mouse anti human ace2 mab  (R&D Systems)

Journal: Viruses

Article Title: A Novel Antiviral Therapeutic Platform: Anchoring IFN-β to the Surface of Infectious Virions Equips Interferon-Evasive Virions with Potent Antiviral Activity

doi: 10.3390/v17050697

Figure Lengend Snippet: Postulated mechanism underlying the IFNβ-ACE2 fusion protein. ( A ) A SARS-CoV-2- or NL63-infected individual is given the IFNβ-ACE2 via nebulization to the lungs. The sACE2 domain is postulated to bind the Spike protein and coat the virion with a surface array of IFN-β. Based on this strategy, the IFN-β domain will drive IFN-β signaling pathways and antiviral activity in the target cell before viral entry. IFNβ-ACE2 is postulated to provide a more concentrated and targeted approach to delivering IFN-β to the exact site and time of imminent viral infection . ( B ) Schematic diagram of the fusion protein construct consisting of an IFN-β domain and a sACE2(18-611) domain. ( C ) Schematic diagrams of the soluble control proteins: sACE2(18-611), sACE2(18-740), and IFN-β. ( D ) Schematic diagrams of the transmembrane proteins expressed in HEK cells for the ACE2-binding assay. SDS-PAGE gels showing purity of IFNβ-ACE2 ( E ), sACE2(18-740) ( F ), sACE2(18-611) ( G ), and IFN-β ( H ) proteins.

Article Snippet: After washing with PBS with 5% FBS, Vero E6-TMPRSS2-T2A-ACE2 cells were surface-stained with 1:1600 dilution of PE-conjugated mouse anti-human TMPRSS2 (378403, Biolegend) and 1:100 dilution of FITC-conjugated mouse anti-human ACE2 (10108-MM36-F, Sino Biological) for 1 h at 4 °C and then were washed twice with PBS with 5% FBS.

Techniques: Infection, Protein-Protein interactions, Activity Assay, Construct, Control, Binding Assay, SDS Page

Journal: Viruses

Article Title: A Novel Antiviral Therapeutic Platform: Anchoring IFN-β to the Surface of Infectious Virions Equips Interferon-Evasive Virions with Potent Antiviral Activity

doi: 10.3390/v17050697

Figure Lengend Snippet: The IFN-β and sACE2 domains of IFNβ-ACE2 exhibited predicted bioactivities. ( A ) To assay the IFN-β domain, TF-1 cells were incubated with GM-CSF and either IFN-β, sACE2(18-611), or IFNβ-ACE2 then pulsed with [ 3 H]thymidine during the last 24 h of a 3-day culture. The y -axis represents counts per minute (CPM), and error bars represent the SD. Statistical significance was analyzed by use of two-way ANOVA with Tukey’s multiple comparisons test comparing the three control groups to the IFNβ-ACE2 treatment group at each concentration (ns nonsignificant, * p < 0.05, **** p < 0.0001). ( B – E ) To assay the ACE2 domain, HEK-Spike or HEK-control cells were incubated with designated concentrations of either sACE2(18-611) or IFNβ-ACE2 for 1 h at 4 °C. After washing, cells were stained with AF647-conjugated anti-human ACE2 antibody for 1 h at 4 °C. Cells were analyzed for ACE2 binding by flow cytometry. ( B ) Viable, single, live, and GFP + stably transfected cells (parental gate representing all cells in plot) were subgated to show the ACE2 + subset. Representative dot plots show binding of either sACE2(18-611) or IFNβ-ACE2 (2 μM each) to HEK-Spike or HEK-control cells. ( C ) Shown are percentages of ACE2 + HEK-Spike cells (ACE2 + gate/parental gate). ( D ) The MFIs of anti-ACE2 fluorescence are shown for the parental gate. ( E ) Bar graphs show mean percentages of HEK-Spike or HEK-control cells bound to ACE2 (ACE2 + gate/parental gate). Each data point represents the mean value (n = 2), and error bars represent SD. These data are representative of three independent experiments.

Article Snippet: After washing with PBS with 5% FBS, Vero E6-TMPRSS2-T2A-ACE2 cells were surface-stained with 1:1600 dilution of PE-conjugated mouse anti-human TMPRSS2 (378403, Biolegend) and 1:100 dilution of FITC-conjugated mouse anti-human ACE2 (10108-MM36-F, Sino Biological) for 1 h at 4 °C and then were washed twice with PBS with 5% FBS.

Techniques: Incubation, Control, Concentration Assay, Staining, Binding Assay, Flow Cytometry, Stable Transfection, Transfection, Fluorescence

Journal: Viruses

Article Title: A Novel Antiviral Therapeutic Platform: Anchoring IFN-β to the Surface of Infectious Virions Equips Interferon-Evasive Virions with Potent Antiviral Activity

doi: 10.3390/v17050697

Figure Lengend Snippet: The sACE2 domain of IFNβ-ACE2 targeted IFN-β to the surface of NL63. NL63 was incubated at 4 °C with designated concentrations of IFNβ-ACE2, sACE2(18-611), recombinant IFN-β, or IFN-β (Peprotech). After a 1 h incubation, NL63 was washed of any unbound protein using 300kD centrifugal filters. NL63-protein complexes were then added to Vero E6-TMPRSS2-T2A-ACE2 cultures (100 μL) in a 96-well plate. The cells were harvested after a 2-day incubation at 33 °C, stained with LIVE/DEAD Fixable Blue Dead Cell Stain, and then surface-labeled with FITC-conjugated anti-human ACE2 and PE-conjugated anti-human TMPRSS2. After fixation and permeabilization, cells were stained with AF647-conjugated rabbit anti-NL63 nucleocapsid antibody. Cells were then analyzed for viral infection by flow cytometry. Cells were gated on viable, single, and live cells (parental gate) before subgating on nucleocapsid + , ACE2 high , or TMPRSS2 high cells. Shown are representative dot plots (( A , D , I ), x -axis = FSC-A as in ( A )) when NL63 was incubated with 1 nM IFNβ-ACE2 or controls. The IFNβ-ACE2 versus sACE2(18-611) groups were compared based on percentages of nucleocapsid + cells ( B ), percentages of ACE2 high cells ( E ), MFI of anti-ACE2 staining ( F ), percentages of TMPRSS2 high cells ( J ), and MFI of anti-TMRSS2 staining ( K ). The IFNβ-ACE2 versus IFN-β groups were compared based on percentages of nucleocapsid cells ( C ), percentages of ACE2 high cells ( G ), MFI of anti-ACE2 staining ( H ), percentages of TMPRSS2 high cells ( L ), and MFI of anti-TMPRSS2 staining ( M ). Cell percentages were calculated by dividing events in the positive subgate by the parental gate, and MFI values represent all events in the parental gate. Each data point represents the mean value (n = 2), and error bars represent SD. Statistical significance comparing IFN-β and ACE2 treatment groups to the IFNβ-ACE2 treatment group at each concentration was analyzed by use of two-way ANOVA with Tukey’s multiple comparisons test (** p < 0.01, *** p < 0.001, **** p < 0.0001). Statistical significance comparing each protein group at each concentration to blank was analyzed by use of two-way ANOVA with Tukey’s multiple comparisons test (° p < 0.05, °° p < 0.01, °°° p < 0.001, °°°° p < 0.0001). These data are representative of three independent experiments.

