Journal: Journal of Virology

Article Title: Variations in Cell Surface ACE2 Levels Alter Direct Binding of SARS-CoV-2 Spike Protein and Viral Infectivity: Implications for Measuring Spike Protein Interactions with Animal ACE2 Orthologs

doi: 10.1128/jvi.00256-22

Figure Lengend Snippet: Recombinant SARS-CoV-2 RBD protein binds to human ACE2 and facilitates pseudoviral infectivity. (A) Schematic of the bicistronic mRNA (3,537 bp) for the production of human ACE2 (2,415 bp; 805 amino acids [aa]) and the reporter cell surface protein mouse Thy1.1 (489 bp; 163 aa). (B) HRT-18G cells were stably transfected with a plasmid containing cDNA of hACE2 and magnetically sorted for Thy1.1 expression. HRT-18G/hACE2 (blue trace) and parental HRT-18G (shaded histogram) cells were stained with fluorescent anti-Thy1.1 antibody, and expression was analyzed by flow cytometry. (C) HRT-18G cells stably expressing ACE2 (blue trace) and parent cells (shaded histogram) were incubated with fluorescent anti-ACE2 antibody and analyzed by flow cytometry. (D) HRT-18G parental cells (shaded histogram) and HRT-18G/hACE2 cells (blue trace) were incubated with the Alexa Fluor 647-labeled SARS-CoV-2 S-protein RBD and analyzed for protein binding by flow cytometry. (E) Same as panel D except that cells were incubated with a range of concentrations of the Alexa Fluor 647-labeled SARS-CoV-2 S-protein RBD and analyzed by flow cytometry. (F) HRT-18G/hACE2 and HRT-18G cells were infected with GFP-expressing SARS-CoV-2 pseudovirus at an MOI of 10 for the indicated times, washed to remove excess virus, and incubated at 37°C overnight. Infectivity was analyzed by flow cytometry 16 h later, and the percentage of GFP-positive (GFP + ) cells is reported. (G) HRT-18G/hACE2 and parent cells were infected with a GFP-expressing SARS-CoV-2 pseudovirus at an MOI of 0.5 and cultured for 36 h prior to analysis by fluorescence microscopy. Cells were stained with DAPI to delineate the nucleus. All data presented are representative of results from three independent experiments. Statistical significance is indicated (***, P < 0.0001). MFI, mean fluorescence intensity.

Article Snippet: The following primary antibodies were used for Western blot analysis: mouse monoclonal β-actin (clone AC-15; Sigma), rabbit polyclonal ACE2 (catalog number 21115-1-AP; Thermo Fisher), mouse monoclonal E-cadherin (clone 4A2C7; Life Technologies), and rabbit polyclonal LC3B (clone D11; Cell Signaling).

Techniques: Recombinant, Infection, Stable Transfection, Transfection, Plasmid Preparation, Expressing, Staining, Flow Cytometry, Incubation, Labeling, Protein Binding, Virus, Cell Culture, Fluorescence, Microscopy

Journal: Journal of Virology

Article Title: Variations in Cell Surface ACE2 Levels Alter Direct Binding of SARS-CoV-2 Spike Protein and Viral Infectivity: Implications for Measuring Spike Protein Interactions with Animal ACE2 Orthologs

doi: 10.1128/jvi.00256-22

Figure Lengend Snippet: Increased ACE2 expression potentiates SARS-CoV-2 RBD binding and pseudoviral infectivity. (A) HRT-18G cells were transiently transfected with cDNA encoding hACE2 and Thy1.1 in a bicistronic cassette, and 2 days later, they were stained with fluorescently labeled anti-Thy1.1 and -ACE2 antibodies and analyzed by flow cytometry. Single-antibody labeling controls are also depicted. (B) Cells were stained with fluorescently labeled anti-Thy1.1 antibody and the Alexa Fluor 647-labeled SARS-CoV-2 S-protein RBD and analyzed by flow cytometry. Histograms of individual protein-labeled cells are also depicted. (C to E) HRT-18G/hACE2 cells (blue traces) were fluorescently sorted based on Thy1.1 expression to generate the cell line HRT-18G/hACE2++ (orange traces). HRT-18G/hACE2 (H) and HRT-18G/hACE2++ (H ++ ) cells and parental HRT-18G cells (P) (shaded histograms) were then incubated with anti-Thy1.1 (C) or anti-ACE2 (D) antibody or the Alexa Fluor 647-labeled SARS-CoV-2 S-protein RBD (E) and analyzed by flow cytometry. Histograms and quantitative MFI measurements are representative of results from three independent biological replicates. (F) HRT-18G/hACE2 and HRT-18G/hACE2++ cells were infected with GFP-expressing SARS-CoV-2 pseudovirus for the indicated times, washed to remove excess virus, incubated in complete medium for 16 h, and analyzed by flow cytometry for GFP expression. All data presented are representative of results from three independent biological experiments. Statistical significance is indicated (***, P < 0.0001).

