Journal: Frontiers in Molecular Biosciences

Article Title: Heparan Sulfate Facilitates Spike Protein-Mediated SARS-CoV-2 Host Cell Invasion and Contributes to Increased Infection of SARS-CoV-2 G614 Mutant and in Lung Cancer

doi: 10.3389/fmolb.2021.649575

Figure Lengend Snippet: Cell surface SV2-S binding depends on and correlates with expression levels of HS. (A,B) Cell surface expressions of ACE2 and HS were assessed by flow cytometry after incubation with anti-ACE2 and anti-HS antibody 10E4, respectively, and sequential Alexa Fluor 488-tagged secondary antibody. A portion of the cells was treated with heparinases I-III (HSase) before the antibody staining. (C) The binding of SV2-S on cell surface was assessed by flow cytometry after sequential incubation with his-tagged SV2-S and Alexa Fluor 488-tagged anti-his antibody. A portion of the cells was treated with HSase before SV2-S binding. Median fluorescence intensity (MFI) was calculated by subtracting MFI of BSA binding or isotype-match naive control antibody binding. The experiments were repeated 3–6 times and an unpaired t -test was performed for comparison of the same cells without vs. with HSase treatment. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. (D–G) The Pearson’s correlation coefficient between SV2-S binding and cell surface expression level of HS or ACE2 was determined by analyzing the data collected in (A–C) using Prism 8. The cells analyzed in (F,G) were pre-treated with HSase.

Article Snippet: SV2-S binding on cell surface was measured by incubating cells with 250 nM His-tagged SV2-S (Sino Biological, 40591-V08B1).

Techniques: Binding Assay, Expressing, Flow Cytometry, Incubation, Staining, Fluorescence

Journal: Frontiers in Molecular Biosciences

Article Title: Heparan Sulfate Facilitates Spike Protein-Mediated SARS-CoV-2 Host Cell Invasion and Contributes to Increased Infection of SARS-CoV-2 G614 Mutant and in Lung Cancer

doi: 10.3389/fmolb.2021.649575

Figure Lengend Snippet: Heparan sulfate (HS) plays a major role in mediating cell surface SV2-S protein binding only when ACE2 expresses at a low level. 293T cells were transfected with ACE2-GFP or scramble-GFP plasmid. In SV2-S binding analysis, the transient 293T-ACE2-GFP-expressing cells were gated into two populations: the ACE2-GFP-low and -high expression cells based on GFP expression. (A) Cell surface ACE2 expression was assessed by flow cytometry after staining with anti-ACE2 antibody and Alexa Fluor 647-conjugated secondary antibody. A portion of the cells was treated with HSase before the specific antibody staining. (B) The binding of SV2-S on cell surface was assessed by flow cytometry after sequential incubation with his-tagged SV2-S and Alexa Fluor 647-tagged anti-his antibody. A portion of the cells was treated with HSase before SV2-S binding. (C) Cell surface HS expression was assessed by flow cytometry after staining with anti-HS antibody and Alexa Fluor 647-conjugated secondary antibody. A portion of the cells was treated with HSase before the specific antibody staining. MFI was calculated by subtracting MFI of BSA binding or isotype-match naive control antibody staining. The experiments were repeated 6- to 12- times and an unpaired t -test was performed for comparison of the same cells without vs. with HSase treatment. Data are presented as mean ± SD. ns, not significant; * p < 0.05, *** p < 0.001, **** p < 0.0001.

Article Snippet: SV2-S binding on cell surface was measured by incubating cells with 250 nM His-tagged SV2-S (Sino Biological, 40591-V08B1).

Techniques: Protein Binding, Transfection, Plasmid Preparation, Binding Assay, Expressing, Flow Cytometry, Staining, Incubation

