-G614 mutant in the presence of various concentrations of sivelestat sodium. RLU was measured at 72 hpi. n = 3, ±SD. * P
    Figure Legend Snippet: The S-G614 protein pseudotyped virus showed increased infectivity. a HEK 293T and 293T-ACE2 (human angiotensin-converting enzyme 2) cells were infected with lentiviruses pseudotyped with VSV-G and SARS-CoV-2 S protein variants. Virus titers were quantified by RT-qPCR and adjusted to 3.8 × 10 4 copies in 50 μL to normalize input virus doses. The relative luminescence units (RLU) detected 72 h post-infection (hpi). b Inhibition of pseudoviral entry by ACE2-Ig. Pseudoviruses were pre-incubated with ACE2-Ig and added to 293T-ACE2 cells, then RLU was measured at 72 hpi. c Viral entry efficiency meditated by S variants. The RLU was measured at 24-72 hpi. d-e D614G mutation facilitates elastase-2 induced pseudoviral entry. 293T-ACE2 cells were treated with elastase for 5 min and then infected with pseudotyped viruses containing the S-D614 or S-G614 mutant in the presence of various concentrations of sivelestat sodium. RLU was measured at 72 hpi. n = 3, ±SD. * P

    Techniques Used: Infection, Quantitative RT-PCR, Inhibition, Incubation, Mutagenesis

    8) Product Images from "Bacterial expression and purification of functional recombinant SARS-CoV-2 spike receptor binding domain"

    Article Title: Bacterial expression and purification of functional recombinant SARS-CoV-2 spike receptor binding domain

    Journal: bioRxiv

    doi: 10.1101/2021.02.03.429601

    Activity assessment of purified recombinant RBD-MBP. A) SPR binding assay of RBD-MBP to hACE2. B) Microsphere immunoassay of RBD-MBP binding to IgG antibodies in patient sera [(+) = SARS-CoV-2 IgG positive, (-) = SARS-CoV-2 IgG negative]. MFI: mean fluorescence intensity.
    Figure Legend Snippet: Activity assessment of purified recombinant RBD-MBP. A) SPR binding assay of RBD-MBP to hACE2. B) Microsphere immunoassay of RBD-MBP binding to IgG antibodies in patient sera [(+) = SARS-CoV-2 IgG positive, (-) = SARS-CoV-2 IgG negative]. MFI: mean fluorescence intensity.

    Techniques Used: Activity Assay, Purification, Recombinant, SPR Assay, Binding Assay, Fluorescence

    Ribbon diagram of SARS-CoV-2 spike RBD showing intramolecular disulfide bonds in yellow. As the structures solved for the RBD had missing gaps in the loops, we fed our sequence into I-Tasser, which generated the complete model ( 14 - 16 ). The ribbon was drawn using UCSF Chimera ( 17 ), is colored from blue (N-terminus) to red (C-terminus), and cysteines forming disulfide bonds are labeled..
    Figure Legend Snippet: Ribbon diagram of SARS-CoV-2 spike RBD showing intramolecular disulfide bonds in yellow. As the structures solved for the RBD had missing gaps in the loops, we fed our sequence into I-Tasser, which generated the complete model ( 14 - 16 ). The ribbon was drawn using UCSF Chimera ( 17 ), is colored from blue (N-terminus) to red (C-terminus), and cysteines forming disulfide bonds are labeled..

    Techniques Used: Sequencing, Generated, Labeling

    9) Product Images from "SARS-CoV-2 entry into human airway organoids is serine protease-mediated and facilitated by the multibasic cleavage site"

    Article Title: SARS-CoV-2 entry into human airway organoids is serine protease-mediated and facilitated by the multibasic cleavage site

    Journal: eLife

    doi: 10.7554/eLife.64508

    A GFP complementation based assay for assessing coronavirus fusogenicity. ( A ) HEK-293T cells expressing an empty vector or S protein together with GFP-11-tagged beta-actin and a BFP containing a nuclear localization signal were added to cells stably expressing GFP1-10. Fusion of these two cell types allowed GFP complementation in cells expressing a nuclear BFP, facilitating easy quantification of nuclei per syncytial cell. Unfused cells only expressed BFP in the nucleus. Fusion with VeroE6 GFP1-10 cells 18 hr after addition of the fusogenic HEK-293T is shown as an example. ( B–D ) Full well scans of the complemented GFP signal 18 hr after addition of the fusogenic HEK-293T cells to Calu-3 GFP1-10 ( B ), VeroE6 GFP1-10 ( C ), and VeroE6-TMPRSS2 GFP1-10 ( D ) cells are shown. Dashed areas are enlarged next to each well. Scale bars indicate 50 μm. ( E and F ) Fusogenicity of SARS-CoV-2 S and SARS-CoV S was assessed after 18 hr by measuring the sum of all GFP+ pixels per well in VeroE6 cells ( E ) and VeroE6 TMPRSS2 cells ( F ). Statistical analysis was performed by one-way ANOVA on SARS-CoV-2 S-mediated fusion compared with SARS-CoV S. *p
    Figure Legend Snippet: A GFP complementation based assay for assessing coronavirus fusogenicity. ( A ) HEK-293T cells expressing an empty vector or S protein together with GFP-11-tagged beta-actin and a BFP containing a nuclear localization signal were added to cells stably expressing GFP1-10. Fusion of these two cell types allowed GFP complementation in cells expressing a nuclear BFP, facilitating easy quantification of nuclei per syncytial cell. Unfused cells only expressed BFP in the nucleus. Fusion with VeroE6 GFP1-10 cells 18 hr after addition of the fusogenic HEK-293T is shown as an example. ( B–D ) Full well scans of the complemented GFP signal 18 hr after addition of the fusogenic HEK-293T cells to Calu-3 GFP1-10 ( B ), VeroE6 GFP1-10 ( C ), and VeroE6-TMPRSS2 GFP1-10 ( D ) cells are shown. Dashed areas are enlarged next to each well. Scale bars indicate 50 μm. ( E and F ) Fusogenicity of SARS-CoV-2 S and SARS-CoV S was assessed after 18 hr by measuring the sum of all GFP+ pixels per well in VeroE6 cells ( E ) and VeroE6 TMPRSS2 cells ( F ). Statistical analysis was performed by one-way ANOVA on SARS-CoV-2 S-mediated fusion compared with SARS-CoV S. *p

    Techniques Used: Expressing, Plasmid Preparation, Stable Transfection

    10) Product Images from "Oral delivery of SARS-CoV-2 DNA vaccines using attenuated Salmonella typhimurium as a carrier in rat"

    Article Title: Oral delivery of SARS-CoV-2 DNA vaccines using attenuated Salmonella typhimurium as a carrier in rat

    Journal: bioRxiv

    doi: 10.1101/2020.07.23.217174

    pcDNA3.1(+)-CMV-SARS-CoV-2-S-GFP plasmid map.
    Figure Legend Snippet: pcDNA3.1(+)-CMV-SARS-CoV-2-S-GFP plasmid map.

