Journal: Viruses

Article Title: Population-Specific ACE2 Single-Nucleotide Polymorphisms Have Limited Impact on SARS-CoV-2 Infectivity In Vitro

doi: 10.3390/v13010067

Figure Lengend Snippet: ACE2 single-nucleotide polymorphisms (SNPs) found in different populations.

Article Snippet: To generate the plasmid (pCMV6-hACE2-FLAG) expressing C-terminally FLAG-tagged wild-type human ACE2 (WT ACE2), a DNA fragment encoding the full-length human ACE2 open reading frame (ORF) was amplified by PCR using a human ACE2-expressing plasmid (HG10108-UT; Sino Biological, Beijing, China) as a template and then inserted in-frame into the pCMV6-Entry vector (OriGene, Rockville, MD, USA) between the SgfI and EcoRV sites connecting the ACE2 ORF and FLAG-tag cDNA sequences.

Techniques:

Journal: Viruses

Article Title: Population-Specific ACE2 Single-Nucleotide Polymorphisms Have Limited Impact on SARS-CoV-2 Infectivity In Vitro

doi: 10.3390/v13010067

Figure Lengend Snippet: Mapping of the amino acid residues corresponding to the selected SNPs used in this study. The positions of the amino acid residues corresponding to the selected SNPs are marked in red on the crystal structure of ACE2 (1R42). Three amino acid positions (N637, L656, and N720) are not shown here because the regions containing these amino acids are not present in this crystal structure. The three separate regions reported to be important for binding to SARS-CoV-S are colored in yellow, magenta, and orange.

Article Snippet: To generate the plasmid (pCMV6-hACE2-FLAG) expressing C-terminally FLAG-tagged wild-type human ACE2 (WT ACE2), a DNA fragment encoding the full-length human ACE2 open reading frame (ORF) was amplified by PCR using a human ACE2-expressing plasmid (HG10108-UT; Sino Biological, Beijing, China) as a template and then inserted in-frame into the pCMV6-Entry vector (OriGene, Rockville, MD, USA) between the SgfI and EcoRV sites connecting the ACE2 ORF and FLAG-tag cDNA sequences.

Techniques: Binding Assay

Journal: Viruses

Article Title: Population-Specific ACE2 Single-Nucleotide Polymorphisms Have Limited Impact on SARS-CoV-2 Infectivity In Vitro

doi: 10.3390/v13010067

Figure Lengend Snippet: Effects of amino acid substitutions corresponding to SNPs in ACE2 on binding to SARS-2-S and cell entry mediated by SARS-2-S in vitro. ( A ) Schematic diagram of the construct used to express the ACE2 variants in this study. Amino acid substitutions corresponding to the SNPs were independently introduced into C-terminally FLAG-tagged human ACE2. Red, signal peptide; yellow, ADAM17 cleavage site; gray, transmembrane and cytoplasmic regions. ( B ) HEK293T cells were transfected with plasmids encoding either FLAG-tagged WT or variant ACE2 proteins. The pCMV6-Entry vector only (Empty) was used as the negative control. ACE2 and GAPDH levels in the total cell lysates were analyzed by western blotting. ( C ) HEK293T cells transfected with plasmids expressing either FLAG-tagged WT or variant ACE2 proteins were biotin-labeled and the cell-surface proteins were pulled down with streptavidin beads. The levels of WT ACE2 and ACE2 variants on the cell surface (in the pulled down samples) and in the total cell lysate were then analyzed by western blotting. The levels of transferrin receptor 1 (TFRC) on the cell surface and in the total cell lysate were also analyzed as an internal control. The ratios of the levels of the protein on the cell surface to that in the total cell lysate were calculated for the WT and each variant after first normalizing the data to the corresponding ratios for TFRC. The value for WT ACE2 was set to 1. ( D ) HEK293T cells transfected with plasmids expressing either FLAG-tagged WT or variant ACE2 proteins were incubated with rSARS-2-S1(D614G), and the levels of rSARS-2-S1(D614G) binding to these cells were analyzed by flow cytometry. The value for WT ACE2 was set to 100%. ( E ) HEK293T cells transfected with plasmids expressing either FLAG-tagged WT or variant ACE2 proteins were inoculated with rVSV∆G-GFP/SARS-2-S(D614G). After 16 h, the proportions of GFP-positive cells were determined with a high-content imaging system. (C–E) Data represent the means + S.D. of at least three independent experiments. White bars, controls (vector-only transfected or WT ACE2); black bars, East Asian population-specific SNPs; gray, non-East Asian population-specific SNPs. Empty, transfected with pCMV6-Entry vector only as the negative control; ND, not detected; WT, wild-type ACE2. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: To generate the plasmid (pCMV6-hACE2-FLAG) expressing C-terminally FLAG-tagged wild-type human ACE2 (WT ACE2), a DNA fragment encoding the full-length human ACE2 open reading frame (ORF) was amplified by PCR using a human ACE2-expressing plasmid (HG10108-UT; Sino Biological, Beijing, China) as a template and then inserted in-frame into the pCMV6-Entry vector (OriGene, Rockville, MD, USA) between the SgfI and EcoRV sites connecting the ACE2 ORF and FLAG-tag cDNA sequences.