Article Snippet: After washing with PBS with 5% FBS, Vero E6-TMPRSS2-T2A-ACE2 cells were surface-stained with 1:1600 dilution of PE-conjugated mouse anti-human TMPRSS2 (378403, Biolegend) and 1:100 dilution of FITC-conjugated mouse anti-human ACE2 (10108-MM36-F, Sino Biological) for 1 h at 4 °C and then were washed twice with PBS with 5% FBS.

Techniques: Incubation, Recombinant, Staining, Labeling, Infection, Flow Cytometry, Concentration Assay

Journal: Viruses

Article Title: A Novel Antiviral Therapeutic Platform: Anchoring IFN-β to the Surface of Infectious Virions Equips Interferon-Evasive Virions with Potent Antiviral Activity

doi: 10.3390/v17050697

Figure Lengend Snippet: The covalent linkage of IFN-β and ACE2 was required for IFN-β targeting to NL63. NL63 was incubated at 4 °C with either IFNβ-ACE2 or the unlinked combination of sACE2(18-611) and IFN-β. After a 1 h incubation, NL63 was repeatedly washed with 300kD centrifugal filters to remove proteins that lacked binding to virions. The retentates, which included virions and virion-bound proteins, were added to Vero E6-TMPRSS2-T2A-ACE2 cells in a 96-well plate. Cells were harvested after a 2-day incubation at 33 °C and stained with LIVE/DEAD Fixable Blue Dead Cell Stain. Cells were surface-stained with PE-conjugated mouse anti-human TMPRSS2 and FITC-conjugated mouse anti-human ACE2. After fixation and permeabilization, cells were stained with AF647-conjugated rabbit anti-NL63 nucleocapsid antibody. Cells were analyzed for viral infection by flow cytometry. Viable, single, and live cells in the parental gate were subgated as the nucleocapsid + subset ( A ), the ACE2 high subset ( C ), and the TMPRSS2 high subset ( F ) as shown for the 1 nM concentration value. Shown are the percentages of nucleocapsid + , ACE2 high , and TMPRSS2 high subsets together with the respective MFI values ( B , D , E ), and ( G , H ), respectively. Cell percentages were calculated by dividing the events in the subset-positive/high subgate by those in the parental gate. MFI values were gated on all viable, single, and live cells (i.e., cells in the parental gate). Each data point represents the mean value (n = 2), and error bars represent SD. Statistical significance of the IFNβ-ACE2 versus the ‘IFN-β + sACE2’ treatment group at each concentration was analyzed by use of two-way ANOVA with Tukey’s multiple comparisons test (** p < 0.01, *** p < 0.001, **** p < 0.0001). Statistical significance was also assessed for treatment groups at each concentration compared to the ‘blank’ control via two-way ANOVA with Tukey’s multiple comparisons test (°° p < 0.01, °°° p < 0.001, °°°° p < 0.0001). These data are representative of three independent experiments.

Article Snippet: After washing with PBS with 5% FBS, Vero E6-TMPRSS2-T2A-ACE2 cells were surface-stained with 1:1600 dilution of PE-conjugated mouse anti-human TMPRSS2 (378403, Biolegend) and 1:100 dilution of FITC-conjugated mouse anti-human ACE2 (10108-MM36-F, Sino Biological) for 1 h at 4 °C and then were washed twice with PBS with 5% FBS.

Techniques: Incubation, Binding Assay, Staining, Infection, Flow Cytometry, Concentration Assay, Control

Journal: Viruses

Article Title: A Novel Antiviral Therapeutic Platform: Anchoring IFN-β to the Surface of Infectious Virions Equips Interferon-Evasive Virions with Potent Antiviral Activity

doi: 10.3390/v17050697

Figure Lengend Snippet: In a non-washed in vitro infection system, IFNβ-ACE2 exhibited enhanced antiviral activity compared to IFN-β alone, ACE2 alone, or the unlinked combination. NL63 was incubated for 1 h at 4 °C with either IFNβ-ACE2, sACE2(18-611), sACE2(18-740), recombinant IFN-β, IFN-β (Peprotech), or the unlinked combination of sACE2(18-611) and IFN-β. In contrast to experiments shown in and , we omitted the virus-washing step. The NL63 + protein mixtures were added to Vero E6-TMPRSS2-T2A-ACE2 cells in a 96-well plate. The cells were harvested after a 2-day incubation at 33 °C and stained with LIVE/DEAD Fixable Blue Dead Cell Stain. Cells were surface-stained with FITC-conjugated mouse anti-human ACE2, were fixed and permeabilized, and then were intracellularly stained with AF647-conjugated rabbit anti-NL63 nucleocapsid antibody. Cells were then analyzed for viral infection by flow cytometry. Cells gated as viable, single, and live cells (parent gate) were subgated to define nucleocapsid + and ACE2 high subsets. Shown ( A , D ) are representative dot plots showing percentages of the nucleocapsid + subset at the 1 pM concentration. Shown ( B , F ) are the percentages of the nucleocapsid + subset for each group over concentrations ranging from 100 fM to 1 μM. Bar graph ( C ) shows mean percentage values of nucleocapsid + cells at the 1 pM concentration. Shown ( E ) are representative dot plots showing percentages of the ACE2 high subset at the 1 pM concentration. Shown ( G ) are the percentages of ACE2 high subset for each group over concentrations ranging from 100 fM to 1 μM. Each data point represents the mean value (n = 2), and error bars represent SD. Statistical significance was analyzed by ( C ) one-way ANOVA with the Dunnett multiple comparisons test or ( F , G ) two-way ANOVA with Tukey’s multiple comparisons test comparing the unlinked combination of IFN-β and ACE2 treatment groups to the IFNβ-ACE2 treatment group at each concentration (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). These data are representative of three independent experiments.

Article Snippet: After washing with PBS with 5% FBS, Vero E6-TMPRSS2-T2A-ACE2 cells were surface-stained with 1:1600 dilution of PE-conjugated mouse anti-human TMPRSS2 (378403, Biolegend) and 1:100 dilution of FITC-conjugated mouse anti-human ACE2 (10108-MM36-F, Sino Biological) for 1 h at 4 °C and then were washed twice with PBS with 5% FBS.

