fitc conjugated mouse anti human ace2  (Sino Biological)

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: Journal of Virology

Article Title: Host cell lectins ASGR1 and DC-SIGN jointly with TMEM106B confer ACE2 independence and imdevimab resistance to SARS-CoV-2 pseudovirus with spike mutation E484D

doi: 10.1128/jvi.01230-24

Figure Lengend Snippet: Huh-7 and NCI-H522 cells allow for ACE2-independent entry of mutant E484D. ( A ) ACE2 dependence of host cell entry. The indicated cell lines were grown in 96-well plates, incubated with ACE2 antibody (10108-MM36, Sino Biological) for 30 min and inoculated with pseudotypes bearing the indicated viral glycoproteins. Luciferase activity in cell lysates was quantified at 16–20 h postinoculation. The average of three independent experiments ± SEM is shown. Data were normalized against the assay background (i.e., particles bearing no glycoprotein, set as 1). Statistical significance was assessed by two-tailed Student’s t -test with Welch’s correction ( P > 0.05, not significant [ns]; P ≤ 0.05, *; P ≤ 0.01, **; P ≤ 0.001, ***; not determined [nd]). ( B ) Mutation E484D does not allow for S protein binding to ACE2 in complex with an entry-inhibiting antibody. The indicated S proteins were transiently expressed in 293T cells and the cells incubated with soluble ACE2 preincubated with the indicated concentrations of anti-ACE2 antibody. ACE2 binding was detected by incubation with a secondary antibody and the cells analyzed by flow cytometry. Soluble ACE2 binding to cells transfected with empty plasmid served as control. The average ±SEM of three biological replicates conducted with unicate samples is shown. Data were normalized against the assay background (i.e., cells incubated with secondary antibody alone). Statistical significance was assessed by two-tailed Student’s t -test with Welch’s correction ( P > 0.05, not significant [ns]; P ≤ 0.05, *; P ≤ 0.01, **).

Article Snippet: Cells incubated with medium without antibody served as controls. (iii) In order to determine ACE2-independence following directed expression of ASGR1 or DC-SIGN, 293T cells transfected to express ASGR1, DC-SIGN, or empty plasmid were incubated with anti-ACE2 antibody at 7 µg/mL for 30 min and subsequently inoculated with pseudotypes bearing SARS-2-S WT or mutant E484D. (iv) In order to determine imdevimab resistance following directed expression of ASGR1 or DC-SIGN, pseudotype particles were incubated with imdevimab at 1 µg/mL for 30 min and subsequently added to 293T cells transfected to express ASGR1, DC-SIGN, or empty plasmid. (v) In order to determine the contribution of ASGR1 (and related proteins) to ACE2-independent entry into Huh-7 cells, the cells were preincubated with medium containing anti-ACE2 antibody (7 µg/mL, 10108-MM36, Sino Biological), EGTA (5 mM), anti-ACE2 antibody combined with EGTA, or medium only for 1 h followed by inoculation with equal volumes of pseudotypes bearing SARS-2-S WT, SARS-2-S E484D, or NiV F/G.

Techniques: Mutagenesis, Incubation, Luciferase, Activity Assay, Two Tailed Test, Protein Binding, Binding Assay, Flow Cytometry, Transfection, Plasmid Preparation, Control

Journal: Journal of Virology

Article Title: Host cell lectins ASGR1 and DC-SIGN jointly with TMEM106B confer ACE2 independence and imdevimab resistance to SARS-CoV-2 pseudovirus with spike mutation E484D

doi: 10.1128/jvi.01230-24

Figure Lengend Snippet: ACE2-independent entry correlates with resistance to imdevimab and bebtelovimab. Pseudotyped particles were preincubated with the indicated antibodies at the indicated concentrations for 1 h before addition to the indicated target cells. Luciferase activity in cell lysates was quantified at 16–20 h postinoculation. The average of three (two in case of NCI-H522 cells) independent experiments ± SEM is shown. Data were normalized against signals measured in the absence of antibody (= 0% inhibition). Curves were calculated using a non-linear regression model (variable slope).

Article Snippet: Cells incubated with medium without antibody served as controls. (iii) In order to determine ACE2-independence following directed expression of ASGR1 or DC-SIGN, 293T cells transfected to express ASGR1, DC-SIGN, or empty plasmid were incubated with anti-ACE2 antibody at 7 µg/mL for 30 min and subsequently inoculated with pseudotypes bearing SARS-2-S WT or mutant E484D. (iv) In order to determine imdevimab resistance following directed expression of ASGR1 or DC-SIGN, pseudotype particles were incubated with imdevimab at 1 µg/mL for 30 min and subsequently added to 293T cells transfected to express ASGR1, DC-SIGN, or empty plasmid. (v) In order to determine the contribution of ASGR1 (and related proteins) to ACE2-independent entry into Huh-7 cells, the cells were preincubated with medium containing anti-ACE2 antibody (7 µg/mL, 10108-MM36, Sino Biological), EGTA (5 mM), anti-ACE2 antibody combined with EGTA, or medium only for 1 h followed by inoculation with equal volumes of pseudotypes bearing SARS-2-S WT, SARS-2-S E484D, or NiV F/G.