Techniques: Binding Assay, In Vitro, Construct, Transfection, Variant Assay, Plasmid Preparation, Negative Control, Western Blot, Expressing, Labeling, Incubation, Flow Cytometry, Imaging

Journal: Viruses

Article Title: Population-Specific ACE2 Single-Nucleotide Polymorphisms Have Limited Impact on SARS-CoV-2 Infectivity In Vitro

doi: 10.3390/v13010067

Figure Lengend Snippet: Neutralization assay of shedded ACE2 variants. HEK293T/ACE2 cells were inoculated with rVSV∆G-GFP/SARS-2-S(D614G) pre-incubated for 1 h at 37 °C with serially diluted tissue culture supernatants (TCS) from cells transfected with wild-type (WT) ACE2- or ACE2 variant-expressing plasmids. After 16 h, the levels of virally infected (GFP + ) cells were measured with a high-content imaging system. The mean number of GFP + cells inoculated with rVSV∆G-GFP/SARS-2-S(D614G) pre-incubated with the TCS from empty vector-transfected cells was set to 100%. The dilution factors required to attain 50% inhibition of viral infection (IC 50 ) were determined using GraphPad Prism 9 software. Data represent the mean ± S.D. of three independent experiments.

Article Snippet: To generate the plasmid (pCMV6-hACE2-FLAG) expressing C-terminally FLAG-tagged wild-type human ACE2 (WT ACE2), a DNA fragment encoding the full-length human ACE2 open reading frame (ORF) was amplified by PCR using a human ACE2-expressing plasmid (HG10108-UT; Sino Biological, Beijing, China) as a template and then inserted in-frame into the pCMV6-Entry vector (OriGene, Rockville, MD, USA) between the SgfI and EcoRV sites connecting the ACE2 ORF and FLAG-tag cDNA sequences.

Techniques: Neutralization, Incubation, Transfection, Variant Assay, Expressing, Infection, Imaging, Plasmid Preparation, Inhibition, Software

Journal: JACS Au

Article Title: Engineered Receptor Capture Combined with Mass Spectrometry Enables High-Throughput Detection and Quantitation of SARS-CoV-2 Spike Protein

doi: 10.1021/jacsau.4c00980

Figure Lengend Snippet: Directed evolution of ACE2 ectodomain. (A) Schematic representation of cell surface expressed ACE2 ectodomain. Full-length ACE2 was expressed with an amino-terminal FLAG tag and short linker region. (B) Selection of ACE2 for increased affinity. Flow cytometry plots are shown for cells incubated with RBD and stained for bound RBD using anti-His-phycoerythrin, and ACE2 expression using anti-FLAG-allophycocyanin. Three rounds of selection at the indicated RBD concentrations and diversification were performed. The sort windows are indicated for each round of selection. (C) Amino acid substitutions of evolved ACE2 are shown in red. (D) Position of substitutions in ACE2 structure (PDB entry ID: 6M0J ). Left panel shows part of ACE2 structure (blue) in complex with RBD (yellow) with substitutions in evolved ACE2 shown in red. Position of A368L is shown in orange. Right panel shows structure of RBD binding interface of ACE2 and the position of substitutions. Position of RBD-interacting residues in Wt-ACE2 are indicated in green. (E) Kinetic binding constants for RBD binding by Wt-ACE2, evolved ACE2 and evolved ACE2 with A386L, measured by biolayer interferometry with soluble ACE2 (19–615) and immobilized monomeric RBD. Data are shown as means and SEM for at least two experiments.