Techniques: In Vitro, Infection, Activity Assay, Incubation, Recombinant, Virus, Staining, Flow Cytometry, Concentration Assay

Journal: Viruses

Article Title: A Novel Antiviral Therapeutic Platform: Anchoring IFN-β to the Surface of Infectious Virions Equips Interferon-Evasive Virions with Potent Antiviral Activity

doi: 10.3390/v17050697

Figure Lengend Snippet: IFNβ-ACE2 exhibited virus-specific targeting in accordance with viral host receptor specificity . The NL63 or 229E viruses were incubated at 4 °C with either IFNβ-ACE2, sACE2(18-611), IFN-β, or the unlinked combination of IFN-β and sACE2(18-611). After a 1 h incubation, the virus + protein mixtures were washed, and the retentate containing virion–protein complexes was used for infection of Vero E6-TMPRSS2-T2A-ACE2 or A549 cells, respectively, in a 96-well plate ( A , B ). After the washing step, IFNβ-ACE2 or the unlinked combination of IFN-β and sACE2(18-611) were directly added to designated groups ( B ). Alternatively, the virus + protein mixtures were not subjected to a virus-washing step and the mixtures were used for infection of the respective host cells ( C – E ). The cells were harvested after a 2-day incubation and stained with LIVE/DEAD Fixable Blue Dead Cell Stain. After fixation and permeabilization, Vero E6-TMPRSS2-T2A-ACE2 cells were stained with AF647-conjugated rabbit anti-NL63 nucleocapsid antibody and A549 cells were stained with AF647-conjugated rabbit anti-229E nucleocapsid antibody. Cells were then analyzed for viral infection by flow cytometry. Cells were gated on viable, single, and live cells before subgating on nucleocapsid + cells. Shown ( A ) are the percentages of nucleocapsid + cells for each treatment group normalized to the ‘no protein’ control group (1 nM concentrations). ( B ) Bar graph shows mean percentages of nucleocapsid + 229E-infected cells when proteins were or were not added after the washing step (1 nM concentrations). Shown ( C ) are representative dot plots including percentages of nucleocapsid + 229E-infected cells (1 nM concentrations). Shown ( D ) are the percentages of nucleocapsid + cells for each treatment group normalized to the ‘no protein’ group for each virus (1 μM concentrations). Shown ( E ) are the percentages of nucleocapsid + 229E-infected cells for each treatment group at designated concentrations (100 fM to 100 nM). Each data point represents the mean value (n = 2), and error bars represent SD. Statistical significance was analyzed by use of two-way ANOVA with Tukey’s multiple comparisons test comparing the IFN-β and/or ACE2 treatment groups to the IFNβ-ACE2 treatment group at each concentration (unless otherwise noted in the figure) (ns nonsignificant, **** p < 0.0001). Experiments shown are representative of three independent experiments.

Article Snippet: After washing with PBS with 5% FBS, Vero E6-TMPRSS2-T2A-ACE2 cells were surface-stained with 1:1600 dilution of PE-conjugated mouse anti-human TMPRSS2 (378403, Biolegend) and 1:100 dilution of FITC-conjugated mouse anti-human ACE2 (10108-MM36-F, Sino Biological) for 1 h at 4 °C and then were washed twice with PBS with 5% FBS.

Techniques: Virus, Incubation, Infection, Staining, Flow Cytometry, Control, Concentration Assay

Journal: bioRxiv

Article Title: An infectivity-enhancing site on the SARS-CoV-2 spike protein is targeted by COVID-19 patient antibodies

doi: 10.1101/2020.12.18.423358

Figure Lengend Snippet: a , The HEK293 cells transfected with vectors expressing NTD-TM, RBD-TM, and S2-TM were stained with anti-spike antibodies. The mean fluorescence intensity (MFI) of the stained cells was calculated (top three columns). The binding of ACE2-Fc-fusion protein to full-length spike transfectants was analyzed in the presence of the indicated antibodies at 1 μg/ml (bottom column). The antibodies that enhanced ACE2-Fc binding to the spike transfectants by more than 1.9 times are indicated in red. b , ACE2-Fc binding to the spike transfectants in the various concentrations of antibodies. c , ACE2-Fc binding to the wild-type or D614G spike protein in the presence of 3 μg/ml of 2490 mAb. The statistical significance derived from an unpaired t -test is indicated. d , ACE2-Fc binding to wild-type spike protein in the presence of the indicated antibodies at 3 μg/ml and various concentrations of anti-RBD neutralizing antibody C144 (red line). ACE2-Fc binding in the absence of the enhancing antibodies was shown as the control (black line). The data from triplicates are presented as mean ± SD. The representative data from three independent experiments are shown.

Article Snippet: Mouse anti-human ACE2 mAb (R&D Systems, USA), rat anti-Flag mAb (L5, Biolegend), allophycocyanin (APC)-conjugated donkey anti-mouse IgG Fc fragment Ab, APC-conjugated anti-human IgG Fc fragment specific Ab, APC-conjugated anti-rat IgG specific Ab, and APC-conjugated streptavidin (Jackson ImmunoResearch, USA) were used.

Techniques: Transfection, Expressing, Staining, Fluorescence, Binding Assay, Derivative Assay, Control

Journal: bioRxiv

Article Title: An infectivity-enhancing site on the SARS-CoV-2 spike protein is targeted by COVID-19 patient antibodies

doi: 10.1101/2020.12.18.423358

Figure Lengend Snippet: The plasmids expressing the full-length spike, NTD-TM, RBD-TM, and mock were transfected into HEK293T cells with the GFP vector, and the transfectants were mixed with the indicated anti-NTD antibodies at 10 μg/ml. 4A8 is a non-enhancing antibody and the remaining is enhancing antibodies. Transfectants not mixed with antibodies were used as a control (shaded histogram). Afterward, the cells were stained with biotin-labeled ACE2-Fc fusion protein, followed by APC-labeled streptavidin. The fluorescence intensities of APC on the GFP-expressing cells are shown (red line). Mean fluorescent intensities (MFI) of red lines were shown in the figure.

Article Snippet: Mouse anti-human ACE2 mAb (R&D Systems, USA), rat anti-Flag mAb (L5, Biolegend), allophycocyanin (APC)-conjugated donkey anti-mouse IgG Fc fragment Ab, APC-conjugated anti-human IgG Fc fragment specific Ab, APC-conjugated anti-rat IgG specific Ab, and APC-conjugated streptavidin (Jackson ImmunoResearch, USA) were used.

Techniques: Expressing, Transfection, Plasmid Preparation, Control, Staining, Labeling, Fluorescence

Journal: bioRxiv

Article Title: An infectivity-enhancing site on the SARS-CoV-2 spike protein is targeted by COVID-19 patient antibodies

doi: 10.1101/2020.12.18.423358

Figure Lengend Snippet: a , The ACE2 -expressing HEK293 cells (MOI: 0.3) were infected by a SARS-CoV-2-spike pseudovirus carrying a GFP reporter gene in the presence of various concentrations of indicated antibodies. The proportion of GFP-positive infected cells is shown. b , The ACE2 -expressing HEK293 cells were infected with a SARS-CoV-2-spike pseudovirus carrying a GFP reporter gene at different MOIs with (red line, 3 μg/ml) or without (black line) the indicated antibodies. c , HEK293 cells, ACE2-expressing HEK293 cells, and Huh7 cells were infected with authentic SARS-CoV-2 virus in the presence (+) or absence (–) of enhancing antibody 2490 at 1 μg/ml. The amounts of SARS-CoV-2 virus produced in the cell culture supernatants were analyzed 48 h after infection. The statistical significance derived from an unpaired t -test is indicated. NS: Not significant. The data are presented as mean ± SD. The representative data from three independent experiments are shown.