Journal: Frontiers in Molecular Biosciences

Article Title: Heparan Sulfate Facilitates Spike Protein-Mediated SARS-CoV-2 Host Cell Invasion and Contributes to Increased Infection of SARS-CoV-2 G614 Mutant and in Lung Cancer

doi: 10.3389/fmolb.2021.649575

Figure Lengend Snippet: The modification of heparin and cell surface HS involved in SV2-S binding. (A,B) Inhibition on the binding of SV2-S to cultured A549 cells. After incubation in the absence or presence of different concentrations of heparin (HP), NDS-HP; 2DS-HP, 6DS-HP, or OS-HP, SV2-S that bound to A549 cells was quantified by cell-based ELISA and normalized to the cell density. The SV2-S binding was further normalized to the wells without inhibitors. The experiments were repeated at least three times. Data are presented as mean ± SEM. (C) SV2-S binding on MLEC surface. The tested HS mutant MLEC lines include wildtype (WT) control, Hs2st1 − /− (lack of 2S), Hs6st1 − /− (reduced 6S), Hs6st2 − /− (normal 6S), Hs6st1 − /− ;2 − /− (no 6S), Sulf1 − /− ;2 − /− (increased 6S), Hs3st1 − /− (reduced 3S), Hs3st4 − /− (reduced 3S), and Hs3st1 − /− ;4 − /− (greatly reduced 3S). Cell surface SV2-S protein binding was assessed by cell-based ELISA after incubation of fixed cells with his-tagged SV2-S and normalization to the cell density. The experiments were repeated 6- to 12- times and the unpaired t-test was performed for comparison between the wildtype and the mutants. Data are presented as mean ± SD. Ns, not significant; * p < 0.05, ** p < 0.01.

Article Snippet: SV2-S binding on cell surface was measured by incubating cells with 250 nM His-tagged SV2-S (Sino Biological, 40591-V08B1).

Techniques: Modification, Binding Assay, Inhibition, Cell Culture, Incubation, In-Cell ELISA, Mutagenesis, Protein Binding

Journal: Frontiers in Molecular Biosciences

Article Title: Heparan Sulfate Facilitates Spike Protein-Mediated SARS-CoV-2 Host Cell Invasion and Contributes to Increased Infection of SARS-CoV-2 G614 Mutant and in Lung Cancer

doi: 10.3389/fmolb.2021.649575

Figure Lengend Snippet: Heparinases, surfen, heparin, and chemically modified non-anticoagulant heparins block SV2-S pseudotyped virus infection of 293T-ACE2 cells. The 293T-ACE2 cells were infected with SV2-S pseudotyped virus expressing luciferase after the cells were treated with HSase (5 mU/ml) (A) or in the presence of surfen (20 μM) (B) , heparin (HP, C ), HS or chemically-modified heparins at 2- and 20 μM (D,E) . The luciferase activity was measured 48 h after the virus infection. VSV pseudotyped virus was used as a control to make sure each well has comparable cell numbers (data are not shown). The experiments were repeated at least 3 times and an unpaired t -test was performed for the two-group comparison. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. RLU, relative light unit.

Article Snippet: SV2-S binding on cell surface was measured by incubating cells with 250 nM His-tagged SV2-S (Sino Biological, 40591-V08B1).

Techniques: Modification, Blocking Assay, Infection, Expressing, Luciferase, Activity Assay

Journal: Frontiers in Molecular Biosciences

Article Title: Heparan Sulfate Facilitates Spike Protein-Mediated SARS-CoV-2 Host Cell Invasion and Contributes to Increased Infection of SARS-CoV-2 G614 Mutant and in Lung Cancer

doi: 10.3389/fmolb.2021.649575

Figure Lengend Snippet: Summary of kinetic data of wild type spike and mutant spike protein-heparin interactions*.

Article Snippet: SV2-S binding on cell surface was measured by incubating cells with 250 nM His-tagged SV2-S (Sino Biological, 40591-V08B1).

Techniques: Mutagenesis

Journal: Frontiers in Molecular Biosciences

Article Title: Heparan Sulfate Facilitates Spike Protein-Mediated SARS-CoV-2 Host Cell Invasion and Contributes to Increased Infection of SARS-CoV-2 G614 Mutant and in Lung Cancer