Article Snippet: DNA encoding full-length human ACE2 was a gift from Hyeryun Choe (Addgene plasmid # 1786; http://n2t.net/addgene:1786 ; RRID:Addgene_1786).

Techniques: FLAG-tag, Selection, Flow Cytometry, Incubation, Staining, Expressing, Binding Assay

Journal: JACS Au

Article Title: Engineered Receptor Capture Combined with Mass Spectrometry Enables High-Throughput Detection and Quantitation of SARS-CoV-2 Spike Protein

doi: 10.1021/jacsau.4c00980

Figure Lengend Snippet: ACE2 multimerization increases binding to RBD. (A) Schematic representation of monomeric (ACE2–615), dimeric (ACE2–740), and pentameric (ACE2-COMP) ACE2. (B) Negative stain electron microscopy of monomeric, dimeric, and pentameric ACE2 (left to right). Scale bar, 50 nm. (C) Kinetic binding constants for RBD binding by Wt-ACE2 dimer and pentamer measured by biolayer interferometry with soluble ACE2 and immobilized RBD. Data are shown as means and SEM for two experiments. (D) Kinetic association and dissociation curves for monomeric, dimeric, and pentameric ACE2 demonstrating decreased dissociation kinetics for dimer and pentamer, and retention of bound RBD for more than 8 h for pentamer. (E) Negative stain electron microscopy image of ACE2-COMP bound to SARS-CoV-2 virus. Scale bar, 100 nm.

Article Snippet: DNA encoding full-length human ACE2 was a gift from Hyeryun Choe (Addgene plasmid # 1786; http://n2t.net/addgene:1786 ; RRID:Addgene_1786).

Techniques: Binding Assay, Staining, Electron Microscopy, Virus

Journal: JACS Au

Article Title: Engineered Receptor Capture Combined with Mass Spectrometry Enables High-Throughput Detection and Quantitation of SARS-CoV-2 Spike Protein

doi: 10.1021/jacsau.4c00980

Figure Lengend Snippet: Binding of ACE2-COMP to RBD variants. (A) Kinetic binding constants for RBD binding by ACE2-COMP measured by biolayer interferometry with soluble ACE2 and immobilized monomeric RBD. Data are shown as means and SEM for two experiments. (B) Kinetic association and dissociation curves for binding of ACE2-COMP to B.1.617.1/3 and B.1.351 variant RBD, demonstrating retention of bound RBD for more than 8 h.

Article Snippet: DNA encoding full-length human ACE2 was a gift from Hyeryun Choe (Addgene plasmid # 1786; http://n2t.net/addgene:1786 ; RRID:Addgene_1786).

Techniques: Binding Assay, Variant Assay

Journal: JACS Au

Article Title: Engineered Receptor Capture Combined with Mass Spectrometry Enables High-Throughput Detection and Quantitation of SARS-CoV-2 Spike Protein

doi: 10.1021/jacsau.4c00980

Figure Lengend Snippet: ACE2-COMP trap capture-based mass spectrometric assay for the resolution and detection of SARS-CoV-2 spike peptides. (A) Overlayed chromatogram of saliva with spike protein (157 nM) extracted with the trap assay but with plates that lack ACE2-COMP capture protein. (B) Overlayed chromatogram of saliva with spike protein (157 nM) extracted using plates coated with the ACE2-COMP capture protein. (C) Concentration dependence of spike protein detection in PBS and saliva (as indicated).