    Techniques Used: Plasmid Preparation

    Micrographs of 293T cells transfected with pSARS-CoV-2-S (X 100). (A1, A2) 293T cells transfected with pSARS-CoV-2-S (pcDNA3.1(+)-CMV-SARS-CoV-2-S-GFP) at 48 hours after transfection. A1 fluorescence micrograph with GFP expression in cells and light micrograph with the same visual field as A2. (B) 293T cells transfected with pSARS-CoV-2-S at 48 hours after transfection. The SARS-CoV-2-S protein showed about 141 kDa.
    Figure Legend Snippet: Micrographs of 293T cells transfected with pSARS-CoV-2-S (X 100). (A1, A2) 293T cells transfected with pSARS-CoV-2-S (pcDNA3.1(+)-CMV-SARS-CoV-2-S-GFP) at 48 hours after transfection. A1 fluorescence micrograph with GFP expression in cells and light micrograph with the same visual field as A2. (B) 293T cells transfected with pSARS-CoV-2-S at 48 hours after transfection. The SARS-CoV-2-S protein showed about 141 kDa.

    Techniques Used: Transfection, Fluorescence, Expressing

    Humoral responses to SARS-CoV-2-S protein antigen in the rat after immunizationon day 0, day 14, and day 28 with Salmonella carrying the control vector or pSARS-CoV-2-S (as described in the methods). (A) After immunization with the control vector, test SARS-CoV-2-S protein antigen binding of IgG in serial serum dilutions from a rat at day (0, 14, 28). Data shown represent test mean OD450 nm values (mean±SD) for each of 9 rats, or (B) After immunization with the pSARS-CoV-2-S vector, SARS-CoV-2-S protein antigen binding of IgG in serial serum dilutions from a rat at day (0, 14, 28). Data shown represent mean OD450 nm values (3 times measurement, mean±SD) for each of 9 rats.
    Figure Legend Snippet: Humoral responses to SARS-CoV-2-S protein antigen in the rat after immunizationon day 0, day 14, and day 28 with Salmonella carrying the control vector or pSARS-CoV-2-S (as described in the methods). (A) After immunization with the control vector, test SARS-CoV-2-S protein antigen binding of IgG in serial serum dilutions from a rat at day (0, 14, 28). Data shown represent test mean OD450 nm values (mean±SD) for each of 9 rats, or (B) After immunization with the pSARS-CoV-2-S vector, SARS-CoV-2-S protein antigen binding of IgG in serial serum dilutions from a rat at day (0, 14, 28). Data shown represent mean OD450 nm values (3 times measurement, mean±SD) for each of 9 rats.

    Techniques Used: Plasmid Preparation, Binding Assay

    11) Product Images from "Development and Optimization of In-house ELISA for Detection of Human IgG Antibody to SARS-CoV-2 Full Length Spike Protein"

    Article Title: Development and Optimization of In-house ELISA for Detection of Human IgG Antibody to SARS-CoV-2 Full Length Spike Protein

    Journal: Pathogens

    doi: 10.3390/pathogens9100803

    The cut-off value of the developed SARS-CoV-2 full-length S (S1 + S2)-based indirect ELISA. Plates were coated overnight at 4 ℃ with 100 ng/well of SARS-CoV-2 full-length S (S1 + S2) ECD-His recombinant protein. Following three washes with PBS containing 0.1% Tween 20 (PBST), positive (green) and negative (red) samples based on micro-neutralization assay were diluted 1:100 and added. PBST was added as blank (empty circles). Following an hour of incubation at 37 ℃, three washes with PBST were performed followed by incubation with the conjugate (peroxidase-labelled anti-human IgG secondary antibody at 1:64,000 dilution) for 1 h at 37 ℃. Then, three washes with PBST were performed followed by the addition of 3,3’,5,5’-Tetramethylbenzidine (TMB) substrate for 5 min. Hydrochloric acid (HCL) was added as a stop solution. The optical density was measured at 450 nm (OD 450 ). The actual values for each sample is shown. Dashed lines represent the cut-off value (Mean + 3 x standard deviation).
    Figure Legend Snippet: The cut-off value of the developed SARS-CoV-2 full-length S (S1 + S2)-based indirect ELISA. Plates were coated overnight at 4 ℃ with 100 ng/well of SARS-CoV-2 full-length S (S1 + S2) ECD-His recombinant protein. Following three washes with PBS containing 0.1% Tween 20 (PBST), positive (green) and negative (red) samples based on micro-neutralization assay were diluted 1:100 and added. PBST was added as blank (empty circles). Following an hour of incubation at 37 ℃, three washes with PBST were performed followed by incubation with the conjugate (peroxidase-labelled anti-human IgG secondary antibody at 1:64,000 dilution) for 1 h at 37 ℃. Then, three washes with PBST were performed followed by the addition of 3,3’,5,5’-Tetramethylbenzidine (TMB) substrate for 5 min. Hydrochloric acid (HCL) was added as a stop solution. The optical density was measured at 450 nm (OD 450 ). The actual values for each sample is shown. Dashed lines represent the cut-off value (Mean + 3 x standard deviation).

    Techniques Used: Indirect ELISA, Recombinant, Neutralization, Incubation, Standard Deviation