doi: 10.3389/fmolb.2021.649575

Figure Lengend Snippet: The SV2-S variant G614 protein has a higher binding affinity to heparin than the wildtype SV2-S D614 protein, and the entry of SAS-Cov-2 G614 pseudotyped virus into host cells depends on cell surface HS and can be inhibited by heparin. (A,B) SPR binding kinetics sensorgrams between heparin and SV2-S D614 or SV2-S G614. Concentrations of D614 were (from top to bottom): 1,000, 800, 600, 400, and 200 nM, respectively (A) , and G614 were (from top to bottom): 1,000, 600, 400, 200, and 100 nM, respectively (B) . The black curves are the fitting curves using a 1:1 Langmuir model from BIAevaluate 4.0.1. (C) Infection of 293T-ACE2 cells by the same titer of SARS-Cov2-D614 and SARS-Cov2-G614 pseudovirus. (D) SARS-Cov2-G614 pseudovirus depends on HS to infect host cells. The 293T-ACE2 cells were infected with SARS-Cov2-G614 pseudovirus expressing luciferase after the cells were treated with heparinases I-III (5 mU/ml) or in the presence of heparin (2 μM). The luciferase activity was measured 48 h after the virus infection. VSV pseudotyped virus was used as a control to make sure each well has comparable cell numbers (data are not shown). The experiments were repeated at least 3 times and an unpaired t-test was performed for two-group comparison. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01.

Article Snippet: SV2-S binding on cell surface was measured by incubating cells with 250 nM His-tagged SV2-S (Sino Biological, 40591-V08B1).

Techniques: Variant Assay, Binding Assay, Infection, Expressing, Luciferase, Activity Assay

Journal: Frontiers in Molecular Biosciences

Article Title: Heparan Sulfate Facilitates Spike Protein-Mediated SARS-CoV-2 Host Cell Invasion and Contributes to Increased Infection of SARS-CoV-2 G614 Mutant and in Lung Cancer

doi: 10.3389/fmolb.2021.649575

Figure Lengend Snippet: Cell surface SV2-S binding depends on and correlates with expression levels of HS. (A,B) Cell surface expressions of ACE2 and HS were assessed by flow cytometry after incubation with anti-ACE2 and anti-HS antibody 10E4, respectively, and sequential Alexa Fluor 488-tagged secondary antibody. A portion of the cells was treated with heparinases I-III (HSase) before the antibody staining. (C) The binding of SV2-S on cell surface was assessed by flow cytometry after sequential incubation with his-tagged SV2-S and Alexa Fluor 488-tagged anti-his antibody. A portion of the cells was treated with HSase before SV2-S binding. Median fluorescence intensity (MFI) was calculated by subtracting MFI of BSA binding or isotype-match naive control antibody binding. The experiments were repeated 3–6 times and an unpaired t -test was performed for comparison of the same cells without vs. with HSase treatment. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. (D–G) The Pearson’s correlation coefficient between SV2-S binding and cell surface expression level of HS or ACE2 was determined by analyzing the data collected in (A–C) using Prism 8. The cells analyzed in (F,G) were pre-treated with HSase.

Article Snippet: For direct binding analysis, SV2-S D614 (Sino Biological, 40591-V08H) and SV2-S G614 (Sino Biological, 40591-V08H3) were diluted in a running HBS-EP buffer containing 0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.005% surfactant P20, at pH 7.4.

Techniques: Binding Assay, Expressing, Flow Cytometry, Incubation, Staining, Fluorescence

Journal: Frontiers in Molecular Biosciences

Article Title: Heparan Sulfate Facilitates Spike Protein-Mediated SARS-CoV-2 Host Cell Invasion and Contributes to Increased Infection of SARS-CoV-2 G614 Mutant and in Lung Cancer

doi: 10.3389/fmolb.2021.649575

Figure Lengend Snippet: Heparan sulfate (HS) plays a major role in mediating cell surface SV2-S protein binding only when ACE2 expresses at a low level. 293T cells were transfected with ACE2-GFP or scramble-GFP plasmid. In SV2-S binding analysis, the transient 293T-ACE2-GFP-expressing cells were gated into two populations: the ACE2-GFP-low and -high expression cells based on GFP expression. (A) Cell surface ACE2 expression was assessed by flow cytometry after staining with anti-ACE2 antibody and Alexa Fluor 647-conjugated secondary antibody. A portion of the cells was treated with HSase before the specific antibody staining. (B) The binding of SV2-S on cell surface was assessed by flow cytometry after sequential incubation with his-tagged SV2-S and Alexa Fluor 647-tagged anti-his antibody. A portion of the cells was treated with HSase before SV2-S binding. (C) Cell surface HS expression was assessed by flow cytometry after staining with anti-HS antibody and Alexa Fluor 647-conjugated secondary antibody. A portion of the cells was treated with HSase before the specific antibody staining. MFI was calculated by subtracting MFI of BSA binding or isotype-match naive control antibody staining. The experiments were repeated 6- to 12- times and an unpaired t -test was performed for comparison of the same cells without vs. with HSase treatment. Data are presented as mean ± SD. ns, not significant; * p < 0.05, *** p < 0.001, **** p < 0.0001.