Article Snippet: DNA encoding full-length human ACE2 was a gift from Hyeryun Choe (Addgene plasmid # 1786; http://n2t.net/addgene:1786 ; RRID:Addgene_1786).

Techniques: TRAP Assay, Concentration Assay

Journal: JACS Au

Article Title: Engineered Receptor Capture Combined with Mass Spectrometry Enables High-Throughput Detection and Quantitation of SARS-CoV-2 Spike Protein

doi: 10.1021/jacsau.4c00980

Figure Lengend Snippet: Binding of Wt and Evolved ACE2 to RBD Variants

Article Snippet: DNA encoding full-length human ACE2 was a gift from Hyeryun Choe (Addgene plasmid # 1786; http://n2t.net/addgene:1786 ; RRID:Addgene_1786).

Techniques: Binding Assay

Journal: Viruses

Article Title: Population-Specific ACE2 Single-Nucleotide Polymorphisms Have Limited Impact on SARS-CoV-2 Infectivity In Vitro

doi: 10.3390/v13010067

Figure Lengend Snippet: Effects of amino acid substitutions corresponding to SNPs in ACE2 on binding to SARS-2-S and cell entry mediated by SARS-2-S in vitro. ( A ) Schematic diagram of the construct used to express the ACE2 variants in this study. Amino acid substitutions corresponding to the SNPs were independently introduced into C-terminally FLAG-tagged human ACE2. Red, signal peptide; yellow, ADAM17 cleavage site; gray, transmembrane and cytoplasmic regions. ( B ) HEK293T cells were transfected with plasmids encoding either FLAG-tagged WT or variant ACE2 proteins. The pCMV6-Entry vector only (Empty) was used as the negative control. ACE2 and GAPDH levels in the total cell lysates were analyzed by western blotting. ( C ) HEK293T cells transfected with plasmids expressing either FLAG-tagged WT or variant ACE2 proteins were biotin-labeled and the cell-surface proteins were pulled down with streptavidin beads. The levels of WT ACE2 and ACE2 variants on the cell surface (in the pulled down samples) and in the total cell lysate were then analyzed by western blotting. The levels of transferrin receptor 1 (TFRC) on the cell surface and in the total cell lysate were also analyzed as an internal control. The ratios of the levels of the protein on the cell surface to that in the total cell lysate were calculated for the WT and each variant after first normalizing the data to the corresponding ratios for TFRC. The value for WT ACE2 was set to 1. ( D ) HEK293T cells transfected with plasmids expressing either FLAG-tagged WT or variant ACE2 proteins were incubated with rSARS-2-S1(D614G), and the levels of rSARS-2-S1(D614G) binding to these cells were analyzed by flow cytometry. The value for WT ACE2 was set to 100%. ( E ) HEK293T cells transfected with plasmids expressing either FLAG-tagged WT or variant ACE2 proteins were inoculated with rVSV∆G-GFP/SARS-2-S(D614G). After 16 h, the proportions of GFP-positive cells were determined with a high-content imaging system. (C–E) Data represent the means + S.D. of at least three independent experiments. White bars, controls (vector-only transfected or WT ACE2); black bars, East Asian population-specific SNPs; gray, non-East Asian population-specific SNPs. Empty, transfected with pCMV6-Entry vector only as the negative control; ND, not detected; WT, wild-type ACE2. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: To generate the plasmid (pCMV6-hACE2-FLAG) expressing C-terminally FLAG-tagged wild-type human ACE2 (WT ACE2), a DNA fragment encoding the full-length human ACE2 open reading frame (ORF) was amplified by PCR using a human ACE2-expressing plasmid (HG10108-UT; Sino Biological, Beijing, China) as a template and then inserted in-frame into the pCMV6-Entry vector (OriGene, Rockville, MD, USA) between the SgfI and EcoRV sites connecting the ACE2 ORF and FLAG-tag cDNA sequences.