Techniques: Luciferase, Activity Assay, Inhibition

Journal: Journal of Virology

Article Title: Host cell lectins ASGR1 and DC-SIGN jointly with TMEM106B confer ACE2 independence and imdevimab resistance to SARS-CoV-2 pseudovirus with spike mutation E484D

doi: 10.1128/jvi.01230-24

Figure Lengend Snippet: Directed expression of ASGR1/DC-SIGN jointly with TMEM106B allows for efficient entry into otherwise non-susceptible cells. ( A ) 293T cells were transfected to express the indicated soluble S proteins (S1 domain protein of B.1 [WT] and B.1 [E484D] S proteins fused to the Fc portion of human immunoglobulin G), and S protein levels in cell lysates and supernatants were analyzed by immunoblot using anti-Fc antibody. The results of a representative experiment are shown and were confirmed in two additional experiments. ( B ) 293T cells transfected to express ASGR1, DC-SIGN, TMEM106B, or ACE2 were analyzed by immunoblot using anti-c-Myc (DC-SIGN, TMEM106B, and ACE2) and anti-AU1 (ASGR1) antibody. The results of a representative experiment are shown and were confirmed in a separate experiment. ( C ) 293T cells transfected to express ASGR1, DC-SIGN, TMEM106B, or ACE2 were incubated with the indicated soluble S proteins, and S protein binding was analyzed by flow cytometry. Presented are the average (mean) data from three (DC-SIGN, TMEM106B) or six (ASGR1, ACE2) biological replicates (each conducted with single samples). Statistical significance was assessed by two-tailed Student’s t -test with Welch’s correction ( P > 0.05, not significant [ns]; P ≤ 0.05, *; P ≤ 0.01, **). ( D ) BHK-21 cells were transfected with plasmids encoding ASGR1, DC-SIGN, TMEM106B, and ACE2 either alone or in combination and transduced with SARS-2-S pp and E484D pp followed by quantification of luciferase activity in cell lysates. Presented are the average (mean) data from three biological replicates (each conducted with four technical replicates), for which transduction was normalized against signals obtained from control-transfected cells inoculated with the respective pseudotyped particles (background, set as 1). Statistical significance was assessed by two-tailed Student’s t -test with Welch’s correction ( P > 0.05, not significant [ns]; P ≤ 0.05, *; P ≤ 0.01, **).

Article Snippet: Cells incubated with medium without antibody served as controls. (iii) In order to determine ACE2-independence following directed expression of ASGR1 or DC-SIGN, 293T cells transfected to express ASGR1, DC-SIGN, or empty plasmid were incubated with anti-ACE2 antibody at 7 µg/mL for 30 min and subsequently inoculated with pseudotypes bearing SARS-2-S WT or mutant E484D. (iv) In order to determine imdevimab resistance following directed expression of ASGR1 or DC-SIGN, pseudotype particles were incubated with imdevimab at 1 µg/mL for 30 min and subsequently added to 293T cells transfected to express ASGR1, DC-SIGN, or empty plasmid. (v) In order to determine the contribution of ASGR1 (and related proteins) to ACE2-independent entry into Huh-7 cells, the cells were preincubated with medium containing anti-ACE2 antibody (7 µg/mL, 10108-MM36, Sino Biological), EGTA (5 mM), anti-ACE2 antibody combined with EGTA, or medium only for 1 h followed by inoculation with equal volumes of pseudotypes bearing SARS-2-S WT, SARS-2-S E484D, or NiV F/G.

Techniques: Expressing, Transfection, Western Blot, Incubation, Protein Binding, Flow Cytometry, Two Tailed Test, Transduction, Luciferase, Activity Assay, Control

Journal: Journal of Virology

Article Title: Host cell lectins ASGR1 and DC-SIGN jointly with TMEM106B confer ACE2 independence and imdevimab resistance to SARS-CoV-2 pseudovirus with spike mutation E484D

doi: 10.1128/jvi.01230-24

Figure Lengend Snippet: Directed expression of DC-SIGN or ASGR1 allows for imdevimab resistance and ACE2 independence of mutant E484D. For analysis of imdevimab resistance (A), 293T cells were transfected with ASGR1 or DC-SIGN encoding plasmids or empty plasmid as control and inoculated with pseudotypes that harbored the indicated S proteins and were pre-incubated with 1 µg/mL of imdevimab. Luciferase activities in cell lysates were quantified at 16–20 h postinoculation. For analysis of ACE2-independent entry (B), the experiment was conducted as described above, but cells were preincubated with 7 µg/mL anti-ACE2 antibody. Presented are the average (mean) data from three biological replicates (each conducted with four technical replicates), for which transduction was normalized against samples that did not contain antibody (= 100% pseudotype entry). Error bars indicate the SEM. Statistical significance was assessed by two-tailed Student’s t -test with Welch’s correction ( P > 0.05, not significant [ns]; P ≤ 0.05, *; P ≤ 0.01, **; P ≤ 0.001, ***).