    Optimization of an indirect ELISA utilizing SARS-CoV-2 full-length S (S1 + S2) recombinant protein. ( A ) and ( B ) Plates were coated overnight at 4 ℃ with different concentrations of either SARS-CoV-2 full-length S (S1 + S2) extracellular domain with a polyhistidine tag (ECD-His) recombinant protein, or bovine serum albumin (BSA) as a coating control. Following three washes with PBS containing 0.1% Tween 20 (PBST), positive (green) and negative (red) samples based on micro-neutralization assay were serially diluted (as indicated) and added. PBST was used as blank (empty black circles). Following an hour of incubation at 37 ℃, three washes with PBST were performed followed by incubation with conjugate (peroxidase-labelled anti-human IgG secondary antibody) for 1 h at 37 ℃. Then, three washes with PBST were performed followed by the addition of 3,3’,5,5’-Tetramethylbenzidine (TMB) substrate for 5 min. Hydrochloric acid (HCL) was added as a stop solution. The optical density was measured at 450 nm (OD 450 ). ( C ) Plates were coated with 100 ng/well of viral antigen, samples were used at 1:100 dilution, and conjugate dilutions ranged from 1:1000 to 1:128,000. The protocol was performed as described above.
    Figure Legend Snippet: Optimization of an indirect ELISA utilizing SARS-CoV-2 full-length S (S1 + S2) recombinant protein. ( A ) and ( B ) Plates were coated overnight at 4 ℃ with different concentrations of either SARS-CoV-2 full-length S (S1 + S2) extracellular domain with a polyhistidine tag (ECD-His) recombinant protein, or bovine serum albumin (BSA) as a coating control. Following three washes with PBS containing 0.1% Tween 20 (PBST), positive (green) and negative (red) samples based on micro-neutralization assay were serially diluted (as indicated) and added. PBST was used as blank (empty black circles). Following an hour of incubation at 37 ℃, three washes with PBST were performed followed by incubation with conjugate (peroxidase-labelled anti-human IgG secondary antibody) for 1 h at 37 ℃. Then, three washes with PBST were performed followed by the addition of 3,3’,5,5’-Tetramethylbenzidine (TMB) substrate for 5 min. Hydrochloric acid (HCL) was added as a stop solution. The optical density was measured at 450 nm (OD 450 ). ( C ) Plates were coated with 100 ng/well of viral antigen, samples were used at 1:100 dilution, and conjugate dilutions ranged from 1:1000 to 1:128,000. The protocol was performed as described above.

    Techniques Used: Indirect ELISA, Recombinant, Neutralization, Incubation

    Assessment of the cross-reactivity of the developed SARS-CoV-2 S1 + S2 indirect ELISA. Plates were coated overnight at 4 ℃ with 100 ng/well of SARS-CoV-2 full-length S (S1 + S2) ECD-His recombinant protein. Following three washes with PBST, Serum samples containing anti-SARS-CoV-2 antibodies (green), anti-Middle East respiratory syndrome coronavirus (MERS-CoV) antibodies (purple), and anti-human coronavirus HKU1 (HCoV HKU1) antibodies (blue) were diluted 1:100 and added. Serum from healthy donors were used as a negative control (red). PBS containing 0.1% Tween 20 (PBST) was added as blank (empty circles). Following an hour of incubation at 37 ℃, three washes with PBST were performed followed by incubation with the conjugate (peroxidase-labelled anti-human IgG secondary antibody at 1:64,000 dilution) for 1 h at 37 ℃. Then, three washes with PBST were performed followed by the addition of 3,3’,5,5’-Tetramethylbenzidine (TMB) substrate for 5 min. Hydrochloric acid (HCL) was added as a stop solution. The optical density was measured at 450 nm (OD 450 ).
    Figure Legend Snippet: Assessment of the cross-reactivity of the developed SARS-CoV-2 S1 + S2 indirect ELISA. Plates were coated overnight at 4 ℃ with 100 ng/well of SARS-CoV-2 full-length S (S1 + S2) ECD-His recombinant protein. Following three washes with PBST, Serum samples containing anti-SARS-CoV-2 antibodies (green), anti-Middle East respiratory syndrome coronavirus (MERS-CoV) antibodies (purple), and anti-human coronavirus HKU1 (HCoV HKU1) antibodies (blue) were diluted 1:100 and added. Serum from healthy donors were used as a negative control (red). PBS containing 0.1% Tween 20 (PBST) was added as blank (empty circles). Following an hour of incubation at 37 ℃, three washes with PBST were performed followed by incubation with the conjugate (peroxidase-labelled anti-human IgG secondary antibody at 1:64,000 dilution) for 1 h at 37 ℃. Then, three washes with PBST were performed followed by the addition of 3,3’,5,5’-Tetramethylbenzidine (TMB) substrate for 5 min. Hydrochloric acid (HCL) was added as a stop solution. The optical density was measured at 450 nm (OD 450 ).

    Techniques Used: Indirect ELISA, Recombinant, Negative Control, Incubation

    Evaluation of the developed SARS-CoV-2 full-length S (S1 + S2)-based indirect ELISA. Plates were coated overnight at 4 ℃ with 100 ng/well of SARS-CoV-2 full-length S (S1 + S2) ECD-His recombinant protein. Following three washes with PBST, positive (green) and negative (red) samples based on micro-neutralization assay were diluted 1:100 and added. PBS containing 0.1% Tween 20 (PBST) was added as blank (empty circles). Following an hour of incubation at 37 ℃, three washes with PBST were performed followed by incubation with the conjugate (peroxidase-labelled anti-human IgG secondary antibody at 1:64,000 dilution) for 1 h at 37 ℃. Then, three washes with PBST was performed followed by the addition of 3,3’,5,5’-Tetramethylbenzidine (TMB) substrate for 5 min. Hydrochloric acid (HCL) was added as a stop solution. The optical density was measured at 450 nm (OD 450 ). Dashed lines represent the cut-off value (Mean + 3 x standard deviation). (A) shows the actual values for each sample, and (B) demonstrates the correlation between OD 450 readings and micro-neutralization (MN) titer. Boxes show the 25th–75th percentile range, the black line represents median, and whiskers are the minimum and maximum values.
    Figure Legend Snippet: Evaluation of the developed SARS-CoV-2 full-length S (S1 + S2)-based indirect ELISA. Plates were coated overnight at 4 ℃ with 100 ng/well of SARS-CoV-2 full-length S (S1 + S2) ECD-His recombinant protein. Following three washes with PBST, positive (green) and negative (red) samples based on micro-neutralization assay were diluted 1:100 and added. PBS containing 0.1% Tween 20 (PBST) was added as blank (empty circles). Following an hour of incubation at 37 ℃, three washes with PBST were performed followed by incubation with the conjugate (peroxidase-labelled anti-human IgG secondary antibody at 1:64,000 dilution) for 1 h at 37 ℃. Then, three washes with PBST was performed followed by the addition of 3,3’,5,5’-Tetramethylbenzidine (TMB) substrate for 5 min. Hydrochloric acid (HCL) was added as a stop solution. The optical density was measured at 450 nm (OD 450 ). Dashed lines represent the cut-off value (Mean + 3 x standard deviation). (A) shows the actual values for each sample, and (B) demonstrates the correlation between OD 450 readings and micro-neutralization (MN) titer. Boxes show the 25th–75th percentile range, the black line represents median, and whiskers are the minimum and maximum values.