Article Snippet: Mouse anti-human ACE2 mAb (R&D Systems, USA), rat anti-Flag mAb (L5, Biolegend), allophycocyanin (APC)-conjugated donkey anti-mouse IgG Fc fragment Ab, APC-conjugated anti-human IgG Fc fragment specific Ab, APC-conjugated anti-rat IgG specific Ab, and APC-conjugated streptavidin (Jackson ImmunoResearch, USA) were used.

Techniques: Expressing, Infection, Virus, Produced, Cell Culture, Derivative Assay

Journal: bioRxiv

Article Title: An infectivity-enhancing site on the SARS-CoV-2 spike protein is targeted by COVID-19 patient antibodies

doi: 10.1101/2020.12.18.423358

Figure Lengend Snippet: Parental HEK293T cells and ACE2-transfected HEK293T cells were stained with anti-ACE2 mAb (red line). Control stainings were shown as shaded histogram.

Article Snippet: Mouse anti-human ACE2 mAb (R&D Systems, USA), rat anti-Flag mAb (L5, Biolegend), allophycocyanin (APC)-conjugated donkey anti-mouse IgG Fc fragment Ab, APC-conjugated anti-human IgG Fc fragment specific Ab, APC-conjugated anti-rat IgG specific Ab, and APC-conjugated streptavidin (Jackson ImmunoResearch, USA) were used.

Techniques: Transfection, Staining, Control

Journal: bioRxiv

Article Title: An infectivity-enhancing site on the SARS-CoV-2 spike protein is targeted by COVID-19 patient antibodies

doi: 10.1101/2020.12.18.423358

Figure Lengend Snippet: a , The binding of the enhancing antibodies (binder) to full-length spike transfectants was analyzed in the presence of the indicated antibodies (competitor). The effect of competitors on ACE2-Fc binding to the spike transfectants was also analyzed. The non-enhancing anti-S2 antibody, 2147, was used as a control. Relative antibody or ACE2 binding lebels observed in the presence of competitor are shown. b , Relative antibody binding levels to a series of NTD mutants compared to wild-type NTD are shown. Non-enhancing anti-NTD antibody 4A8 was used as a control. The most affected resideus were shown as red. c , The full-length mutant spike proteins were stained with the indicated enhancing antibodies (red line). Staining of wild-type spike were shown as shaded histogram. d . Amino acid residues that affected the binding of each enhancing antibodies are shown as a heatmap based on their percent reduction of the MFIs in b ), with higher reduction indicated by darker shades. NTD: dark grey, RBD: medium grey, other regions: light grey. e , The MFIs reduction of the affected residues are averaged across the six antibodies and shown as a heatmap.

Article Snippet: Mouse anti-human ACE2 mAb (R&D Systems, USA), rat anti-Flag mAb (L5, Biolegend), allophycocyanin (APC)-conjugated donkey anti-mouse IgG Fc fragment Ab, APC-conjugated anti-human IgG Fc fragment specific Ab, APC-conjugated anti-rat IgG specific Ab, and APC-conjugated streptavidin (Jackson ImmunoResearch, USA) were used.

Techniques: Binding Assay, Control, Mutagenesis, Staining

Journal: bioRxiv

Article Title: Vitamin C is an efficient natural product for prevention of SARS-CoV-2 infection by targeting ACE2 in both cell and in vivo mouse models

doi: 10.1101/2022.07.14.499651

Figure Lengend Snippet: (A) Western blot analysis of endogenous ACE2 in various types of cell lines, including HEK293T transfected with either control shRNAs (shCtrl) or shRNAs against ACE2 (shACE2), 2fTGH, Caco-2 and A549 cells. (B) HEK293T cells were transfected with shCtrl or increasing amounts of shACE2. Then cells were subjected to RT-qPCR analysis of Ace2 mRNA levels (right), or were infected with SARS-CoV-2 GFP/ΔN (MOI = 0.1) for 2 hrs. RT-qPCR was used to analyze SARS-CoV-2 RNA levels (left). (C) Western blot analysis of ACE2 in Caco-2 cells treated with vitamin (Vit) compounds (VitB1, 500 µM; VitB6, 500 µM; VitB12, 50 nM; VitC, 5 mM; VitD3, 25 µM; VitK1, 0.5 µM) for 24 hrs. (D) Western blot analysis of ACE2 in HEK293T, 2fTGH, Caco-2 and A549 cells treated with VitC at indicated concentrations for 24 hrs. (E) Western blot analysis of ACE2 in HEK293T cells treated with 5 mM or 0.2 mM of VitC for different durations. (F) Immunofluorescence analysis of ACE2 proteins in HeLa cells treated with VitC (5 mM) for 24 hrs. DAPI was used for the nucleus. Scale bars, 1 μm. (G) Western blot analysis of ACE2, IRF3 and STAT1 in A549 cells treated with VitC at indicated concentrations for 24 hrs. (H) Fluorescence microscopy of the SARS-CoV-2 GFP/ΔN or VSV-GFP viruses in Caco-2-N cells pretreated with VitC (5 mM and 10 mM) for 24 hrs, and then infected with SARS-CoV-2 GFP/ΔN (MOI = 0.1) or VSV-GFP (MOI = 0.1) viruses for 24 hrs. Scale bar: 100 µm. (I) RT-qPCR analysis of SARS-CoV-2 GFP/ΔN or VSV RNA levels in Caco-2 cells pretreated with VitC as (H), and then infected with SARS-CoV-2 GFP/ΔN (MOI = 0.1) or VSV (MOI = 0.1) for 2 hrs. Data are representative of three independent experiments (A, C-F), or are shown as mean and s.d. of three biological replicates (B, I). N.S, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001 (two-tailed unpaired Student’s t -test). See also Figure S1.

Article Snippet: The antibodies with the indicated dilutions were as follows: anti-human ACE2 (Cell Signaling Technology, #74512, 1:1000), anti-ACE2 (Affinity, AF5165, 1:1000), anti-USP50 (Affinity, AF9225, 1:1000), anti-USP50 (Proteintech, 20374-1-AP, 1:1000), anti-Flag (Sigma, F7425, 1:5000), anti-HA (Abcam, ab9110, 1:3000), anti-Myc (Abmart, M20002H, 1:3000), anti-IRF3 (Santa Cruz, sc-33641, 1:1000), anti-STAT1 (Cell Signaling Technology, 9172, 1:1000), anti-Ubiquitin (Ub) (Santa Cruz, 12987-1-AP, 1:1000), anti-K48 Ub (Cell Signaling Technology, 4289S, 1:1000), anti-VSV-G (Santa Cruz, sc-66180, 1:2000) and anti-Tubulin (Proteintech, 66031-1-Ig, 1:3000).