Article Snippet: Cells incubated with medium without antibody served as controls. (iii) In order to determine ACE2-independence following directed expression of ASGR1 or DC-SIGN, 293T cells transfected to express ASGR1, DC-SIGN, or empty plasmid were incubated with anti-ACE2 antibody at 7 µg/mL for 30 min and subsequently inoculated with pseudotypes bearing SARS-2-S WT or mutant E484D. (iv) In order to determine imdevimab resistance following directed expression of ASGR1 or DC-SIGN, pseudotype particles were incubated with imdevimab at 1 µg/mL for 30 min and subsequently added to 293T cells transfected to express ASGR1, DC-SIGN, or empty plasmid. (v) In order to determine the contribution of ASGR1 (and related proteins) to ACE2-independent entry into Huh-7 cells, the cells were preincubated with medium containing anti-ACE2 antibody (7 µg/mL, 10108-MM36, Sino Biological), EGTA (5 mM), anti-ACE2 antibody combined with EGTA, or medium only for 1 h followed by inoculation with equal volumes of pseudotypes bearing SARS-2-S WT, SARS-2-S E484D, or NiV F/G.

Techniques: Expressing, Mutagenesis, Transfection, Plasmid Preparation, Control, Incubation, Luciferase, Transduction, Two Tailed Test

Journal: Journal of Virology

Article Title: Host cell lectins ASGR1 and DC-SIGN jointly with TMEM106B confer ACE2 independence and imdevimab resistance to SARS-CoV-2 pseudovirus with spike mutation E484D

doi: 10.1128/jvi.01230-24

Figure Lengend Snippet: Endogenous expression of TMEM106B is required for DC-SIGN-dependent imdevimab resistance and ACE2 independence of mutant E484D. For analysis of ACE2-independent entry (A), 293T control (EV) or TMEM106B KO cells were transiently transfected with EV (control), or DC-SIGN plasmid or cotransfected with DC-SIGN and TMEM106B plasmids, incubated with 7 µg/mL of anti-ACE2 antibody and inoculated with pseudotypes bearing the indicated S proteins followed by quantification of luciferase activities in cell lysates at 16–20 h postinoculation. For analysis of imdevimab resistance (B), the experiment was conducted as described above, but pseudotyped particles were preincubated with 1 µg/mL imdevimab. Presented are the results from a representative experiment carried out with four technical replicates; transduction was normalized against samples that did not contain antibody. Error bars indicate SD, similar results were obtained in a separate experiment. Statistical significance was assessed by two-tailed Student’s t -test with Welch’s correction ( P > 0.05, not significant [ns]; P ≤ 0.05, *; P ≤ 0.01, **; P ≤ 0.001, ***).

Article Snippet: Cells incubated with medium without antibody served as controls. (iii) In order to determine ACE2-independence following directed expression of ASGR1 or DC-SIGN, 293T cells transfected to express ASGR1, DC-SIGN, or empty plasmid were incubated with anti-ACE2 antibody at 7 µg/mL for 30 min and subsequently inoculated with pseudotypes bearing SARS-2-S WT or mutant E484D. (iv) In order to determine imdevimab resistance following directed expression of ASGR1 or DC-SIGN, pseudotype particles were incubated with imdevimab at 1 µg/mL for 30 min and subsequently added to 293T cells transfected to express ASGR1, DC-SIGN, or empty plasmid. (v) In order to determine the contribution of ASGR1 (and related proteins) to ACE2-independent entry into Huh-7 cells, the cells were preincubated with medium containing anti-ACE2 antibody (7 µg/mL, 10108-MM36, Sino Biological), EGTA (5 mM), anti-ACE2 antibody combined with EGTA, or medium only for 1 h followed by inoculation with equal volumes of pseudotypes bearing SARS-2-S WT, SARS-2-S E484D, or NiV F/G.

Techniques: Expressing, Mutagenesis, Control, Transfection, Plasmid Preparation, Incubation, Luciferase, Transduction, Two Tailed Test

Journal: Journal of Virology

Article Title: Host cell lectins ASGR1 and DC-SIGN jointly with TMEM106B confer ACE2 independence and imdevimab resistance to SARS-CoV-2 pseudovirus with spike mutation E484D

doi: 10.1128/jvi.01230-24

Figure Lengend Snippet: EGTA blocks ACE2-independent entry into Huh-7 cells. ( A ) Expression and N-glycosylation of ASGR1 in Huh-7 cells. Untransfected Huh-7 cells and 293T and BHK21 cells transfected with EV or ASGR1 encoding vector were control treated or treated with PNGaseF and analyzed for ASGR1 expression by immunoblot. Expression of β-actin served as loading control. Similar results were obtained in two (all cell lines) to four (293T, BHK21 cells) independent experiments. ( B ) Huh-7 cells were preincubated with medium alone (control) or medium containing anti-ACE2 antibody, EGTA, or anti-ACE2 antibody combined with EGTA for 1 h followed by inoculation with pseudotypes bearing the indicated S proteins. Luciferase activity in cell lysates was quantified at 16–20 h postinoculation. The results ± SD of a single experiment performed with technical quadruplicates are shown. Similar results were obtained in a separate experiment.