Techniques: Binding Assay, In Vitro, Construct, Transfection, Variant Assay, Plasmid Preparation, Negative Control, Western Blot, Expressing, Labeling, Incubation, Flow Cytometry, Imaging

Journal: iScience

Article Title: Enhanced reverse zoonotic potential and immune evasion by omicron JN.1 variant

doi: 10.1016/j.isci.2025.112824

Figure Lengend Snippet: Receptor binding and fusogenicity of S proteins of omicron variants (A) Amino Acid sequence alignment of RBDs from different omicron variants. Dashed lines represent residues identical to WT. Residues of RBM are shown in red. (B and C) Cleavage of S proteins from different omicron variants. Plasmids encoding codon-optimized S proteins were transfected into HEK293T cells and the cells were then lysed at 40 h post-transfection. The expression of S proteins was detected by western blot using rabbit polyclonal anti-S2 antibodies. Actin served as the loading control. Experiments were done three times and one representative was shown. Band densities were quantified using ImageLab software to determine the cleaved/FL ratio and normalized to WT. FL S, full-length spike protein. (D) Binding of soluble hACE2 to S proteins of omicron variants. HEK293T cells transiently expressing the indicated S proteins were incubated with biotinylated hACE2 followed by Alexa Fluor 488-conjugated streptavidin and then analyzed by flow cytometry. Experiments were done three times, and one representative was shown. (E and F) Cell-cell fusion mediated by S proteins of omicron variants. HEK293T cells were co-transfected with indicated S protein and eGFP. The cells were detached with trypsin at 48h post-transfection and overlaid on the HEK293 cells expressing hACE2 or Calu3 cells. After 2 h of incubation, five randomly selected images of syncytia were captured using a fluorescent microscope. Fusion areas were quantified using ImageJ software and normalized to WT as fusion efficiency. Experiments were done three times in triplicate and one representative is shown. The statistical difference relative to WT was determined by one-way ANOVA with Dunnett’s multiple testing correction ( n = 3 for (C) and (D); n = 5 for (E) and (F)). The statistical difference between BA.2.86 and JN.1 was determined by a two-tailed t-test ( n = 3 for (C) and (D); n = 5 for (E) and (F)). Error bars indicate SEM. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. Scale bar = 200μm. See also .

Article Snippet: The plasmids encoding animal ACE2 orthologs and hACE2 were synthesized by Sango Biotech (Shanghai, China) and cloned into the p3×Flag-CMV14 vector between the Hind III and BamH I sites.

Techniques: Binding Assay, Sequencing, Transfection, Expressing, Western Blot, Control, Software, Incubation, Flow Cytometry, Microscopy, Two Tailed Test

Journal: iScience

Article Title: Enhanced reverse zoonotic potential and immune evasion by omicron JN.1 variant

doi: 10.1016/j.isci.2025.112824

Figure Lengend Snippet: Transduction of various omicron S pseudovirions on 293/hACE2 and Calu3 cells (A) S proteins incorporation in pseudovirions. Pseudovirions with different omicron S proteins were pelleted down by centrifugation through 20% sucrose cushion and separated in a 10% SDS-PAGE. Detection of S proteins in pseudovirions was performed by Western blot using rabbit polyclonal anti-S2 antibodies. The p24 served as the loading controls. Experiments were done three times and one representative was shown. (B and C) Entry of pseudovirions of omicron variants. HEK 293/hACE2 and Calu3 cells were transduced with omicron S pseudovirions and lysed at 40 h post-transduction. The transduction efficiencies were determined according to luciferase activities. Experiments were done three times in triplicate and one representative is shown. The statistical difference relative to WT was determined by one-way ANOVA with Dunnett’s multiple testing correction ( n = 3). The statistical difference between BA.2.86 and JN.1 was determined by a two-tailed t-test ( n = 3). Error bars indicate SEM. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: The plasmids encoding animal ACE2 orthologs and hACE2 were synthesized by Sango Biotech (Shanghai, China) and cloned into the p3×Flag-CMV14 vector between the Hind III and BamH I sites.