Article Snippet: For direct binding analysis, SV2-S D614 (Sino Biological, 40591-V08H) and SV2-S G614 (Sino Biological, 40591-V08H3) were diluted in a running HBS-EP buffer containing 0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.005% surfactant P20, at pH 7.4.

Techniques: Protein Binding, Transfection, Plasmid Preparation, Binding Assay, Expressing, Flow Cytometry, Staining, Incubation

Journal: Frontiers in Molecular Biosciences

Article Title: Heparan Sulfate Facilitates Spike Protein-Mediated SARS-CoV-2 Host Cell Invasion and Contributes to Increased Infection of SARS-CoV-2 G614 Mutant and in Lung Cancer

doi: 10.3389/fmolb.2021.649575

Figure Lengend Snippet: The modification of heparin and cell surface HS involved in SV2-S binding. (A,B) Inhibition on the binding of SV2-S to cultured A549 cells. After incubation in the absence or presence of different concentrations of heparin (HP), NDS-HP; 2DS-HP, 6DS-HP, or OS-HP, SV2-S that bound to A549 cells was quantified by cell-based ELISA and normalized to the cell density. The SV2-S binding was further normalized to the wells without inhibitors. The experiments were repeated at least three times. Data are presented as mean ± SEM. (C) SV2-S binding on MLEC surface. The tested HS mutant MLEC lines include wildtype (WT) control, Hs2st1 − /− (lack of 2S), Hs6st1 − /− (reduced 6S), Hs6st2 − /− (normal 6S), Hs6st1 − /− ;2 − /− (no 6S), Sulf1 − /− ;2 − /− (increased 6S), Hs3st1 − /− (reduced 3S), Hs3st4 − /− (reduced 3S), and Hs3st1 − /− ;4 − /− (greatly reduced 3S). Cell surface SV2-S protein binding was assessed by cell-based ELISA after incubation of fixed cells with his-tagged SV2-S and normalization to the cell density. The experiments were repeated 6- to 12- times and the unpaired t-test was performed for comparison between the wildtype and the mutants. Data are presented as mean ± SD. Ns, not significant; * p < 0.05, ** p < 0.01.

Article Snippet: For direct binding analysis, SV2-S D614 (Sino Biological, 40591-V08H) and SV2-S G614 (Sino Biological, 40591-V08H3) were diluted in a running HBS-EP buffer containing 0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.005% surfactant P20, at pH 7.4.

Techniques: Modification, Binding Assay, Inhibition, Cell Culture, Incubation, In-Cell ELISA, Mutagenesis, Protein Binding

Journal: Frontiers in Molecular Biosciences

Article Title: Heparan Sulfate Facilitates Spike Protein-Mediated SARS-CoV-2 Host Cell Invasion and Contributes to Increased Infection of SARS-CoV-2 G614 Mutant and in Lung Cancer

doi: 10.3389/fmolb.2021.649575

Figure Lengend Snippet: Heparinases, surfen, heparin, and chemically modified non-anticoagulant heparins block SV2-S pseudotyped virus infection of 293T-ACE2 cells. The 293T-ACE2 cells were infected with SV2-S pseudotyped virus expressing luciferase after the cells were treated with HSase (5 mU/ml) (A) or in the presence of surfen (20 μM) (B) , heparin (HP, C ), HS or chemically-modified heparins at 2- and 20 μM (D,E) . The luciferase activity was measured 48 h after the virus infection. VSV pseudotyped virus was used as a control to make sure each well has comparable cell numbers (data are not shown). The experiments were repeated at least 3 times and an unpaired t -test was performed for the two-group comparison. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. RLU, relative light unit.

Article Snippet: For direct binding analysis, SV2-S D614 (Sino Biological, 40591-V08H) and SV2-S G614 (Sino Biological, 40591-V08H3) were diluted in a running HBS-EP buffer containing 0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.005% surfactant P20, at pH 7.4.