Article Snippet: Cells incubated with medium without antibody served as controls. (iii) In order to determine ACE2-independence following directed expression of ASGR1 or DC-SIGN, 293T cells transfected to express ASGR1, DC-SIGN, or empty plasmid were incubated with anti-ACE2 antibody at 7 µg/mL for 30 min and subsequently inoculated with pseudotypes bearing SARS-2-S WT or mutant E484D. (iv) In order to determine imdevimab resistance following directed expression of ASGR1 or DC-SIGN, pseudotype particles were incubated with imdevimab at 1 µg/mL for 30 min and subsequently added to 293T cells transfected to express ASGR1, DC-SIGN, or empty plasmid. (v) In order to determine the contribution of ASGR1 (and related proteins) to ACE2-independent entry into Huh-7 cells, the cells were preincubated with medium containing anti-ACE2 antibody (7 µg/mL, 10108-MM36, Sino Biological), EGTA (5 mM), anti-ACE2 antibody combined with EGTA, or medium only for 1 h followed by inoculation with equal volumes of pseudotypes bearing SARS-2-S WT, SARS-2-S E484D, or NiV F/G.

Techniques: Expressing, Transfection, Plasmid Preparation, Control, Western Blot, Luciferase, Activity Assay

Journal: Journal of Virology

Article Title: Host cell lectins ASGR1 and DC-SIGN jointly with TMEM106B confer ACE2 independence and imdevimab resistance to SARS-CoV-2 pseudovirus with spike mutation E484D

doi: 10.1128/jvi.01230-24

Figure Lengend Snippet: Huh-7 and NCI-H522 cells allow for ACE2-independent entry of mutant E484D. ( A ) ACE2 dependence of host cell entry. The indicated cell lines were grown in 96-well plates, incubated with ACE2 antibody (10108-MM36, Sino Biological) for 30 min and inoculated with pseudotypes bearing the indicated viral glycoproteins. Luciferase activity in cell lysates was quantified at 16–20 h postinoculation. The average of three independent experiments ± SEM is shown. Data were normalized against the assay background (i.e., particles bearing no glycoprotein, set as 1). Statistical significance was assessed by two-tailed Student’s t -test with Welch’s correction ( P > 0.05, not significant [ns]; P ≤ 0.05, *; P ≤ 0.01, **; P ≤ 0.001, ***; not determined [nd]). ( B ) Mutation E484D does not allow for S protein binding to ACE2 in complex with an entry-inhibiting antibody. The indicated S proteins were transiently expressed in 293T cells and the cells incubated with soluble ACE2 preincubated with the indicated concentrations of anti-ACE2 antibody. ACE2 binding was detected by incubation with a secondary antibody and the cells analyzed by flow cytometry. Soluble ACE2 binding to cells transfected with empty plasmid served as control. The average ±SEM of three biological replicates conducted with unicate samples is shown. Data were normalized against the assay background (i.e., cells incubated with secondary antibody alone). Statistical significance was assessed by two-tailed Student’s t -test with Welch’s correction ( P > 0.05, not significant [ns]; P ≤ 0.05, *; P ≤ 0.01, **).

Article Snippet: Particles bearing EBOV-GP, VSV-G, or no viral surface protein served as controls. (ii) In order to assess ACE2-independent entry into cell lines, Huh-7, Li-7, and NCI-H522 cells were preincubated (30 min, 37°C) with medium containing 7 µg/mL of anti-ACE2 neutralizing mouse monoclonal antibody (10108-MM36, Sino Biological), before addition of equal volumes of pseudotyped particles.

Techniques: Mutagenesis, Incubation, Luciferase, Activity Assay, Two Tailed Test, Protein Binding, Binding Assay, Flow Cytometry, Transfection, Plasmid Preparation, Control

Journal: Journal of Virology

Article Title: Host cell lectins ASGR1 and DC-SIGN jointly with TMEM106B confer ACE2 independence and imdevimab resistance to SARS-CoV-2 pseudovirus with spike mutation E484D

doi: 10.1128/jvi.01230-24

Figure Lengend Snippet: ACE2-independent entry correlates with resistance to imdevimab and bebtelovimab. Pseudotyped particles were preincubated with the indicated antibodies at the indicated concentrations for 1 h before addition to the indicated target cells. Luciferase activity in cell lysates was quantified at 16–20 h postinoculation. The average of three (two in case of NCI-H522 cells) independent experiments ± SEM is shown. Data were normalized against signals measured in the absence of antibody (= 0% inhibition). Curves were calculated using a non-linear regression model (variable slope).

Article Snippet: Particles bearing EBOV-GP, VSV-G, or no viral surface protein served as controls. (ii) In order to assess ACE2-independent entry into cell lines, Huh-7, Li-7, and NCI-H522 cells were preincubated (30 min, 37°C) with medium containing 7 µg/mL of anti-ACE2 neutralizing mouse monoclonal antibody (10108-MM36, Sino Biological), before addition of equal volumes of pseudotyped particles.