Techniques: Transduction, Centrifugation, SDS Page, Western Blot, Luciferase, Two Tailed Test

Journal: iScience

Article Title: Enhanced reverse zoonotic potential and immune evasion by omicron JN.1 variant

doi: 10.1016/j.isci.2025.112824

Figure Lengend Snippet: Entry of different omicron pseudovirions on HEK293 cells expressing various animal ACE2s (A) Analysis of expression of various ACE2s on HEK293 cell surface by cell surface biotinylation assay. HEK293 cells transiently expressing different FLAG-tagged different ACE2s were labeled with EZ-link Sulfo-NHS-LC-LC-biotin on ice, and lysed with RIPA buffer. Biotinylated proteins were enriched with NeutraAvidin beads and detected by Western blot using mouse monoclonal anti-FLAG M2 antibody. Human Leukocyte Antigen (HLA) and actin served as controls. WCL, whole cell lysate. (B) Relative transduction efficiencies of different omicron variant pseudovirions on HEK 293 cells expressing various ACE2 orthologs. HEK293 cells transiently expressing different animal ACE2s were transduced with pseudovirions of WT, BA.1, XBB.1.16, EG.5.1, BA.2.86 and JN.1. Relative transduction efficiencies were normalized to WT/hACE2(100%). Experiments were performed three times in triplicate, and the averages are shown as the heatmap. (C) Statistic analyses of differences of transduction efficiencies between different omicron variant pseudovirion by Wilcoxon test. Significant differences indicated by p < 0.05 are highlighted in green background. See also .

Article Snippet: The plasmids encoding animal ACE2 orthologs and hACE2 were synthesized by Sango Biotech (Shanghai, China) and cloned into the p3×Flag-CMV14 vector between the Hind III and BamH I sites.

Techniques: Expressing, Cell Surface Biotinylation Assay, Labeling, Western Blot, Transduction, Variant Assay

Journal: iScience

Article Title: Enhanced reverse zoonotic potential and immune evasion by omicron JN.1 variant

doi: 10.1016/j.isci.2025.112824

Figure Lengend Snippet: Receptor binding of omicron variants to various animal ACE2s (A) Relative receptor binding efficiencies of RBDs of WT, BA.1, XBB.1.16, EG.5.1, BA.2.86, and JN.1 variants to various animal ACE2s. HEK293T transiently expressing different animal ACE2s were detached with EDTA and incubated with mouse Fc-tagged RBDs of WT, BA.1, XBB.1.16, EG.5.1, BA.2.86, or JN.1, followed by FITC-conjugated polyclonal goat anti-mouse antibodies. The cells were analyzed by flow cytometry. Receptor binding efficiencies were calculated according to WT/hACE2, set as 100%. Experiments were performed three times, and the averages are shown in the heatmap. (B) Statistic analyses of levels of receptor binding between different omicron variant S protein by Wilcoxon test. Significant differences indicated by p < 0.05 are highlighted in green background.

Article Snippet: The plasmids encoding animal ACE2 orthologs and hACE2 were synthesized by Sango Biotech (Shanghai, China) and cloned into the p3×Flag-CMV14 vector between the Hind III and BamH I sites.

Techniques: Binding Assay, Expressing, Incubation, Flow Cytometry, Variant Assay

Journal: iScience

Article Title: Enhanced reverse zoonotic potential and immune evasion by omicron JN.1 variant

doi: 10.1016/j.isci.2025.112824

Figure Lengend Snippet: Cell-cell fusion mediated by different omicron variant S proteins on HEK 293 cells expressing various animal ACE2s (A) Relative fusion efficiencies of WT, BA.1, XBB.1.16, EG.5.1, BA.2.86, and JN.1 on HEK 293 cells expressing animal ACE2 orthologs. HEK 293T cells transiently co-expressing S protein and eGFP were detached with trypsin and overlaid on HEK 293 cells expressing different animal ACE2s. After 2 h of incubation, the images of syncytia were captured using a fluorescent microscope. Fusion areas were quantified using ImageJ software and normalized to WT/hACE2, set as 100%. Experiments were performed three times, and the averages are shown in the heatmap. (B) Statistic analyses of levels of cell-cell fusion between different omicron variant S proteins by Wilcoxon test. Significant differences indicated by p < 0.05 are highlighted in green background.