Techniques: Modification, Blocking Assay, Infection, Expressing, Luciferase, Activity Assay

Journal: Frontiers in Molecular Biosciences

Article Title: Heparan Sulfate Facilitates Spike Protein-Mediated SARS-CoV-2 Host Cell Invasion and Contributes to Increased Infection of SARS-CoV-2 G614 Mutant and in Lung Cancer

doi: 10.3389/fmolb.2021.649575

Figure Lengend Snippet: Summary of kinetic data of wild type spike and mutant spike protein-heparin interactions*.

Article Snippet: For direct binding analysis, SV2-S D614 (Sino Biological, 40591-V08H) and SV2-S G614 (Sino Biological, 40591-V08H3) were diluted in a running HBS-EP buffer containing 0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.005% surfactant P20, at pH 7.4.

Techniques: Mutagenesis

Journal: Frontiers in Molecular Biosciences

Article Title: Heparan Sulfate Facilitates Spike Protein-Mediated SARS-CoV-2 Host Cell Invasion and Contributes to Increased Infection of SARS-CoV-2 G614 Mutant and in Lung Cancer

doi: 10.3389/fmolb.2021.649575

Figure Lengend Snippet: The SV2-S variant G614 protein has a higher binding affinity to heparin than the wildtype SV2-S D614 protein, and the entry of SAS-Cov-2 G614 pseudotyped virus into host cells depends on cell surface HS and can be inhibited by heparin. (A,B) SPR binding kinetics sensorgrams between heparin and SV2-S D614 or SV2-S G614. Concentrations of D614 were (from top to bottom): 1,000, 800, 600, 400, and 200 nM, respectively (A) , and G614 were (from top to bottom): 1,000, 600, 400, 200, and 100 nM, respectively (B) . The black curves are the fitting curves using a 1:1 Langmuir model from BIAevaluate 4.0.1. (C) Infection of 293T-ACE2 cells by the same titer of SARS-Cov2-D614 and SARS-Cov2-G614 pseudovirus. (D) SARS-Cov2-G614 pseudovirus depends on HS to infect host cells. The 293T-ACE2 cells were infected with SARS-Cov2-G614 pseudovirus expressing luciferase after the cells were treated with heparinases I-III (5 mU/ml) or in the presence of heparin (2 μM). The luciferase activity was measured 48 h after the virus infection. VSV pseudotyped virus was used as a control to make sure each well has comparable cell numbers (data are not shown). The experiments were repeated at least 3 times and an unpaired t-test was performed for two-group comparison. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01.

Article Snippet: For direct binding analysis, SV2-S D614 (Sino Biological, 40591-V08H) and SV2-S G614 (Sino Biological, 40591-V08H3) were diluted in a running HBS-EP buffer containing 0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.005% surfactant P20, at pH 7.4.

Techniques: Variant Assay, Binding Assay, Infection, Expressing, Luciferase, Activity Assay

Journal: Frontiers in Molecular Biosciences

Article Title: Heparan Sulfate Facilitates Spike Protein-Mediated SARS-CoV-2 Host Cell Invasion and Contributes to Increased Infection of SARS-CoV-2 G614 Mutant and in Lung Cancer

doi: 10.3389/fmolb.2021.649575

Figure Lengend Snippet: Cell surface SV2-S binding depends on and correlates with expression levels of HS. (A,B) Cell surface expressions of ACE2 and HS were assessed by flow cytometry after incubation with anti-ACE2 and anti-HS antibody 10E4, respectively, and sequential Alexa Fluor 488-tagged secondary antibody. A portion of the cells was treated with heparinases I-III (HSase) before the antibody staining. (C) The binding of SV2-S on cell surface was assessed by flow cytometry after sequential incubation with his-tagged SV2-S and Alexa Fluor 488-tagged anti-his antibody. A portion of the cells was treated with HSase before SV2-S binding. Median fluorescence intensity (MFI) was calculated by subtracting MFI of BSA binding or isotype-match naive control antibody binding. The experiments were repeated 3–6 times and an unpaired t -test was performed for comparison of the same cells without vs. with HSase treatment. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. (D–G) The Pearson’s correlation coefficient between SV2-S binding and cell surface expression level of HS or ACE2 was determined by analyzing the data collected in (A–C) using Prism 8. The cells analyzed in (F,G) were pre-treated with HSase.

Article Snippet: For direct binding analysis, SV2-S D614 (Sino Biological, 40591-V08H) and SV2-S G614 (Sino Biological, 40591-V08H3) were diluted in a running HBS-EP buffer containing 0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.005% surfactant P20, at pH 7.4.