Techniques: Luciferase, Activity Assay, Inhibition

Journal: Journal of Virology

Article Title: Host cell lectins ASGR1 and DC-SIGN jointly with TMEM106B confer ACE2 independence and imdevimab resistance to SARS-CoV-2 pseudovirus with spike mutation E484D

doi: 10.1128/jvi.01230-24

Figure Lengend Snippet: Directed expression of ASGR1/DC-SIGN jointly with TMEM106B allows for efficient entry into otherwise non-susceptible cells. ( A ) 293T cells were transfected to express the indicated soluble S proteins (S1 domain protein of B.1 [WT] and B.1 [E484D] S proteins fused to the Fc portion of human immunoglobulin G), and S protein levels in cell lysates and supernatants were analyzed by immunoblot using anti-Fc antibody. The results of a representative experiment are shown and were confirmed in two additional experiments. ( B ) 293T cells transfected to express ASGR1, DC-SIGN, TMEM106B, or ACE2 were analyzed by immunoblot using anti-c-Myc (DC-SIGN, TMEM106B, and ACE2) and anti-AU1 (ASGR1) antibody. The results of a representative experiment are shown and were confirmed in a separate experiment. ( C ) 293T cells transfected to express ASGR1, DC-SIGN, TMEM106B, or ACE2 were incubated with the indicated soluble S proteins, and S protein binding was analyzed by flow cytometry. Presented are the average (mean) data from three (DC-SIGN, TMEM106B) or six (ASGR1, ACE2) biological replicates (each conducted with single samples). Statistical significance was assessed by two-tailed Student’s t -test with Welch’s correction ( P > 0.05, not significant [ns]; P ≤ 0.05, *; P ≤ 0.01, **). ( D ) BHK-21 cells were transfected with plasmids encoding ASGR1, DC-SIGN, TMEM106B, and ACE2 either alone or in combination and transduced with SARS-2-S pp and E484D pp followed by quantification of luciferase activity in cell lysates. Presented are the average (mean) data from three biological replicates (each conducted with four technical replicates), for which transduction was normalized against signals obtained from control-transfected cells inoculated with the respective pseudotyped particles (background, set as 1). Statistical significance was assessed by two-tailed Student’s t -test with Welch’s correction ( P > 0.05, not significant [ns]; P ≤ 0.05, *; P ≤ 0.01, **).

Article Snippet: Particles bearing EBOV-GP, VSV-G, or no viral surface protein served as controls. (ii) In order to assess ACE2-independent entry into cell lines, Huh-7, Li-7, and NCI-H522 cells were preincubated (30 min, 37°C) with medium containing 7 µg/mL of anti-ACE2 neutralizing mouse monoclonal antibody (10108-MM36, Sino Biological), before addition of equal volumes of pseudotyped particles.

Techniques: Expressing, Transfection, Western Blot, Incubation, Protein Binding, Flow Cytometry, Two Tailed Test, Transduction, Luciferase, Activity Assay, Control

Journal: Journal of Virology

Article Title: Host cell lectins ASGR1 and DC-SIGN jointly with TMEM106B confer ACE2 independence and imdevimab resistance to SARS-CoV-2 pseudovirus with spike mutation E484D

doi: 10.1128/jvi.01230-24

Figure Lengend Snippet: Directed expression of DC-SIGN or ASGR1 allows for imdevimab resistance and ACE2 independence of mutant E484D. For analysis of imdevimab resistance (A), 293T cells were transfected with ASGR1 or DC-SIGN encoding plasmids or empty plasmid as control and inoculated with pseudotypes that harbored the indicated S proteins and were pre-incubated with 1 µg/mL of imdevimab. Luciferase activities in cell lysates were quantified at 16–20 h postinoculation. For analysis of ACE2-independent entry (B), the experiment was conducted as described above, but cells were preincubated with 7 µg/mL anti-ACE2 antibody. Presented are the average (mean) data from three biological replicates (each conducted with four technical replicates), for which transduction was normalized against samples that did not contain antibody (= 100% pseudotype entry). Error bars indicate the SEM. Statistical significance was assessed by two-tailed Student’s t -test with Welch’s correction ( P > 0.05, not significant [ns]; P ≤ 0.05, *; P ≤ 0.01, **; P ≤ 0.001, ***).

Article Snippet: Particles bearing EBOV-GP, VSV-G, or no viral surface protein served as controls. (ii) In order to assess ACE2-independent entry into cell lines, Huh-7, Li-7, and NCI-H522 cells were preincubated (30 min, 37°C) with medium containing 7 µg/mL of anti-ACE2 neutralizing mouse monoclonal antibody (10108-MM36, Sino Biological), before addition of equal volumes of pseudotyped particles.

Techniques: Expressing, Mutagenesis, Transfection, Plasmid Preparation, Control, Incubation, Luciferase, Transduction, Two Tailed Test

Journal: Journal of Virology

Article Title: Host cell lectins ASGR1 and DC-SIGN jointly with TMEM106B confer ACE2 independence and imdevimab resistance to SARS-CoV-2 pseudovirus with spike mutation E484D

doi: 10.1128/jvi.01230-24

Figure Lengend Snippet: Endogenous expression of TMEM106B is required for DC-SIGN-dependent imdevimab resistance and ACE2 independence of mutant E484D. For analysis of ACE2-independent entry (A), 293T control (EV) or TMEM106B KO cells were transiently transfected with EV (control), or DC-SIGN plasmid or cotransfected with DC-SIGN and TMEM106B plasmids, incubated with 7 µg/mL of anti-ACE2 antibody and inoculated with pseudotypes bearing the indicated S proteins followed by quantification of luciferase activities in cell lysates at 16–20 h postinoculation. For analysis of imdevimab resistance (B), the experiment was conducted as described above, but pseudotyped particles were preincubated with 1 µg/mL imdevimab. Presented are the results from a representative experiment carried out with four technical replicates; transduction was normalized against samples that did not contain antibody. Error bars indicate SD, similar results were obtained in a separate experiment. Statistical significance was assessed by two-tailed Student’s t -test with Welch’s correction ( P > 0.05, not significant [ns]; P ≤ 0.05, *; P ≤ 0.01, **; P ≤ 0.001, ***).