Article Snippet: The following primary antibodies were used for Western blot analysis: mouse monoclonal β-actin (clone AC-15; Sigma), rabbit polyclonal ACE2 (catalog number 21115-1-AP; Thermo Fisher), mouse monoclonal E-cadherin (clone 4A2C7; Life Technologies), and rabbit polyclonal LC3B (clone D11; Cell Signaling).

Techniques: Expressing, Binding Assay, Infection, Transfection, Staining, Labeling, Flow Cytometry, Antibody Labeling, Incubation, Virus

Journal: Journal of Virology

Article Title: Variations in Cell Surface ACE2 Levels Alter Direct Binding of SARS-CoV-2 Spike Protein and Viral Infectivity: Implications for Measuring Spike Protein Interactions with Animal ACE2 Orthologs

doi: 10.1128/jvi.00256-22

Figure Lengend Snippet: Human (h), feline (f), and mouse (m) ACE2 orthologs are glycosylated, metabolically stable, and detected at the cell surface in stably transfected HRT-18G cells. (A) HRT-18G cells were stably transfected with IRES-Thy1.1 plasmids containing cDNAs of human (blue), feline (orange), and mouse (purple) ACE2 and magnetically sorted until equivalent levels of the reporter protein Thy1.1 were expressed on all three cell lines. Staining of parental HRT-18G cells is shown as a negative control (shaded histogram). (B) RNA was isolated from equal numbers of HRT-18G/hACE2, HRT-18G/fACE2, and HRT-18G/mACE2 cells. cDNAs were made from extracted RNAs of the tested cell lines, and qPCR was performed using GAPDH as a housekeeping gene. C T values from ACE2 amplifications were normalized to C T values from GAPDH, and the average fold changes from three independent experiments are shown. (C) To confirm the specificity of the ACE2 antibody for human ACE2, HRT-18G/hACE2 (blue), HRT-18G/fACE2 (orange), HRT-18G/mACE2 (purple), and HRT-18G (shaded histogram) cells were simultaneously incubated with fluorescently labeled ACE2 antibody and analyzed by flow cytometry. (D) HRT-18G cells expressing the indicated ACE2 orthologs were lysed and either mock treated or deglycosylated by incubation with PNGase F. Cell lysates were then resolved by SDS-PAGE and immunoblotted (IB) with polyclonal antibodies to ACE2 or β-actin. Molecular weight markers are indicated. (E) Cells were cultured with CHX for 0, 5, or 10 h, followed by lysis, SDS-PAGE, and Western blot analysis for either ACE2, β-actin, or LC3B. C, control: HRT-18G cells which do not express any ACE2 ortholog. (F) Cell surface proteins were biotinylated prior to cell lysis. Biotin-conjugated proteins were isolated using a streptavidin agarose slurry and analyzed by Western blotting for ACE2, E-cadherin, and β-actin.

Article Snippet: The following primary antibodies were used for Western blot analysis: mouse monoclonal β-actin (clone AC-15; Sigma), rabbit polyclonal ACE2 (catalog number 21115-1-AP; Thermo Fisher), mouse monoclonal E-cadherin (clone 4A2C7; Life Technologies), and rabbit polyclonal LC3B (clone D11; Cell Signaling).

Techniques: Metabolic Labelling, Stable Transfection, Transfection, Staining, Negative Control, Isolation, Incubation, Labeling, Flow Cytometry, Expressing, SDS Page, Molecular Weight, Cell Culture, Lysis, Western Blot, Control

Journal: Journal of Virology

Article Title: Variations in Cell Surface ACE2 Levels Alter Direct Binding of SARS-CoV-2 Spike Protein and Viral Infectivity: Implications for Measuring Spike Protein Interactions with Animal ACE2 Orthologs

doi: 10.1128/jvi.00256-22

Figure Lengend Snippet: SARS-CoV-2 RBD binding and viral infectivity with different ACE2 orthologs. (A) HRT-18G (shaded histogram), HRT-18G/hACE2 (blue trace), HRT-18G/fACE2 (orange trace), and HRT-18G/mACE2 (purple trace) cell lines were incubated with 7.8 ng of the Alexa Fluor 647-labeled SARS-CoV-2 S-protein RBD and analyzed for SARS-CoV-2 S-protein RBD/ACE2 binding by flow cytometry. (B) Same as panel A except that cells were incubated with different concentrations of the Alexa Fluor 647-labeled RBD. The MFI of the population is reported on the y axis. (C) HRT-18G/hACE2, HRT-18G/fACE2, HRT-18G/mACE2, and HRT-18G cells were infected with GFP-expressing SARS-CoV-2 pseudovirus for the specified times, washed to remove excess virus, and incubated for 16 h, and GFP-expressing cells were quantified by flow cytometry. All data presented are representative of results from three independent biological experiments.

Article Snippet: The following primary antibodies were used for Western blot analysis: mouse monoclonal β-actin (clone AC-15; Sigma), rabbit polyclonal ACE2 (catalog number 21115-1-AP; Thermo Fisher), mouse monoclonal E-cadherin (clone 4A2C7; Life Technologies), and rabbit polyclonal LC3B (clone D11; Cell Signaling).

Techniques: Binding Assay, Infection, Incubation, Labeling, Flow Cytometry, Expressing, Virus