Techniques: Western Blot, Transfection, Control, Quantitative RT-PCR, Infection, Immunofluorescence, Fluorescence, Microscopy, Two Tailed Test

Journal: bioRxiv

Article Title: Vitamin C is an efficient natural product for prevention of SARS-CoV-2 infection by targeting ACE2 in both cell and in vivo mouse models

doi: 10.1101/2022.07.14.499651

Figure Lengend Snippet: (A) RT-qPCR analysis of Ace2 mRNA in 2fTGH cells treated with VitC (5 mM) as indicated. (B) Western blot analysis of ACE2 in 2fTGH cells pretreated with ddH 2 O (Ctrl) or VitC (5 mM) for 12 hrs and then treated with CHX (50 μM) for 6 and 12 hrs. (C) Western blot analysis of Myc-ACE2 levels in HEK293T cells transfected with Myc-ACE2 and then treated with VitC at indicated concentrations for 12 hrs. (D) Western blot analysis of ACE2 in HEK293T cells pretreated with MG132 (10 µM) or MA (10 µM) for 2 hrs, followed by VitC treatment (5 mM) for 6 hrs. (E) Immunoprecipitation (IP)-immunoblotting (IB) analysis of ubiquitination (Ub) of endogenous ACE2 in 2fTGH cells treated with VitC at indicated concentrations for 12 hrs. (F) IP-IB analysis of ubiquitination types of Myc-ACE2 in HEK293T cells cotransfected with Myc-ACE2 and different types of HA-Ub, and then treated with VitC (5 mM) for 12 hrs. (G) IP-IB analysis of K48-linked polyubiquitination (K48-Ub) of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2 and then treated with VitC (2.5 mM and 5 mM) for 12 hrs, using a specific anti-K48-Ub antibody. (H) IP-IB analysis of K48-Ub of endogenous ACE2 in 2fTGH cells treated with VitC (2.5 mM and 5 mM) for 12 hrs. Data are representative of three independent experiments (B-H), or are shown as mean and s.d. of three biological replicates (A, B). N.S, not significant, ** p < 0.01 (two-tailed unpaired Student’s t -test). See also Figure S2.

Article Snippet: The antibodies with the indicated dilutions were as follows: anti-human ACE2 (Cell Signaling Technology, #74512, 1:1000), anti-ACE2 (Affinity, AF5165, 1:1000), anti-USP50 (Affinity, AF9225, 1:1000), anti-USP50 (Proteintech, 20374-1-AP, 1:1000), anti-Flag (Sigma, F7425, 1:5000), anti-HA (Abcam, ab9110, 1:3000), anti-Myc (Abmart, M20002H, 1:3000), anti-IRF3 (Santa Cruz, sc-33641, 1:1000), anti-STAT1 (Cell Signaling Technology, 9172, 1:1000), anti-Ubiquitin (Ub) (Santa Cruz, 12987-1-AP, 1:1000), anti-K48 Ub (Cell Signaling Technology, 4289S, 1:1000), anti-VSV-G (Santa Cruz, sc-66180, 1:2000) and anti-Tubulin (Proteintech, 66031-1-Ig, 1:3000).

Techniques: Quantitative RT-PCR, Western Blot, Transfection, Immunoprecipitation, Ubiquitin Proteomics, Two Tailed Test

Journal: bioRxiv

Article Title: Vitamin C is an efficient natural product for prevention of SARS-CoV-2 infection by targeting ACE2 in both cell and in vivo mouse models

doi: 10.1101/2022.07.14.499651

Figure Lengend Snippet: (A) Western blot analysis of ACE2 in HEK293T cells pretreated with PR619 (50 µM, 2 hrs) and then treated with VitC (5 mM) for 12 hrs. (B) HEK293T cells were individually transfected with the plasmids from the human DUBs expression library. Western blot was used to identify the key deubiquitinase that significantly increases ACE2 levels. (C) IP-IB analysis of the interaction between Flag-USP50 and Myc-ACE2 in HEK293T cells cotransfected with these two constructs. (D) Immunoprecipitation analysis of the interaction between endogenous USP50 and ACE2 in 2fTGH cells. (E) Western blot analysis of ACE2 in HEK293T cells transfected with increasing amounts of Flag-USP50. (F) Western blot analysis of ACE2 in HEK293T cells transfected with shCtrl or shUSP50 (#1 or #2). (G) Western blot analysis of ACE2 in HEK293T cells transfected with Flag-USP50 and then treated with CHX (50 μM) as indicated. (H) Western blot analysis of ACE2 in Usp50 +/+ and Usp50 -/- HEK293T cells. (I) Western blot analysis of ACE2 in Usp50 +/+ and Usp50 -/- HEK293T cells treated with VitC (5 mM) for 12 hrs. (J) RT-qPCR analysis of SARS-CoV-2 GFP/ΔN RNA levels in HEK293T cells transfected with Flag-USP50 and then infected with the SARS-CoV-2 GFP/ΔN virus (MOI = 0.1) for 24 hrs. (K) Fluorescence microscopy of the SARS-CoV-2-S pseudovirus in Usp50 +/+ and Usp50 -/- HEK293T cells pretreated with or without VitC (5 mM) for 12 hrs, followed by infection with SARS-CoV-2-S pseudovirus (MOI = 0.1) for 24 hrs. Scale bar: 100 µm. Data are representative of three independent experiments (A-I), or are shown as mean and s.d. of three biological replicates (J, K). N.S, not significant, * p < 0.05, *** p < 0.001 (two-tailed unpaired Student’s t -test). See also Figure S3.

Article Snippet: The antibodies with the indicated dilutions were as follows: anti-human ACE2 (Cell Signaling Technology, #74512, 1:1000), anti-ACE2 (Affinity, AF5165, 1:1000), anti-USP50 (Affinity, AF9225, 1:1000), anti-USP50 (Proteintech, 20374-1-AP, 1:1000), anti-Flag (Sigma, F7425, 1:5000), anti-HA (Abcam, ab9110, 1:3000), anti-Myc (Abmart, M20002H, 1:3000), anti-IRF3 (Santa Cruz, sc-33641, 1:1000), anti-STAT1 (Cell Signaling Technology, 9172, 1:1000), anti-Ubiquitin (Ub) (Santa Cruz, 12987-1-AP, 1:1000), anti-K48 Ub (Cell Signaling Technology, 4289S, 1:1000), anti-VSV-G (Santa Cruz, sc-66180, 1:2000) and anti-Tubulin (Proteintech, 66031-1-Ig, 1:3000).