    Techniques Used: Indirect ELISA, Recombinant, Neutralization, Incubation, Standard Deviation

    12) Product Images from "Trimeric SARS-CoV-2 Spike proteins produced from CHO-cells in bioreactors are high quality antigens"

    Article Title: Trimeric SARS-CoV-2 Spike proteins produced from CHO-cells in bioreactors are high quality antigens

    Journal: bioRxiv

    doi: 10.1101/2020.11.15.382044

    Inhibition of SARS-CoV-2 infection in Vero E6 cells (a) Inhibitory effect of S trimer and RBD on infection of Vero E6 cells. SARS-CoV-2 antigen-positive cells were visualized by immunofluorescent staining. Virus nucleocapsid antigen staining (red), cell nucleus staining (blue). All methods as described previously[ 10 ]. (b) Inhibitory effect of S trimer and RBD on infection of Vero E6 cells. SARS-CoV-2 antigen-positivity fluorescence was quantified as described [ 10 , 11 ].
    Figure Legend Snippet: Inhibition of SARS-CoV-2 infection in Vero E6 cells (a) Inhibitory effect of S trimer and RBD on infection of Vero E6 cells. SARS-CoV-2 antigen-positive cells were visualized by immunofluorescent staining. Virus nucleocapsid antigen staining (red), cell nucleus staining (blue). All methods as described previously[ 10 ]. (b) Inhibitory effect of S trimer and RBD on infection of Vero E6 cells. SARS-CoV-2 antigen-positivity fluorescence was quantified as described [ 10 , 11 ].

    Techniques Used: Inhibition, Infection, Staining, Fluorescence

    Schematic representation of SARS-CoV-2 Spike protein designs. SP: Signal Peptide, NTD: N Terminal Domain, RBD: Receptor Binding Domain, FCS: Furin Cleavage Site, FP: Fusion Peptide, HR1: Heptad Repeat 1, HR2: Heptad Repeat 2, TM: Transmembrane Domain, CT: C terminal Tail.
    Figure Legend Snippet: Schematic representation of SARS-CoV-2 Spike protein designs. SP: Signal Peptide, NTD: N Terminal Domain, RBD: Receptor Binding Domain, FCS: Furin Cleavage Site, FP: Fusion Peptide, HR1: Heptad Repeat 1, HR2: Heptad Repeat 2, TM: Transmembrane Domain, CT: C terminal Tail.

    Techniques Used: Binding Assay

    Characterization of the recombinant trimeric S trimer and RBD. Size-exclusion chromatography (SEC) analysis plot with a single peak at 4.0 minutes and 6.4 minutes. SDS-PAGE gel showing bands for S trimer at different dilutions (c) and (d) for RBD monomer, NR=non-reduced, R=reduced. Negative stain electron microscopy of SARS-CoV-2 S trimer (Wuhan, d,e, D614G, f-g). White bars in d and f: 50 nm. Six 2D class averages are shown to the right of each representative micrograph. 3D reconstructions are shown in (e) and (g).
    Figure Legend Snippet: Characterization of the recombinant trimeric S trimer and RBD. Size-exclusion chromatography (SEC) analysis plot with a single peak at 4.0 minutes and 6.4 minutes. SDS-PAGE gel showing bands for S trimer at different dilutions (c) and (d) for RBD monomer, NR=non-reduced, R=reduced. Negative stain electron microscopy of SARS-CoV-2 S trimer (Wuhan, d,e, D614G, f-g). White bars in d and f: 50 nm. Six 2D class averages are shown to the right of each representative micrograph. 3D reconstructions are shown in (e) and (g).

    Techniques Used: Recombinant, Size-exclusion Chromatography, SDS Page, Staining, Electron Microscopy

    Related Articles

    other:

    Article Title: D614G mutation of SARS-CoV-2 spike protein enhances viral infectivity
    Article Snippet: Antibodies and inhibitorsThe anti-RBD monoclonal antibody against the SARS-CoV-2 S protein was kindly provided by Prof. Aishun Jin from Chongqing Medical University.

    Article Title: Cathepsin L plays a key role in SARS-CoV-2 infection in humans and humanized mice and is a promising target for new drug development
    Article Snippet: Where indicated, 20 μM E64d or 20 μM SID 26681509 was added in the reaction system with 0.5 μg SARS-CoV-2 S protein and 2 μg/ml CTSL.

    Article Title: Development of cell-based pseudovirus entry assay to identify potential viral entry inhibitors and neutralizing antibodies against SARS-CoV-2
    Article Snippet: The anti-RBD monoclonal antibody against the SARS-CoV-2 S protein was kindly provided by Prof. Aishun Jin (College of Basic Medial, Chongqing Medical University).

    Clone Assay:

    Article Title: Development and structural basis of a two-MAb cocktail for treating SARS-CoV-2 infections
    Article Snippet: To generate ACE2, DNA fragment encoding the extracellular domain of human ACE2 (residues Q18 to S740) was cloned into a modified pcDNA3.4 vector that contains IL-10 signal sequence and C-terminal human IgG1 Fc and His tag, yielding plasmid pcDNA3.4-ACE2-hFc. .. To prepare SARS-CoV-2 S protein, mammalian codon-optimized gene coding S ectodomain (residues 1–1208) with proline substitutions at residues 986 and 987, a “GSAS” substitution at the furin cleavage site (residues 682–685) was cloned into vector pcDNA3.1+. .. A C-terminal T4 fibritin trimerization motif, a TEV protease cleavage site, a FLAG tag and a His tag were cloned downstream of the SARS-CoV-2 S glycoprotein ectodomain.

    Plasmid Preparation:

    Article Title: Development and structural basis of a two-MAb cocktail for treating SARS-CoV-2 infections
    Article Snippet: To generate ACE2, DNA fragment encoding the extracellular domain of human ACE2 (residues Q18 to S740) was cloned into a modified pcDNA3.4 vector that contains IL-10 signal sequence and C-terminal human IgG1 Fc and His tag, yielding plasmid pcDNA3.4-ACE2-hFc. .. To prepare SARS-CoV-2 S protein, mammalian codon-optimized gene coding S ectodomain (residues 1–1208) with proline substitutions at residues 986 and 987, a “GSAS” substitution at the furin cleavage site (residues 682–685) was cloned into vector pcDNA3.1+. .. A C-terminal T4 fibritin trimerization motif, a TEV protease cleavage site, a FLAG tag and a His tag were cloned downstream of the SARS-CoV-2 S glycoprotein ectodomain.

    Recombinant:

    Article Title: SARS-CoV-2 Infection Depends on Cellular Heparan Sulfate and ACE2
    Article Snippet: .. The SARS-CoV-2 Spike Protein Binds Heparin through the RBD To test experimentally if the SARS-CoV-2 S protein interacts with heparin/HS, recombinant ectodomain and RBD proteins were prepared and characterized. .. Initial studies encountered difficulty in stabilizing the S ectodomain protein, a problem that was resolved by raising the concentration of NaCl to 0.3 M in HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer.