Article Snippet: The plasmids encoding animal ACE2 orthologs and hACE2 were synthesized by Sango Biotech (Shanghai, China) and cloned into the p3×Flag-CMV14 vector between the Hind III and BamH I sites.

Techniques: Variant Assay, Expressing, Incubation, Microscopy, Software

Journal: iScience

Article Title: Enhanced reverse zoonotic potential and immune evasion by omicron JN.1 variant

doi: 10.1016/j.isci.2025.112824

Figure Lengend Snippet: Effect of L455S mutation on transduction efficiency and fusogenicity of XBB.1.16 S protein (A) Relative transduction efficiencies of XBB.1.16 and XBB.1.16-L455S on HEK 293 cells expressing different animal ACE2s. HEK 293 cells transiently expressing different animal ACE2s were transduced by either XBB.1.16 WT S or L455S mutant S pseudovirions. The luciferase activities were measured at 40 h post transduction. Relative transduction efficiencies were calculated based on WT/hACE2(100%). Experiments were performed three times in triplicates and the representative one was shown. (B) Cell-cell fusion mediated by XBB.1.16 and XBB.1.16-L455S S proteins. HEK 293T cells transiently co-expressing S protein and eGFP were detached with trypsin and overlaid on HEK 293 cells expressing different animal ACE2s. After 2 h of incubation, the five randomly selected images of syncytia were captured using a fluorescent microscope. Fusion areas were quantified using ImageJ software and normalized to WT/hACE2, set as 100%. Experiments were performed twice and the representative one was shown. Statistical analyses were done by Wilcoxon test. See also .

Article Snippet: The plasmids encoding animal ACE2 orthologs and hACE2 were synthesized by Sango Biotech (Shanghai, China) and cloned into the p3×Flag-CMV14 vector between the Hind III and BamH I sites.

Techniques: Mutagenesis, Transduction, Expressing, Luciferase, Incubation, Microscopy, Software

Journal: iScience

Article Title: Enhanced reverse zoonotic potential and immune evasion by omicron JN.1 variant

doi: 10.1016/j.isci.2025.112824

Figure Lengend Snippet: Effect of L455S mutation on thermal and proteolytic stability on XBB.1.16 and BA.2.86 (A and B) Thermal stability of JN.1 compared to its parental BA.2.86 (left panel) and XBB.1.16–455S to XBB.1.16 (right panel). Pseudovirus of XBB.1.16, XBB.1.16–455S, BA.2.86 and JN.1 were centrifuged through 20% sucrose to remove serum and resuspended in serum-free DMEM, followed by incubation either at 37°C for the indicated time (A) or at the indicated temperature for 2 h (B). The virus suspension was used to transduce 293/hACE2 cells to access their remaining transduction capability. Experiments were performed three times in triplicate, and the representative one was shown. The statistical difference was determined by a multiple t-test ( n = 3). Error bars indicate SEM. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. (C) Proteolytic stability of JN.1 compared to its parental BA.2.86 (left panel) and XBB.1.16–455S to XBB.1.16 (right panel). Pseudovirus of XBB.1.16, XBB.1.16–455S, BA.2.86 and JN.1 were resuspended in serum-free DMEM and incubated with soluble hACE2(20 μg) at 37°C for 30 min, followed by incubation with TPCK-treated trypsin. The mixture was immediately boiled with loading buffer containing DTT and separated in a 10% SDS-PAGE. Detection was carried out using rabbit polyclonal anti-SARS-CoV-2 S2 antibodies (1:3000). The numbers below S protein blot are the relative quantification of ratio of S2’/S2 using Image Lab (Bio-Rad). Experiments were performed at least three times, and the representative one was shown.

Article Snippet: The plasmids encoding animal ACE2 orthologs and hACE2 were synthesized by Sango Biotech (Shanghai, China) and cloned into the p3×Flag-CMV14 vector between the Hind III and BamH I sites.

Techniques: Mutagenesis, Incubation, Virus, Suspension, Transduction, SDS Page, Quantitative Proteomics