Techniques: Western Blot, Transfection, Expressing, Construct, Immunoprecipitation, Quantitative RT-PCR, Infection, Virus, Fluorescence, Microscopy, Two Tailed Test

Journal: bioRxiv

Article Title: Vitamin C is an efficient natural product for prevention of SARS-CoV-2 infection by targeting ACE2 in both cell and in vivo mouse models

doi: 10.1101/2022.07.14.499651

Figure Lengend Snippet: (A) IP-IB analysis of ubiquitination of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2, together with Flag-USP50 (WT) or its deubiquitinase inactive mutant (C53S). (B) IP-IB analysis of ubiquitination of endogenous ACE2 in HEK293T cells transfected with either shCtrl (-) or shUSP50 (#1, #2). (C) IP-IB analysis of ubiquitination types of Myc-ACE2 in HEK293T cells cotransfected with Myc-ACE2, Flag-USP50 and different types of HA-Ub. (D) IP-IB analysis of K48-Ub of endogenous ACE2 in HEK293T cells transfected with shCtrl (-) or shUSP50 (#1, #2). (E) Putative ubiquitination sites of ACE2 in the PhosphoSitePlus database (Upper). Myc-ACE2 K48-linked ubiquitination was analyzed by IP-IB in HEK293T cells cotransfected with Myc-ACE2 (WT or its mutants) and HA-K48-Ub (Lower). (F) IP-IB analysis of Myc-ACE2 K48-linked ubiquitination in HEK293T cells cotransfected with Myc-ACE2 (WT or K788R) and HA-K48, together with shCtrl or shUSP50. (G) Western blot analysis of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2 (WT or K788R) and then treated with CHX (50 μM) as indicated. (H) IP-IB analysis of K48-Ub of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2 (WT or K788R) and then treated with VitC (5 mM) for 12 hrs, by a specific anti-K48-Ub antibody. (I) Western blot analysis of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2 (WT or K788R) and then treated with VitC (5 mM) as indicated. Data are representative of three independent experiments (A-I). See also Figure S3 and S4.

Article Snippet: The antibodies with the indicated dilutions were as follows: anti-human ACE2 (Cell Signaling Technology, #74512, 1:1000), anti-ACE2 (Affinity, AF5165, 1:1000), anti-USP50 (Affinity, AF9225, 1:1000), anti-USP50 (Proteintech, 20374-1-AP, 1:1000), anti-Flag (Sigma, F7425, 1:5000), anti-HA (Abcam, ab9110, 1:3000), anti-Myc (Abmart, M20002H, 1:3000), anti-IRF3 (Santa Cruz, sc-33641, 1:1000), anti-STAT1 (Cell Signaling Technology, 9172, 1:1000), anti-Ubiquitin (Ub) (Santa Cruz, 12987-1-AP, 1:1000), anti-K48 Ub (Cell Signaling Technology, 4289S, 1:1000), anti-VSV-G (Santa Cruz, sc-66180, 1:2000) and anti-Tubulin (Proteintech, 66031-1-Ig, 1:3000).

Techniques: Ubiquitin Proteomics, Transfection, Mutagenesis, Western Blot

Journal: bioRxiv

Article Title: Vitamin C is an efficient natural product for prevention of SARS-CoV-2 infection by targeting ACE2 in both cell and in vivo mouse models

doi: 10.1101/2022.07.14.499651

Figure Lengend Snippet: (A) The procedure for analysis of the binding of VitC to Flag-USP50 or Myc-ACE2 (left) can be seen detailedly in the Methods. The concentrations of VitC that binds with either Flag-USP50 (middle) or Myc-ACE2 (right) proteins were measured by the Vitamin-C Detection Kit. (B) Recombinant human ACE2-IgG-Fc proteins (r-hACE2-Fc) or anti-Tyk2 IgG proteins were incubated with protein-G beads for 2 hrs, and then VitC was added for binding. After washing and centrifuging, the concentrations of VitC that binds to anti-Tyk2 proteins or r-hACE2-Fc proteins were detected as (A). (C) The concentrations of VitC that binds to Myc-ACE2 (WT) or its deletion mutants were detected as (A). (D) VitC docking to the binding pocket of ACE2 by standard precision (SP) using the Glide docking module in the Schrödinger molecular simulation software. (E) The docking diagram between amino acids of ACE2 and VitC based on the SP docking scoring function. (F) IP-IB analysis of the interaction between USP50 and Myc-ACE2 (WT) or its deletion mutants (Δ19-200, Δ201-400, Δ401-600 and Δ601-805) in HEK293T. (G) IP-IB analysis of the interaction between Flag-USP50 and Myc-ACE2 in HEK293T cells transfected with these two constructs and then treated with VitC (2.5 mM and 5 mM) for 12 hrs. (H) IP-IB analysis of the in vivo interaction between endogenous USP50 and ACE2 in 2fTGH cells treated with VitC (5 mM) for 12 hrs. (I) Myc-ACE2 and Flag-USP50 proteins were immunoprecipitated from HEK293T cells transfected with either Myc-ACE2 or Flag-USP50. Flag-USP50 proteins were eluted by the Flag (M2) agarose. After washing, Flag-USP50 proteins were mixed with the Myc beads with Myc-ACE2, together with or without VitC (5 mM) for 2 hrs. After centrifuging, Flag-USP50 proteins interacting with Myc-ACE2 were analyzed by immunoblotting. (J) Flag-USP50 proteins were obtained as (I). Flag-USP50 and r-hACE2 proteins were mixed, together with increasing amounts of VitC or with VitC-Na + (20 mM). HCl is a pH control (pH = 4). After 2 hrs incubation, ACE2 proteins were analyzed by immunoblotting by a specific anti-ACE2 antibody. Data are representative of three independent experiments (F-J), or are shown as mean and s.d. of three biological replicates (A-C). N.S, not significant. *** p < 0.001 (two-tailed unpaired Student’s t -test). See also Figure S5.

Article Snippet: The antibodies with the indicated dilutions were as follows: anti-human ACE2 (Cell Signaling Technology, #74512, 1:1000), anti-ACE2 (Affinity, AF5165, 1:1000), anti-USP50 (Affinity, AF9225, 1:1000), anti-USP50 (Proteintech, 20374-1-AP, 1:1000), anti-Flag (Sigma, F7425, 1:5000), anti-HA (Abcam, ab9110, 1:3000), anti-Myc (Abmart, M20002H, 1:3000), anti-IRF3 (Santa Cruz, sc-33641, 1:1000), anti-STAT1 (Cell Signaling Technology, 9172, 1:1000), anti-Ubiquitin (Ub) (Santa Cruz, 12987-1-AP, 1:1000), anti-K48 Ub (Cell Signaling Technology, 4289S, 1:1000), anti-VSV-G (Santa Cruz, sc-66180, 1:2000) and anti-Tubulin (Proteintech, 66031-1-Ig, 1:3000).

Techniques: Binding Assay, Recombinant, Incubation, Software, Transfection, Construct, In Vivo, Immunoprecipitation, Western Blot, Control, Two Tailed Test

Journal: eLife

Article Title: Inhibition of SARS-CoV-2 viral entry upon blocking N- and O-glycan elaboration

doi: 10.7554/eLife.61552

Figure Lengend Snippet: SARS-CoV-2 Spike protein trimer (pink) bound to ACE2 (green). ( A ) Without glycans. ( B ) With N-glycans (red) identified using LC-MS on Spike and ACE2. ( C ) Molecular dynamics simulation analyzed the range of movement of each glycan. The space sampled by glycans is represented by a gray cloud. Glycans cover the Spike-ACE2 interface. They also surround the putative proteolysis site of furin (‘S1-S2’, yellow) and S2’ (blue).