    Expressing:

    Article Title: Functional evaluation of proteolytic activation for the SARS-CoV-2 variant B.1.1.7: role of the P681H mutation
    Article Snippet: Blocking was performed using 5% heat inactivated goat serum in PBS for 20 minutes and the antibodies for labeling were diluted in the same solution. .. The spike expression was detected using the SARS-CoV-2 spike antibody (Cat: 40591-T62, Sino Biological Inc.) at 1/500 dilution for 1 hour. .. Secondary antibody labeling was performed using AlexaFluorTM 488 goat anti-rabitt IgG antibody (Cat: A32731, Invitrogen Co.) at a 1/500 dilution for 45 minutes.

    Western Blot:

    Article Title: Bacterial expression and purification of functional recombinant SARS-CoV-2 spike receptor binding domain
    Article Snippet: Fractions corresponding to the fusion protein , were pooled and concentrated (using Amicon regenerated cellulose concentrators (Cat # UFC803096). .. RBD-MBP identity after each column was confirmed by Peggy Sue (Protein Simple) western analysis using Sino Biological Anti-Coronavirus spike antibody (Cat # 40591-T62). .. The concentrations were determined using a Fisher NanoDrop1000 using a molecular weight of 74 kDa for the fusion protein, and calculated ε280 =101.19 M−1 cm−1.

    Purification:

    Article Title: Cathepsin L plays a key role in SARS-CoV-2 infection in humans and humanized mice and is a promising target for new drug development
    Article Snippet: After 24 h incubation, the cells were lysed for analysis of the firefly luciferase activity, VSV-P mRNA, and the detection of CTSL and CTSB by ELISA assays. .. Cleavage of SARS-CoV-2 S protein by CTSLThe purified extracellular domain of SARS-CoV-2 S protein (YP_009724390.1. .. Sino Biological, Cat. No. 40589-V08B1) and SARS-CoV S protein (NP_828851.1.

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    Sino Biological sars cov 2 s protein
    CTSL cleaves the <t>SARS-CoV-2</t> spike (S) protein, and this cleavage promotes cell–cell fusion. a Overview of the SARS-CoV-1 and SARS-CoV-2 S1/S2 cleavage sites. FP (fusion peptide), HR1 (heptad repeat 1), and HR2 (heptad repeat 2) are units of the S2 subunit that function in membrane fusion. b Analysis of CTSL-mediated S-protein cleavage. Purified SARS-CoV-1 or SARS-CoV-2 S protein was incubated in the presence or absence (assay buffer, pH = 5.5) of CTSL (2 or 10 μg/ml in assay buffer, pH = 5 .5) at 37 °C for 1 h. The reaction system of 2 μg/ml CTSL was further supplemented with CTSL inhibitors (20 μM E64d or 20 μM SID 26681509), as indicated. Proteins were subjected to SDS-PAGE and detected by silver staining. Representative data from three independent experiments are shown. c Syncytium-formation assay: Huh7 cells were untransfected (Null) or transfected with plasmid to express the SARS-CoV-2 S protein. Cells were incubated in the presence or absence (PBS, pH = 7.4) of trypsin (2 μg/ml in PBS, pH = 7.4) or in the presence or absence (PBS, pH = 5.8) of CTSL (2 or 4 μg/ml in PBS, pH = 5.8) for 20 min. Images were acquired after an additional 16 h incubation in the medium. (scale bars, 50 μm). The black arrowheads indicate syncytia. Representative data from seven independent experiments are shown. d Quantitative analysis of syncytia in panel c . n = 7. Statistical significance was assessed by one-way ANOVA with Tukey’s post hoc test. The data are expressed as the mean ± s.e.m. values. * P
    Sars Cov 2 S Protein, supplied by Sino Biological, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Sino Biological sars cov 2 2019 ncov spike antibody rabbit pab
    LL37 attenuates S1 binding to cells by cloaking ACE2. (A) Binding kinetics for LL37 and ACE2. (B) BLI-based ACE2 blocking assay. (C) Protein bands of S1 binding to A549 cells pretreated with increasing concentrations of LL37. β-actin is the reference. (D) Pseudovirion neutralization assay. Results are shown as the mean ± SD. Compared with the peptide-free group, the cells pretreated with 5 and 10 μg/mL LL37 were less sensitive to <t>SARS-CoV-2</t> S pseudovirion infection. ***, P
    Sars Cov 2 2019 Ncov Spike Antibody Rabbit Pab, supplied by Sino Biological, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    CTSL cleaves the SARS-CoV-2 spike (S) protein, and this cleavage promotes cell–cell fusion. a Overview of the SARS-CoV-1 and SARS-CoV-2 S1/S2 cleavage sites. FP (fusion peptide), HR1 (heptad repeat 1), and HR2 (heptad repeat 2) are units of the S2 subunit that function in membrane fusion. b Analysis of CTSL-mediated S-protein cleavage. Purified SARS-CoV-1 or SARS-CoV-2 S protein was incubated in the presence or absence (assay buffer, pH = 5.5) of CTSL (2 or 10 μg/ml in assay buffer, pH = 5 .5) at 37 °C for 1 h. The reaction system of 2 μg/ml CTSL was further supplemented with CTSL inhibitors (20 μM E64d or 20 μM SID 26681509), as indicated. Proteins were subjected to SDS-PAGE and detected by silver staining. Representative data from three independent experiments are shown. c Syncytium-formation assay: Huh7 cells were untransfected (Null) or transfected with plasmid to express the SARS-CoV-2 S protein. Cells were incubated in the presence or absence (PBS, pH = 7.4) of trypsin (2 μg/ml in PBS, pH = 7.4) or in the presence or absence (PBS, pH = 5.8) of CTSL (2 or 4 μg/ml in PBS, pH = 5.8) for 20 min. Images were acquired after an additional 16 h incubation in the medium. (scale bars, 50 μm). The black arrowheads indicate syncytia. Representative data from seven independent experiments are shown. d Quantitative analysis of syncytia in panel c . n = 7. Statistical significance was assessed by one-way ANOVA with Tukey’s post hoc test. The data are expressed as the mean ± s.e.m. values. * P