Techniques: Binding Assay, Expressing, Flow Cytometry, Incubation, Staining, Fluorescence

Journal: Frontiers in Molecular Biosciences

Article Title: Heparan Sulfate Facilitates Spike Protein-Mediated SARS-CoV-2 Host Cell Invasion and Contributes to Increased Infection of SARS-CoV-2 G614 Mutant and in Lung Cancer

doi: 10.3389/fmolb.2021.649575

Figure Lengend Snippet: Heparan sulfate (HS) plays a major role in mediating cell surface SV2-S protein binding only when ACE2 expresses at a low level. 293T cells were transfected with ACE2-GFP or scramble-GFP plasmid. In SV2-S binding analysis, the transient 293T-ACE2-GFP-expressing cells were gated into two populations: the ACE2-GFP-low and -high expression cells based on GFP expression. (A) Cell surface ACE2 expression was assessed by flow cytometry after staining with anti-ACE2 antibody and Alexa Fluor 647-conjugated secondary antibody. A portion of the cells was treated with HSase before the specific antibody staining. (B) The binding of SV2-S on cell surface was assessed by flow cytometry after sequential incubation with his-tagged SV2-S and Alexa Fluor 647-tagged anti-his antibody. A portion of the cells was treated with HSase before SV2-S binding. (C) Cell surface HS expression was assessed by flow cytometry after staining with anti-HS antibody and Alexa Fluor 647-conjugated secondary antibody. A portion of the cells was treated with HSase before the specific antibody staining. MFI was calculated by subtracting MFI of BSA binding or isotype-match naive control antibody staining. The experiments were repeated 6- to 12- times and an unpaired t -test was performed for comparison of the same cells without vs. with HSase treatment. Data are presented as mean ± SD. ns, not significant; * p < 0.05, *** p < 0.001, **** p < 0.0001.

Article Snippet: For direct binding analysis, SV2-S D614 (Sino Biological, 40591-V08H) and SV2-S G614 (Sino Biological, 40591-V08H3) were diluted in a running HBS-EP buffer containing 0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.005% surfactant P20, at pH 7.4.

Techniques: Protein Binding, Transfection, Plasmid Preparation, Binding Assay, Expressing, Flow Cytometry, Staining, Incubation

Journal: Frontiers in Molecular Biosciences

Article Title: Heparan Sulfate Facilitates Spike Protein-Mediated SARS-CoV-2 Host Cell Invasion and Contributes to Increased Infection of SARS-CoV-2 G614 Mutant and in Lung Cancer

doi: 10.3389/fmolb.2021.649575

Figure Lengend Snippet: The modification of heparin and cell surface HS involved in SV2-S binding. (A,B) Inhibition on the binding of SV2-S to cultured A549 cells. After incubation in the absence or presence of different concentrations of heparin (HP), NDS-HP; 2DS-HP, 6DS-HP, or OS-HP, SV2-S that bound to A549 cells was quantified by cell-based ELISA and normalized to the cell density. The SV2-S binding was further normalized to the wells without inhibitors. The experiments were repeated at least three times. Data are presented as mean ± SEM. (C) SV2-S binding on MLEC surface. The tested HS mutant MLEC lines include wildtype (WT) control, Hs2st1 − /− (lack of 2S), Hs6st1 − /− (reduced 6S), Hs6st2 − /− (normal 6S), Hs6st1 − /− ;2 − /− (no 6S), Sulf1 − /− ;2 − /− (increased 6S), Hs3st1 − /− (reduced 3S), Hs3st4 − /− (reduced 3S), and Hs3st1 − /− ;4 − /− (greatly reduced 3S). Cell surface SV2-S protein binding was assessed by cell-based ELISA after incubation of fixed cells with his-tagged SV2-S and normalization to the cell density. The experiments were repeated 6- to 12- times and the unpaired t-test was performed for comparison between the wildtype and the mutants. Data are presented as mean ± SD. Ns, not significant; * p < 0.05, ** p < 0.01.

Article Snippet: For direct binding analysis, SV2-S D614 (Sino Biological, 40591-V08H) and SV2-S G614 (Sino Biological, 40591-V08H3) were diluted in a running HBS-EP buffer containing 0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.005% surfactant P20, at pH 7.4.