Article Snippet: Particles bearing EBOV-GP, VSV-G, or no viral surface protein served as controls. (ii) In order to assess ACE2-independent entry into cell lines, Huh-7, Li-7, and NCI-H522 cells were preincubated (30 min, 37°C) with medium containing 7 µg/mL of anti-ACE2 neutralizing mouse monoclonal antibody (10108-MM36, Sino Biological), before addition of equal volumes of pseudotyped particles.

Techniques: Expressing, Mutagenesis, Control, Transfection, Plasmid Preparation, Incubation, Luciferase, Transduction, Two Tailed Test

Journal: Journal of Virology

Article Title: Host cell lectins ASGR1 and DC-SIGN jointly with TMEM106B confer ACE2 independence and imdevimab resistance to SARS-CoV-2 pseudovirus with spike mutation E484D

doi: 10.1128/jvi.01230-24

Figure Lengend Snippet: EGTA blocks ACE2-independent entry into Huh-7 cells. ( A ) Expression and N-glycosylation of ASGR1 in Huh-7 cells. Untransfected Huh-7 cells and 293T and BHK21 cells transfected with EV or ASGR1 encoding vector were control treated or treated with PNGaseF and analyzed for ASGR1 expression by immunoblot. Expression of β-actin served as loading control. Similar results were obtained in two (all cell lines) to four (293T, BHK21 cells) independent experiments. ( B ) Huh-7 cells were preincubated with medium alone (control) or medium containing anti-ACE2 antibody, EGTA, or anti-ACE2 antibody combined with EGTA for 1 h followed by inoculation with pseudotypes bearing the indicated S proteins. Luciferase activity in cell lysates was quantified at 16–20 h postinoculation. The results ± SD of a single experiment performed with technical quadruplicates are shown. Similar results were obtained in a separate experiment.

Article Snippet: Particles bearing EBOV-GP, VSV-G, or no viral surface protein served as controls. (ii) In order to assess ACE2-independent entry into cell lines, Huh-7, Li-7, and NCI-H522 cells were preincubated (30 min, 37°C) with medium containing 7 µg/mL of anti-ACE2 neutralizing mouse monoclonal antibody (10108-MM36, Sino Biological), before addition of equal volumes of pseudotyped particles.

Techniques: Expressing, Transfection, Plasmid Preparation, Control, Western Blot, Luciferase, Activity Assay

Journal: PLOS Pathogens

Article Title: ACE2-independent sarbecovirus cell entry can be supported by TMPRSS2-related enzymes and can reduce sensitivity to antibody-mediated neutralization

doi: 10.1371/journal.ppat.1012653

Figure Lengend Snippet: A ) Phylogenetic analysis of human and animal sarbecoviruses. The sarbecoviruses were grouped into five clades, indicated by different colors, based on the full spike sequences. The sarbecoviruses functionally analyzed in the present study are indicated in grey boxes. (See for more details). B ) Structure of RBD. The structure of RBDs was predicted based on homology modeling using SARS-2-S RBD as template. Two loops involved in ACE2 interactions are highlighted (See for more details), RBD-based clades are indicated. C ) Schematic overview of the spike (S) protein domain structure (upper panel) and alignment of the RBM sequences of the S proteins analyzed in panel A. The ACE2 interacting residues of SARS-1-S and SARS-2-S are marked in blue (lower panel). “*” indicates conserved amino acid residues, “-”indicates gaps. The S proteins under study are indicated by circles. Abbreviations: NTD = N-terminal domain; RBD = receptor-binding domain; TD = transmembrane domain; S1/S2 and S2’ = cleavage sites in the S protein.

Article Snippet: To determine whether ACE2 was required for entry, Vero-TMPRSS2 cells were incubated with recombinant anti-ACE2 neutralizing antibody (Sino Biologics, Cat: 10108-MM36) for 30 minutes prior to inoculation with pseudotyped particles.