    Journal: Signal Transduction and Targeted Therapy

    Article Title: Cathepsin L plays a key role in SARS-CoV-2 infection in humans and humanized mice and is a promising target for new drug development

    doi: 10.1038/s41392-021-00558-8

    Figure Lengend Snippet: CTSL cleaves the SARS-CoV-2 spike (S) protein, and this cleavage promotes cell–cell fusion. a Overview of the SARS-CoV-1 and SARS-CoV-2 S1/S2 cleavage sites. FP (fusion peptide), HR1 (heptad repeat 1), and HR2 (heptad repeat 2) are units of the S2 subunit that function in membrane fusion. b Analysis of CTSL-mediated S-protein cleavage. Purified SARS-CoV-1 or SARS-CoV-2 S protein was incubated in the presence or absence (assay buffer, pH = 5.5) of CTSL (2 or 10 μg/ml in assay buffer, pH = 5 .5) at 37 °C for 1 h. The reaction system of 2 μg/ml CTSL was further supplemented with CTSL inhibitors (20 μM E64d or 20 μM SID 26681509), as indicated. Proteins were subjected to SDS-PAGE and detected by silver staining. Representative data from three independent experiments are shown. c Syncytium-formation assay: Huh7 cells were untransfected (Null) or transfected with plasmid to express the SARS-CoV-2 S protein. Cells were incubated in the presence or absence (PBS, pH = 7.4) of trypsin (2 μg/ml in PBS, pH = 7.4) or in the presence or absence (PBS, pH = 5.8) of CTSL (2 or 4 μg/ml in PBS, pH = 5.8) for 20 min. Images were acquired after an additional 16 h incubation in the medium. (scale bars, 50 μm). The black arrowheads indicate syncytia. Representative data from seven independent experiments are shown. d Quantitative analysis of syncytia in panel c . n = 7. Statistical significance was assessed by one-way ANOVA with Tukey’s post hoc test. The data are expressed as the mean ± s.e.m. values. * P

    Article Snippet: Cleavage of SARS-CoV-2 S protein by CTSLThe purified extracellular domain of SARS-CoV-2 S protein (YP_009724390.1.

    Techniques: Purification, Incubation, SDS Page, Silver Staining, Tube Formation Assay, Transfection, Plasmid Preparation

    Cryo-EM structures of the SARS-CoV-2 S trimer in complex with the 3C1 Fab. a , b S-3C1-F3b cryo-EM map ( a ) and pseudo atomic model ( b ). All the three RBDs are up and each of them binds with a 3C1 Fab. The heavy chain of the 3C1 Fab in medium blue and light chain in violet red. c , d S-3C1-F3a cryo-EM map ( c ) and pseudo atomic model ( d ). There are two up RBDs and one down RBD, with each bound with a 3C1 Fab. e Structural alignment of the three up RBDs of S-3C1-F3b (in color) and the only up RBD from S-open (gray), suggesting 3C1 induced outward tilt of the RBDs within the S trimer. f , g Conformational comparation between S-3C1-F1 and S-open ( f ), as well as between S-3C1-F3a and S-3C1-F2 ( g ). h RBD/3C1 interaction interface (take RBD-3/3C1 of S-3C1-F3b as an example), with major involved structural elements labeled. i ACE2 (coral, PDB: 6M0J) would clash with the heavy chain of 3C1 Fab (blue). They share overlapping epitopes on the RBM (dotted black circle); additionally, the framework of 3C1-VH would clash with ACE2 (dotted black frame), which could be enhanced by the presence of an N-linked glycan at site N322 of ACE2. j 3C1 showed two distinct orientations to bind RBD within S trimer, i.e., adopting orientation 1 to associate with up RBD while orientation 2 with down RBD. k Contact footprint variations of 3C1 on up RBD (left) compared with that on down RBD (right), with unique epitopes indicated by dotted black frame. l – m Potential simultaneous binding of RBD by 2H2 and 3C1 cocktail. In 3C1 orientation 1, 3C1 and 2H2 could have minor clash (indicated by black frame, l ); while in origination 2, there is no clash between 3C1 and 2H2 Fabs ( m ).

    Journal: Nature Communications

    Article Title: Development and structural basis of a two-MAb cocktail for treating SARS-CoV-2 infections

    doi: 10.1038/s41467-020-20465-w

    Figure Lengend Snippet: Cryo-EM structures of the SARS-CoV-2 S trimer in complex with the 3C1 Fab. a , b S-3C1-F3b cryo-EM map ( a ) and pseudo atomic model ( b ). All the three RBDs are up and each of them binds with a 3C1 Fab. The heavy chain of the 3C1 Fab in medium blue and light chain in violet red. c , d S-3C1-F3a cryo-EM map ( c ) and pseudo atomic model ( d ). There are two up RBDs and one down RBD, with each bound with a 3C1 Fab. e Structural alignment of the three up RBDs of S-3C1-F3b (in color) and the only up RBD from S-open (gray), suggesting 3C1 induced outward tilt of the RBDs within the S trimer. f , g Conformational comparation between S-3C1-F1 and S-open ( f ), as well as between S-3C1-F3a and S-3C1-F2 ( g ). h RBD/3C1 interaction interface (take RBD-3/3C1 of S-3C1-F3b as an example), with major involved structural elements labeled. i ACE2 (coral, PDB: 6M0J) would clash with the heavy chain of 3C1 Fab (blue). They share overlapping epitopes on the RBM (dotted black circle); additionally, the framework of 3C1-VH would clash with ACE2 (dotted black frame), which could be enhanced by the presence of an N-linked glycan at site N322 of ACE2. j 3C1 showed two distinct orientations to bind RBD within S trimer, i.e., adopting orientation 1 to associate with up RBD while orientation 2 with down RBD. k Contact footprint variations of 3C1 on up RBD (left) compared with that on down RBD (right), with unique epitopes indicated by dotted black frame. l – m Potential simultaneous binding of RBD by 2H2 and 3C1 cocktail. In 3C1 orientation 1, 3C1 and 2H2 could have minor clash (indicated by black frame, l ); while in origination 2, there is no clash between 3C1 and 2H2 Fabs ( m ).

    Article Snippet: To prepare SARS-CoV-2 S protein, mammalian codon-optimized gene coding S ectodomain (residues 1–1208) with proline substitutions at residues 986 and 987, a “GSAS” substitution at the furin cleavage site (residues 682–685) was cloned into vector pcDNA3.1+.

    Techniques: Labeling, Binding Assay

    A proposed model of stepwise binding of 2H2/3C1 Fabs to the RBD of SARS-CoV-2 S trimer. a 2H2 and 3C1 Fabs appear to follow similar pathway to induce generally comparable conformational transitions of the S trimer to neutralize the virus. RBD-1, RBD-2, and RBD-3 are colored in light green, light blue, and gold, respectively; 2H2 and 3C1 Fab in violent red and medium blue, respectively. Red ellipsoid and black ellipsoid indicate Fab bound to up RBD and down RBD, respectively. The maps of S-2H2 and S-3C1 complexes shown here were generated by lowpass filtering of the corresponding models to 10 Å resolution. b Population distribution for the S-2H2 and S-3C1 dataset.