Article Snippet: Goat anti-human ACE2 polyclonal antibody (AF933) and mouse anti-human ACE2 mAb MAB9332 were available from R and D Systems (Minneapolis, MA).

Techniques: Liquid Chromatography with Mass Spectroscopy, Glycoproteomics

Journal: eLife

Article Title: Inhibition of SARS-CoV-2 viral entry upon blocking N- and O-glycan elaboration

doi: 10.7554/eLife.61552

Figure Lengend Snippet: ( A ) Full-length proteins expressed on cells include wild-type Spike-protein [v1] and human ACE2 [v2]. N-glycosylation sites are indicated by lollipop. Fc-his soluble proteins encode for S1-subunit [v3], RBD [v4] and soluble ACE2 [v5]. All constructs were co-expressed with fluorescent reporters separated by P2A. Note that the Fc-section also contains one N-glycosylation site. ( B ) Western blot for purified Fc-proteins from HEK293T probed with anti-Fc, anti-RBD or anti-ACE2 Ab. CD44-Fc is positive control. ( C ) Flow cytometry data showing S1-Fc (1.7 µg/mL) and RBD-Fc (0.35 µg/mL) binding to ACE2 expressed on HEK293T (middle panel). Spike expression enhances ACE2-Fc (1.4 µg/mL) binding (bottom). ( D ) Desialylation of Spike-protein expressed on 293 T/S had minimal effect on ACE2-Fc (0.7 µg/mL) binding. ACE2 desialylation on 293T/ACE2 increased binding of RBD-Fc (0.2 µg/mL) and S1-Fc (1.7 µg/mL) by 26–56% (paired experiments, *p<0.05). ( E ) Pseudovirus with DsRed-reporter were developed with three different envelope proteins. VSVG pseudotyped virus infected both HEK293T (black line) and stable 293T/ACE2 (red line) cells. Virus with Spike-WT and Spike-mutant entered 293T/ACE2 only. ( F ) Same titer of virus (0.3 µg/mL p24-equivalent) were treated with or without sialidase, prior to addition to stable 293T/ACE2 cells. Infection using Spike-mutant was higher compared to Spike-WT. Sialidase treatment of virus had no effect. ( G ) 293T/ACE2 cells were sialidase treated prior to addition of VSVG (0.3 µg/mL p24-equiv.), Spike-WT (1.5 µg/mL p24-equiv.) or Spike-mutant (0.2 µg/mL p24-equiv.) pseudovirus. Sialidase treatment did not affect viral entry. Abbreviations: Spike signal peptide (SP), N-terminal domain (NTD), receptor-binding domain (RBD), receptor-binding motif (RBM), subdomain 1 (SD1), subdomain 2 (SD2), fusion peptide (FP), heptad repeat 1 (HR1), central helix (CH), connector domain (CD), heptad repeat 2 (HR2) transmembrane section (TM), cytoplasmic tail (CT), ACE2: Angiotensin-converting enzyme-2; VSVG: Vesicular stomatitis virus G-protein; WT: wild-type; mut: mutant.

Article Snippet: Goat anti-human ACE2 polyclonal antibody (AF933) and mouse anti-human ACE2 mAb MAB9332 were available from R and D Systems (Minneapolis, MA).

Techniques: Glycoproteomics, Construct, Western Blot, Purification, Positive Control, Flow Cytometry, Binding Assay, Expressing, Virus, Infection, Mutagenesis

Journal: eLife

Article Title: Inhibition of SARS-CoV-2 viral entry upon blocking N- and O-glycan elaboration

doi: 10.7554/eLife.61552

Figure Lengend Snippet: ( A ) Sialidase protocol validation. All lectins were directly conjugated with Alexa dyes. They were incubated with cells at 1–5 µg/mL for 15 min before a quick wash and cytometry measurement. Compared to untreated control (left), sialidase treatment (right) decreased SNA lectin binding to α2,6 sialylated structures by 15-fold and increased ECL binding to desialylated lactosamine chains (Galβ1,4GlcNAcβ) by an order of magnitude. ( B ) Pseudovirus assay. DsRed fluorescence in HEK293T and stable 293T/ACE2 cells upon addition of VSVG, Spike-WT and Spike-mutant pseudotyped virus. ( C ) Sialidase treatment of pseudovirus. % DsRed positive cell data are shown for study in (main manuscript). Viral entry was sialidase independent. ( D ) Sialidase treatment of HEK/ACE2 cells. Pseudovirus expressing VSVG, Spike-WT and Spike-mutant were added to cells under conditions described in (main manuscript). All error bars are standard deviations. Data are representative of 3 independent runs.

Article Snippet: Goat anti-human ACE2 polyclonal antibody (AF933) and mouse anti-human ACE2 mAb MAB9332 were available from R and D Systems (Minneapolis, MA).

Techniques: Biomarker Discovery, Incubation, Cytometry, Control, Binding Assay, Fluorescence, Mutagenesis, Virus, Expressing

Journal: eLife

Article Title: Inhibition of SARS-CoV-2 viral entry upon blocking N- and O-glycan elaboration

doi: 10.7554/eLife.61552

Figure Lengend Snippet: ( A ) Knocking out C1GALT1 and MGAT1 using CRISPR-Cas9 inhibits O- and N-glycan biosynthesis in HEK293Ts. ( B ) Sanger sequencing results of isogenic 293T clones shows indels on all 3 alleles of C1GALT1 (‘[O] - 293T’) and single allele of MGAT1 (‘[N] - 293T’) knockout cells. Wild-type (WT) sequence is on the first line. Lower line shows base deletions (hyphen) and insertions (black fonts) for individual KOs. sgRNA target sequence is in red and protospacer adjacent motif is underlined. ( C ) Increased VVA and reduced PHA-L binding confirm loss of O-linked glycans in [O] - 293Ts and N-glycans in [N] - 293Ts, respectively. ( D ) Knocking out N-glycans on Spike protein reduced ACE2-Fc binding in cytometry based binding studies. Knocking out Spike O-glycans increased ACE-2 binding. ( E–F ) Truncation of ACE2 N- and O-glycans did not affect either S1-Fc (panel E ) or RBD-Fc (panel F ) binding. ( G ) ACE2 was transiently expressed on 293T, [O] - 293T and [N] - 293 T cells. All pseudotyped virus efficiently entered ACE2 expressing cells. Virus was not titered for these runs, and thus comparison between viruses is not possible. *p<0.05 with respect to all other treatments. # p<0.05 with respect to 293T and [N] - 293T/ACE2 in panel G .