Techniques: Binding Assay

Journal: PLOS Pathogens

Article Title: ACE2-independent sarbecovirus cell entry can be supported by TMPRSS2-related enzymes and can reduce sensitivity to antibody-mediated neutralization

doi: 10.1371/journal.ppat.1012653

Figure Lengend Snippet: A ) Binding of soluble human ACE2 to S protein expressing cells. 293T cells transiently expressing the indicated S proteins (or no S protein) were first incubated with soluble ACE2 containing a C-terminal Fc-tag (derived from human immunoglobulin G; solACE2-Fc) and subsequently incubated with an AlexaFluor-488-coupled secondary antibody, before solACE2-Fc binding was analyzed by flow cytometry (see for details on the gating strategy). Presented are the average (mean) mean fluorescence intensity (MFI) data from five biological replicates (each conducted with single samples). Signals obtained from control transfected cells (no S protein expression) that were incubated with solACE2-Fc and secondary antibody were used to determine the background (grey area). Error bars indicate the standard error of the mean (SEM). Statistical significance was assessed by two-tailed Student’s t-tests (p > 0.05, not significant [ns]; p ≤ 0.05, *; p ≤ 0.01, **; p ≤ 0.001, ***). B ) Receptor activity of ACE2 orthologues. BHK-21 cells transiently expressing the indicated ACE2 orthologues (or empty vector) were inoculated with pseudotyped particles bearing the indicated S proteins (or no S protein). S-protein driven cell entry was analyzed by measuring the activity of virus-encoded firefly luciferase in the cell lysate at 16-18h post inoculation. Presented are the average (mean) data from three biological replicates (each conducted with four technical replicates) in which cell entry was normalized against that measured for particles bearing no S protein (set as 1). Error bars show the SEM. Statistical significance was assessed by two-tailed Student’s t-tests (p > 0.05, not significant [ns]; p ≤ 0.05, *; p ≤ 0.01, **; p ≤ 0.001, ***). C) Heat map for the data presented in panel B. Entry into cells expressing ACE2 orthologues was normalized against entry into cells expressing human ACE2 (set as 1).

Article Snippet: To determine whether ACE2 was required for entry, Vero-TMPRSS2 cells were incubated with recombinant anti-ACE2 neutralizing antibody (Sino Biologics, Cat: 10108-MM36) for 30 minutes prior to inoculation with pseudotyped particles.

Techniques: Binding Assay, Expressing, Incubation, Derivative Assay, Flow Cytometry, Fluorescence, Control, Transfection, Two Tailed Test, Activity Assay, Plasmid Preparation, Virus, Luciferase

Journal: PLOS Pathogens

Article Title: ACE2-independent sarbecovirus cell entry can be supported by TMPRSS2-related enzymes and can reduce sensitivity to antibody-mediated neutralization

doi: 10.1371/journal.ppat.1012653

Figure Lengend Snippet: A ) S protein driven cell entry in the presence and absence of trypsin. Particles bearing the indicated S proteins (or no S protein) were preincubated with or without trypsin (50 μg/ml for 30 min at 37°C) before being added to the respective cell lines. S-protein driven cell entry was analyzed by measuring the activity of virus-encoded firefly luciferase in the cell lysate at 16-18h post inoculation. Presented are the average (mean) data from three biological replicates (each conducted with four technical replicates) in which cell entry was normalized against that measured for particles bearing no S protein (set as 1). Error bars show the SEM. Statistical significance was assessed by two-tailed Student’s t-tests (p > 0.05, not significant [ns]; p ≤ 0.05, *; p ≤ 0.01, **; p ≤ 0.001, ***). B ) Trypsin treatment of viral particles but not target cells promotes entry. Vero cells or pseudotyped particles bearing the indicated S proteins were pre-incubated with trypsin (50 μg/ml for 30 min at 37°C) and subsequently trypsin inhibitor (200 μg/ml for 10 min at 37°C) as indicated. The pseudotyped particles were added to the cells. S-protein-driven cell entry was analyzed and data presented as described for panel A. Presented are the average (mean) data of three biological replicates, each performed with four technical replicates. Error bars show SEM. Statistical significance was assessed by two-tailed Student’s t-tests (p > 0.05, not significant [ns]; p ≤ 0.05, *; p ≤ 0.01, **; p ≤ 0.001, ***). C ) Multiple bat sarbecovirus spike proteins can employ an ACE2-independent entry pathway following exposure to trypsin. Particles bearing the indicated S proteins were incubated with trypsin (50 μg/ml for 30 min at 37°C) before addition onto 293T wildtype (293T WT) and 293T ACE2 knockout cells (293T KO-ACE2). S-protein-driven cell entry was analyzed and data presented as described for panel A. Presented are the average (mean) data of three biological replicates, each performed with four technical replicates. Error bars show SEM. Statistical significance was assessed by two-tailed Student’s t-tests (p > 0.05, not significant [ns]; p ≤ 0.05, *; p ≤ 0.01, **; p ≤ 0.001, ***). Of note, entry of particles bearing VSV-G in the presence of trypsin has not been analyzed (n.t. = not tested).

Article Snippet: To determine whether ACE2 was required for entry, Vero-TMPRSS2 cells were incubated with recombinant anti-ACE2 neutralizing antibody (Sino Biologics, Cat: 10108-MM36) for 30 minutes prior to inoculation with pseudotyped particles.