    Journal: Nature Communications

    Article Title: Development and structural basis of a two-MAb cocktail for treating SARS-CoV-2 infections

    doi: 10.1038/s41467-020-20465-w

    Figure Lengend Snippet: A proposed model of stepwise binding of 2H2/3C1 Fabs to the RBD of SARS-CoV-2 S trimer. a 2H2 and 3C1 Fabs appear to follow similar pathway to induce generally comparable conformational transitions of the S trimer to neutralize the virus. RBD-1, RBD-2, and RBD-3 are colored in light green, light blue, and gold, respectively; 2H2 and 3C1 Fab in violent red and medium blue, respectively. Red ellipsoid and black ellipsoid indicate Fab bound to up RBD and down RBD, respectively. The maps of S-2H2 and S-3C1 complexes shown here were generated by lowpass filtering of the corresponding models to 10 Å resolution. b Population distribution for the S-2H2 and S-3C1 dataset.

    Article Snippet: To prepare SARS-CoV-2 S protein, mammalian codon-optimized gene coding S ectodomain (residues 1–1208) with proline substitutions at residues 986 and 987, a “GSAS” substitution at the furin cleavage site (residues 682–685) was cloned into vector pcDNA3.1+.

    Techniques: Binding Assay, Generated

    Cryo-EM structures of the SARS-CoV-2 S trimer in complex with 2H2 Fab. a , b Side and top views of the S-2H2-F3a cryo-EM map ( a ) and pseudo atomic model ( b ). RBD-1 and RBD-2 are in up configuration, while RBD-3 is down, with each of the RBDs bound with a 2H2 Fab. Protomer 1, 2, and 3 are shown in light green, powder blue, and gold, respectively. This color scheme is followed throughout. Heavy chain and light chain of 2H2 Fab in royal blue and violet red, respectively. c , d Side and top views of the S-2H2-F2 cryo-EM map ( c ) and pseudo atomic model ( d ), with two up RBDs (RBD-1 and RBD-2) each bound with a 2H2 Fab. e , f 2H2 Fab-induced conformational changes of the S trimer. Shown is the structural comparation of RBDs between S-2H2-F1 (in color) and S-open (dim gray) ( e ), and between S-2H2-F3a (in color) and S-2H2-F2 (dim gray) ( f ). g 2H2 Fab mainly binds to the RBM (light sea green surface) of RBD, with major involved structural elements labeled. RBD core is rendered as light green surface. h 2H2 Fab (left) and ACE2 (right, gold, PDB: 6M0J) share overlapping epitopes on RBM (second row) and would clash upon binding to the S trimer. i , j The involved regions/residues forming potential contacts between the light chain (in violent red, i ) or heavy chain (in royal blue, j ) of 2H2 and the RBD-1 of S-2H2-F3a. Asterisks highlight residues also involved in the interactions with ACE2. Note that considering the local resolution limitation in the RBD-2H2 portion of the map due to intrinsic dynamic nature in these regions, we analyzed the potential interactions that fulfill criteria of both

    Journal: Nature Communications

    Article Title: Development and structural basis of a two-MAb cocktail for treating SARS-CoV-2 infections

    doi: 10.1038/s41467-020-20465-w

    Figure Lengend Snippet: Cryo-EM structures of the SARS-CoV-2 S trimer in complex with 2H2 Fab. a , b Side and top views of the S-2H2-F3a cryo-EM map ( a ) and pseudo atomic model ( b ). RBD-1 and RBD-2 are in up configuration, while RBD-3 is down, with each of the RBDs bound with a 2H2 Fab. Protomer 1, 2, and 3 are shown in light green, powder blue, and gold, respectively. This color scheme is followed throughout. Heavy chain and light chain of 2H2 Fab in royal blue and violet red, respectively. c , d Side and top views of the S-2H2-F2 cryo-EM map ( c ) and pseudo atomic model ( d ), with two up RBDs (RBD-1 and RBD-2) each bound with a 2H2 Fab. e , f 2H2 Fab-induced conformational changes of the S trimer. Shown is the structural comparation of RBDs between S-2H2-F1 (in color) and S-open (dim gray) ( e ), and between S-2H2-F3a (in color) and S-2H2-F2 (dim gray) ( f ). g 2H2 Fab mainly binds to the RBM (light sea green surface) of RBD, with major involved structural elements labeled. RBD core is rendered as light green surface. h 2H2 Fab (left) and ACE2 (right, gold, PDB: 6M0J) share overlapping epitopes on RBM (second row) and would clash upon binding to the S trimer. i , j The involved regions/residues forming potential contacts between the light chain (in violent red, i ) or heavy chain (in royal blue, j ) of 2H2 and the RBD-1 of S-2H2-F3a. Asterisks highlight residues also involved in the interactions with ACE2. Note that considering the local resolution limitation in the RBD-2H2 portion of the map due to intrinsic dynamic nature in these regions, we analyzed the potential interactions that fulfill criteria of both

    Article Snippet: To prepare SARS-CoV-2 S protein, mammalian codon-optimized gene coding S ectodomain (residues 1–1208) with proline substitutions at residues 986 and 987, a “GSAS” substitution at the furin cleavage site (residues 682–685) was cloned into vector pcDNA3.1+.

    Techniques: Labeling, Binding Assay

    LL37 attenuates S1 binding to cells by cloaking ACE2. (A) Binding kinetics for LL37 and ACE2. (B) BLI-based ACE2 blocking assay. (C) Protein bands of S1 binding to A549 cells pretreated with increasing concentrations of LL37. β-actin is the reference. (D) Pseudovirion neutralization assay. Results are shown as the mean ± SD. Compared with the peptide-free group, the cells pretreated with 5 and 10 μg/mL LL37 were less sensitive to SARS-CoV-2 S pseudovirion infection. ***, P

    Journal: ACS Infectious Diseases

    Article Title: Human Cathelicidin Inhibits SARS-CoV-2 Infection: Killing Two Birds with One Stone

    doi: 10.1021/acsinfecdis.1c00096

    Figure Lengend Snippet: LL37 attenuates S1 binding to cells by cloaking ACE2. (A) Binding kinetics for LL37 and ACE2. (B) BLI-based ACE2 blocking assay. (C) Protein bands of S1 binding to A549 cells pretreated with increasing concentrations of LL37. β-actin is the reference. (D) Pseudovirion neutralization assay. Results are shown as the mean ± SD. Compared with the peptide-free group, the cells pretreated with 5 and 10 μg/mL LL37 were less sensitive to SARS-CoV-2 S pseudovirion infection. ***, P

    Article Snippet: An anti-S rabbit polyclonal primary antibody (40591-T62, Sino Biological, 1:1000) and a goat antirabbit secondary antibody (A0208, Beyotime) were employed to detect S1. β-actin was detected with a rabbit monoclonal antibody (AF5003, Beyotime).