Article Snippet: Goat anti-human ACE2 polyclonal antibody (AF933) and mouse anti-human ACE2 mAb MAB9332 were available from R and D Systems (Minneapolis, MA).

Techniques: CRISPR, Glycoproteomics, Sequencing, Clone Assay, Knock-Out, Binding Assay, Cytometry, Virus, Expressing, Comparison

Journal: eLife

Article Title: Inhibition of SARS-CoV-2 viral entry upon blocking N- and O-glycan elaboration

doi: 10.7554/eLife.61552

Figure Lengend Snippet: ( A ) Surface expression of Spike-protein and ACE2. Full-length Spike (top) and human ACE2 (bottom) were expressed in HEK 293T, [N] - 293T and [O] - 293T. Protein expression was measured in EGFP+ cells in the case of Spike (using anti-RBD), and on BFP+ cells in the case of ACE2 (using anti-ACE2), as these fluorescent reporters are co-expressed with surface proteins. Protein expression was comparable in all cells. Untransfected 293Ts serve as negative control. ( B ) Viral entry assay. Pseudovirus expressing VSVG envelope protein, Spike-WT or Spike-mutant were added to HEK 293 T cells transiently transfected to overexpress ACE2 (both wild-type 293T and glycosylation mutants). An additional control included 293 T cells not expressing ACE2, which only allowed entry of VSVG pseudotyped viral particles, but not Spike bearing virus. % cells that were DsRed (reporter) positive is shown at 72 hr. All treatments were statistically different except as indicated by n.s. (‘not significant’). All data are from of N > 3 repeats.

Article Snippet: Goat anti-human ACE2 polyclonal antibody (AF933) and mouse anti-human ACE2 mAb MAB9332 were available from R and D Systems (Minneapolis, MA).

Techniques: Expressing, Negative Control, Mutagenesis, Transfection, Glycoproteomics, Control, Virus

Journal: eLife

Article Title: Inhibition of SARS-CoV-2 viral entry upon blocking N- and O-glycan elaboration

doi: 10.7554/eLife.61552

Figure Lengend Snippet: ( A ) Pseudovirus expressing VSVG envelope protein, Spike-WT and Spike-mutant were produced in wild-type, [O] - and [N] - 293 T cells. All nine viruses were applied at equal titer to stable 293T/ACE2. ( B–C ) O-glycan truncation of Spike partially reduced viral entry. N-glycan truncation abolished viral entry. In order to combine data from multiple viral preparations and independent runs in a single plot, all data were normalized by setting DsRed signal produced by virus generated in wild-type 293T to 10,000 normalized MFI or 100% normalized DsRed positive value. ( D ) Viral titration study performed with Spike-mutant virus shows complete loss of viral infection over a wide range. ( E ) Western blot of Spike protein using anti-S2 Ab shows reduced proteolysis of Spike-mut compared to Spike-WT. The full Spike protein and free S2-subunit resulting from S1-S2 cleavage is indicated. Molecular mass is reduced in [N] - 293T products due to truncation of glycan biosynthesis. ( F ) Anti-FLAG Ab binds the C-terminus of Spike-mutant. Spike produced in [N] - 293Ts is almost fully proteolyzed during viral production (red arrowhead). *p<0.05 with respect to all other treatments.

Article Snippet: Goat anti-human ACE2 polyclonal antibody (AF933) and mouse anti-human ACE2 mAb MAB9332 were available from R and D Systems (Minneapolis, MA).

Techniques: Expressing, Mutagenesis, Produced, Glycoproteomics, Virus, Generated, Titration, Infection, Western Blot

Journal: eLife

Article Title: Inhibition of SARS-CoV-2 viral entry upon blocking N- and O-glycan elaboration

doi: 10.7554/eLife.61552

Figure Lengend Snippet: ( A ) VSVG, Spike-WT and Spike-mutant pseudovirus were produced in the presence of 15 µM kifunensine or vehicle control. The six viruses were added to 293T/ACE2 at equal titer. ( B–D ) Microscopy (panel B) and cytometry (panel C, D ) show ~90% loss of viral infection in the case of Spike-WT and Spike-mutant virus upon kifunensine treatment (*p<0.05). ( E ) Spike molecular mass is reduced in the western blots due to high-mannose glycan synthesis in runs with kifunensine. Intact Spike is reduced in the presence of kifunensine, in anti-FLAG blot. ( F ) The polybasic furin ‘RRAR’ site was substituted by a single ‘A’ amino acid in Spike-delta. Virus with Spike-delta were expressed both in the presence of vehicle and kifunensine. Western blot shows lack of S1-S2 cleavage in this construct. In viral entry assay, kifunensine reduced DsRed expression in 293T/ACE2 cells, even in the case of Spike-delta pseudovirus (*p<0.05). Similar observation was made at two different viral titers (0.3 and 0.6 μg/mL p24 equivalent).

Article Snippet: Goat anti-human ACE2 polyclonal antibody (AF933) and mouse anti-human ACE2 mAb MAB9332 were available from R and D Systems (Minneapolis, MA).

Techniques: Mutagenesis, Produced, Control, Microscopy, Cytometry, Infection, Virus, Western Blot, Glycoproteomics, Construct, Expressing

Journal: eLife

Article Title: Inhibition of SARS-CoV-2 viral entry upon blocking N- and O-glycan elaboration

doi: 10.7554/eLife.61552

Figure Lengend Snippet: ( A ) ACE2-Fc binding was measured to wild-type or glycoEnzyme-KO 293 T cells expressing Spike. Sialidase treatment of cells was performed in some cases. Similar studies also measured S1-Fc and RBD-Fc binding to cell-surface expressed ACE2. ( B ) SARS-CoV-2 pseudovirus (bearing Spike-WT, Spike-mut, Spike-delta variants) were generated in wild-type or glycoEnzyme-KO 293Ts, in the presence and absence of kifunensine. Main results of binding ( A ) and viral entry ( B ) assay are listed. ( C ) Conceptual model shows that kifunensine can induce S1-S2 site proteolysis on Spike-WT and Spike-mut virus, but not Spike-delta virus. This proteolysis reduces RBD presentation and attenuates viral entry into 293T/ACE2. Without affecting S1-S2 cleavage, kifunensine also partially reduced Spike-delta pseudovirus entry function. The data suggest additional roles for Spike N-glycans during viral entry.

Article Snippet: Goat anti-human ACE2 polyclonal antibody (AF933) and mouse anti-human ACE2 mAb MAB9332 were available from R and D Systems (Minneapolis, MA).

Techniques: Binding Assay, Expressing, Generated, Virus

Journal: eLife

Article Title: Inhibition of SARS-CoV-2 viral entry upon blocking N- and O-glycan elaboration

doi: 10.7554/eLife.61552

Figure Lengend Snippet:

Article Snippet: Goat anti-human ACE2 polyclonal antibody (AF933) and mouse anti-human ACE2 mAb MAB9332 were available from R and D Systems (Minneapolis, MA).

Techniques: Binding Assay, Plasmid Preparation, Recombinant, Knock-Out, Derivative Assay