Techniques: Activity Assay, Virus, Luciferase, Two Tailed Test, Incubation, Knock-Out

Journal: PLOS Pathogens

Article Title: ACE2-independent sarbecovirus cell entry can be supported by TMPRSS2-related enzymes and can reduce sensitivity to antibody-mediated neutralization

doi: 10.1371/journal.ppat.1012653

Figure Lengend Snippet: A ) Alignment of the S1/S2 loop sequences of the indicated S proteins. Amino acid residues were color coded on the basis of biochemical properties. Asterisks indicate conserved residues. B ) Analysis of S protein cleavage. Particles pseudotyped with the indicated S proteins were subjected to immunoblot analysis, using an antibody directed against the S2 subunit of SARS-2-S. Black and red indicate uncleaved precursor respective S (S0) and S2, respectively. Detection of VSV-M served as a loading control. Shown is a representative immunoblot from three independent experiments. C ) Impact of the multibasic cleavage site on S protein-driven entry. Particles bearing the indicated S proteins (or no S protein) were added to 293T-ACE2 or Calu-3-ACE2 cells. The efficiency of S protein-driven cell entry was determined by measuring the activity of virus-encoded firefly luciferase in cell lysates at 16-18h post inoculation. Results for S protein bearing particles were normalized against those obtained for particles bearing no S protein (set as 1). Presented are the average (mean) data of three biological replicates, each performed with four technical replicates. Error bars indicate SEM. Statistical significance was assessed by two-tailed Student’s t-tests (p > 0.05, not significant [ns]; p ≤ 0.05, *; p ≤ 0.01, **; p ≤ 0.001, ***).

Article Snippet: To determine whether ACE2 was required for entry, Vero-TMPRSS2 cells were incubated with recombinant anti-ACE2 neutralizing antibody (Sino Biologics, Cat: 10108-MM36) for 30 minutes prior to inoculation with pseudotyped particles.

Techniques: Western Blot, Control, Activity Assay, Virus, Luciferase, Two Tailed Test

Journal: PLOS Pathogens

Article Title: ACE2-independent sarbecovirus cell entry can be supported by TMPRSS2-related enzymes and can reduce sensitivity to antibody-mediated neutralization

doi: 10.1371/journal.ppat.1012653

Figure Lengend Snippet: Particles bearing the indicated S proteins were preincubated with or without trypsin (50 μg/ml for 30 min at 37°C) and subsequently trypsin inhibitor (200 μg/ml for 10 min at 37°C). Thereafter, the particles were incubated with different concentrations of the pan-sarbecovirus monoclonal antibody S2H97 (30 min at 37°C) before being added to Vero-ACE2-TMPRSS2 cells. S protein-driven cell entry was analyzed by measuring the activity of virus-encoded firefly luciferase in cell lysates at 16-18h post inoculation and normalized to entry in the absence of antibody. Presented are the average (mean) data of three biological replicates, each performed with four technical replicates. Error bars indicate SEM. Statistical significance was assessed by two-way analysis of variance with Sidak’s multiple comparisons test (p > 0.05, not significant [ns]; p ≤ 0.05, *; p ≤ 0.01, **; p ≤ 0.001, ***).

Article Snippet: To determine whether ACE2 was required for entry, Vero-TMPRSS2 cells were incubated with recombinant anti-ACE2 neutralizing antibody (Sino Biologics, Cat: 10108-MM36) for 30 minutes prior to inoculation with pseudotyped particles.

Techniques: Incubation, Activity Assay, Virus, Luciferase

Journal: PLOS Pathogens

Article Title: ACE2-independent sarbecovirus cell entry can be supported by TMPRSS2-related enzymes and can reduce sensitivity to antibody-mediated neutralization

doi: 10.1371/journal.ppat.1012653

Figure Lengend Snippet: A ) Particles bearing the indicated S proteins were preincubated with a 1:25 dilution of plasma from convalescent patients, individuals vaccinated two times with BNT162b2 (BNT/BNT), three times with ChAdOx1-S and BNT162b2 (AZ/BNT/BNT), and four times, including a bivalent, BA.5-adapted booster, before being added to A549-ACE2-TMPRSS2 cells. S protein-driven cell entry was analyzed by measuring the activity of virus-encoded firefly luciferase in cell lysates at 16-18h post inoculation and normalized to entry of in the absence of plasma. Presented are the combined data for 9–10 plasma per group. Please see for detailed information on the plasma samples. B ) Particles bearing the indicated S proteins were preincubated with or without trypsin (50 μg/ml for 30 min at 37°C) and subsequently trypsin inhibitor (200 μg/ml for 10 min at 37°C). Subsequently, the particles were incubated with a fixed 1:25 dilution of plasma from quadruple vaccinated donors (30 min at 37°C) and added to Vero-ACE2-TMPRSS2 cells. S protein-driven cell entry was analyzed by measuring the activity of virus-encoded firefly luciferase in cell lysates at 16-18h post inoculation and normalized to entry of in the absence of plasma. Presented are the combined data for 10 plasma (three technical replicates). Statistical significance was assessed by Mann-Whitney test (p > 0.05, not significant [ns]; p ≤ 0.05, *; p ≤ 0.01, **; p ≤ 0.001, ***). Please see for detailed information on the plasma samples tested.

Article Snippet: To determine whether ACE2 was required for entry, Vero-TMPRSS2 cells were incubated with recombinant anti-ACE2 neutralizing antibody (Sino Biologics, Cat: 10108-MM36) for 30 minutes prior to inoculation with pseudotyped particles.

Techniques: Activity Assay, Virus, Luciferase, Incubation, MANN-WHITNEY