    Techniques: Binding Assay, Blocking Assay, Neutralization, Infection

    Complex structures of LL37 with SARS-CoV-2 RBD (A) and ACE2 (B). RBD and ACE2 (PDB: 6M0J) are shown in the ribbon structure. LL37 is shown in the red ribbon on the right. Salt bridges, hydrogen bonds, and hydrophobic interactions are shown in orange, yellow, and purple, respectively.

    Journal: ACS Infectious Diseases

    Article Title: Human Cathelicidin Inhibits SARS-CoV-2 Infection: Killing Two Birds with One Stone

    doi: 10.1021/acsinfecdis.1c00096

    Figure Lengend Snippet: Complex structures of LL37 with SARS-CoV-2 RBD (A) and ACE2 (B). RBD and ACE2 (PDB: 6M0J) are shown in the ribbon structure. LL37 is shown in the red ribbon on the right. Salt bridges, hydrogen bonds, and hydrophobic interactions are shown in orange, yellow, and purple, respectively.

    Article Snippet: An anti-S rabbit polyclonal primary antibody (40591-T62, Sino Biological, 1:1000) and a goat antirabbit secondary antibody (A0208, Beyotime) were employed to detect S1. β-actin was detected with a rabbit monoclonal antibody (AF5003, Beyotime).

    Techniques:

    Antigenic properties of the purified full-length SARS-CoV-2 S proteins. Bio-layer interferometry (BLI) analysis of the association of prefusion S trimers from the G614 “parent” strain and the B.1.1.7 and B.1.351 variants derived from it with soluble ACE2 constructs and with a panel of antibodies representing five epitopic regions on the RBD and NTD (see Fig. S4A and ref( 35 )). For ACE2 binding, purified S proteins were immobilized to AR2G biosensors and dipped into the wells containing ACE2 at various concentrations. For antibody binding, various antibodies were immobilized to AHC biosensors and dipped into the wells containing each purified S protein at different concentration. Binding kinetics were evaluated using a 1:1 Langmuir model except for dimeric ACE2 and antibody 32B6 targeting the RBD-2, which were analyzed by a bivalent binding model. The sensorgrams are in black and the fits in red. RU, response unit. Binding constants are also summarized here and in Table S1. All experiments were repeated at least twice with essentially identical results.

    Journal: bioRxiv

    Article Title: Structural basis for enhanced infectivity and immune evasion of SARS-CoV-2 variants

    doi: 10.1101/2021.04.13.439709

    Figure Lengend Snippet: Antigenic properties of the purified full-length SARS-CoV-2 S proteins. Bio-layer interferometry (BLI) analysis of the association of prefusion S trimers from the G614 “parent” strain and the B.1.1.7 and B.1.351 variants derived from it with soluble ACE2 constructs and with a panel of antibodies representing five epitopic regions on the RBD and NTD (see Fig. S4A and ref( 35 )). For ACE2 binding, purified S proteins were immobilized to AR2G biosensors and dipped into the wells containing ACE2 at various concentrations. For antibody binding, various antibodies were immobilized to AHC biosensors and dipped into the wells containing each purified S protein at different concentration. Binding kinetics were evaluated using a 1:1 Langmuir model except for dimeric ACE2 and antibody 32B6 targeting the RBD-2, which were analyzed by a bivalent binding model. The sensorgrams are in black and the fits in red. RU, response unit. Binding constants are also summarized here and in Table S1. All experiments were repeated at least twice with essentially identical results.

    Article Snippet: Western blot Western blot was performed using an anti-SARS-COV-2 S antibody following a protocol described previously .

    Techniques: Purification, Derivative Assay, Construct, Binding Assay, Concentration Assay

    Cryo-EM structures of the full-length SARS-CoV-2 S proteins from the B.1.1.7 and B.1.351 variants. ( A-E ) The structures of the closed prefusion conformation, three one RBD-up conformations and a two RBD-up conformation of the B.1.1.7 S trimer are shown in ribbon diagram with one protomer colored as NTD in blue, RBD in cyan, CTD1 in green, CTD2 in light green, S2 in light blue, the 630 loop in red and the FPPR in magenta. ( G ) and ( H ) The structures of the closed prefusion conformation and one RBD-up conformation of the B.1.351 S trimer are shown in ribbon diagram with the same color scheme as in (A). All mutations in the new variants, as compared to the original virus (D614), are highlighted in sphere model. ( F ) and ( I ) Structures, in the B.1.1.7 trimer, of segments (residues 617-644) containing the 630 loop (red) and segments (residues 823-862) containing the FPPR (magenta) from each of the three protomers (a, b and c). The position of each RBD is indicated. Dashed lines indicate gaps in the chain trace (disordered loops).

    Journal: bioRxiv

    Article Title: Structural basis for enhanced infectivity and immune evasion of SARS-CoV-2 variants

    doi: 10.1101/2021.04.13.439709

    Figure Lengend Snippet: Cryo-EM structures of the full-length SARS-CoV-2 S proteins from the B.1.1.7 and B.1.351 variants. ( A-E ) The structures of the closed prefusion conformation, three one RBD-up conformations and a two RBD-up conformation of the B.1.1.7 S trimer are shown in ribbon diagram with one protomer colored as NTD in blue, RBD in cyan, CTD1 in green, CTD2 in light green, S2 in light blue, the 630 loop in red and the FPPR in magenta. ( G ) and ( H ) The structures of the closed prefusion conformation and one RBD-up conformation of the B.1.351 S trimer are shown in ribbon diagram with the same color scheme as in (A). All mutations in the new variants, as compared to the original virus (D614), are highlighted in sphere model. ( F ) and ( I ) Structures, in the B.1.1.7 trimer, of segments (residues 617-644) containing the 630 loop (red) and segments (residues 823-862) containing the FPPR (magenta) from each of the three protomers (a, b and c). The position of each RBD is indicated. Dashed lines indicate gaps in the chain trace (disordered loops).

    Article Snippet: Western blot Western blot was performed using an anti-SARS-COV-2 S antibody following a protocol described previously .

    Techniques: Cryo-EM Sample Prep