Techniques: Modification, Binding Assay, Inhibition, Cell Culture, Incubation, In-Cell ELISA, Mutagenesis, Protein Binding

Journal: Frontiers in Molecular Biosciences

Article Title: Heparan Sulfate Facilitates Spike Protein-Mediated SARS-CoV-2 Host Cell Invasion and Contributes to Increased Infection of SARS-CoV-2 G614 Mutant and in Lung Cancer

doi: 10.3389/fmolb.2021.649575

Figure Lengend Snippet: Heparinases, surfen, heparin, and chemically modified non-anticoagulant heparins block SV2-S pseudotyped virus infection of 293T-ACE2 cells. The 293T-ACE2 cells were infected with SV2-S pseudotyped virus expressing luciferase after the cells were treated with HSase (5 mU/ml) (A) or in the presence of surfen (20 μM) (B) , heparin (HP, C ), HS or chemically-modified heparins at 2- and 20 μM (D,E) . The luciferase activity was measured 48 h after the virus infection. VSV pseudotyped virus was used as a control to make sure each well has comparable cell numbers (data are not shown). The experiments were repeated at least 3 times and an unpaired t -test was performed for the two-group comparison. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. RLU, relative light unit.

Article Snippet: For direct binding analysis, SV2-S D614 (Sino Biological, 40591-V08H) and SV2-S G614 (Sino Biological, 40591-V08H3) were diluted in a running HBS-EP buffer containing 0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.005% surfactant P20, at pH 7.4.

Techniques: Modification, Blocking Assay, Infection, Expressing, Luciferase, Activity Assay

Journal: Frontiers in Molecular Biosciences

Article Title: Heparan Sulfate Facilitates Spike Protein-Mediated SARS-CoV-2 Host Cell Invasion and Contributes to Increased Infection of SARS-CoV-2 G614 Mutant and in Lung Cancer

doi: 10.3389/fmolb.2021.649575

Figure Lengend Snippet: Summary of kinetic data of wild type spike and mutant spike protein-heparin interactions*.

Article Snippet: For direct binding analysis, SV2-S D614 (Sino Biological, 40591-V08H) and SV2-S G614 (Sino Biological, 40591-V08H3) were diluted in a running HBS-EP buffer containing 0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.005% surfactant P20, at pH 7.4.

Techniques: Mutagenesis

Journal: Frontiers in Molecular Biosciences

Article Title: Heparan Sulfate Facilitates Spike Protein-Mediated SARS-CoV-2 Host Cell Invasion and Contributes to Increased Infection of SARS-CoV-2 G614 Mutant and in Lung Cancer

doi: 10.3389/fmolb.2021.649575

Figure Lengend Snippet: The SV2-S variant G614 protein has a higher binding affinity to heparin than the wildtype SV2-S D614 protein, and the entry of SAS-Cov-2 G614 pseudotyped virus into host cells depends on cell surface HS and can be inhibited by heparin. (A,B) SPR binding kinetics sensorgrams between heparin and SV2-S D614 or SV2-S G614. Concentrations of D614 were (from top to bottom): 1,000, 800, 600, 400, and 200 nM, respectively (A) , and G614 were (from top to bottom): 1,000, 600, 400, 200, and 100 nM, respectively (B) . The black curves are the fitting curves using a 1:1 Langmuir model from BIAevaluate 4.0.1. (C) Infection of 293T-ACE2 cells by the same titer of SARS-Cov2-D614 and SARS-Cov2-G614 pseudovirus. (D) SARS-Cov2-G614 pseudovirus depends on HS to infect host cells. The 293T-ACE2 cells were infected with SARS-Cov2-G614 pseudovirus expressing luciferase after the cells were treated with heparinases I-III (5 mU/ml) or in the presence of heparin (2 μM). The luciferase activity was measured 48 h after the virus infection. VSV pseudotyped virus was used as a control to make sure each well has comparable cell numbers (data are not shown). The experiments were repeated at least 3 times and an unpaired t-test was performed for two-group comparison. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01.

Article Snippet: For direct binding analysis, SV2-S D614 (Sino Biological, 40591-V08H) and SV2-S G614 (Sino Biological, 40591-V08H3) were diluted in a running HBS-EP buffer containing 0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.005% surfactant P20, at pH 7.4.

Techniques: Variant Assay, Binding Assay, Infection, Expressing, Luciferase, Activity Assay