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    R&D Systems ace2
    Inhibitory effects of peptides on the SARS-CoV S protein and <t>ACE2</t> interaction by competitive biotinylated ELISA. Biotin-labeled S protein (1 nmol) was mixed with 10 nmol of synthetic peptides or BSA and incubated at 37 °C with shaking. After a 2-h incubation, the mixtures were added to wells, which were coated with 1 ng of ACE2 and incubated at 37 °C for 1 h. Following three washes, peroxidase-conjugated avidin and chromatic substrate were sequentially added. The absorbance was read at 405 nm in an ELISA plate reader. The results are expressed as inhibition described in Section 2 . Values are mean ± standard error of six independent assays. ** p
    Ace2, supplied by R&D Systems, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ace2/product/R&D Systems
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ace2 - by Bioz Stars, 2021-06
    86/100 stars
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    99
    Addgene inc human ace2
    Increased transduction of SARS-CoV-2 Spike-pseudotyped lentivirus in human cells that constitutively overexpress the human <t>ACE2</t> receptor. ( a ) Percent of EGFP+ cells at 6 days post-transduction with 100 μL of supernatant SARS-CoV-2 Spike (D614) pseudotyped lentivirus and unpseudotyped lentivirus in human liver Huh7.5 with and without ACE2 overexpression. ( b ) Flow cytometry gating for GFP quantification. Cells were first gated by the forward and side scatter area to exclude debris and then doublets were excluded by gating on forward scatter area and width.
    Human Ace2, supplied by Addgene inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/human ace2/product/Addgene inc
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    human ace2 - by Bioz Stars, 2021-06
    99/100 stars
      Buy from Supplier

    96
    Addgene inc pcep4 myc ace2
    Increased transduction of SARS-CoV-2 Spike-pseudotyped lentivirus in human cells that constitutively overexpress the human <t>ACE2</t> receptor. ( a ) Percent of EGFP+ cells at 6 days post-transduction with 100 μL of supernatant SARS-CoV-2 Spike (D614) pseudotyped lentivirus and unpseudotyped lentivirus in human liver Huh7.5 with and without ACE2 overexpression. ( b ) Flow cytometry gating for GFP quantification. Cells were first gated by the forward and side scatter area to exclude debris and then doublets were excluded by gating on forward scatter area and width.
    Pcep4 Myc Ace2, supplied by Addgene inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcep4 myc ace2/product/Addgene inc
    Average 96 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pcep4 myc ace2 - by Bioz Stars, 2021-06
    96/100 stars
      Buy from Supplier

    86
    Addgene inc human ace2 expression plasmid
    Receptor screening using surrogate entry assays identifies SARS-CoV-2 Spike as a pan-tropic viral attachment protein. (A) A heatmap illustrating the receptor usage profile of SARS-CoV-2 and SARS-CoV in pseudotype entry and cell–cell fusion assays with various mammalian and bird ACE2s. The data in each row are normalised to the signal seen for human <t>ACE2</t> (top), with results representing the mean percentage calculated from 3 separate experiments performed on different days. A vector-only control (pDISPLAY) was added to demonstrate specificity. Mammalian and bird ACE2s are organised, top to bottom, based on their phylogenetic relationship (rectangular cladogram, left). The inter-experimental standard error of the mean for the pseudotype and cell–cell fusion assays ranged from 0.01% to 47.92% (median 10.73%) and 0.12% to 32.97% (median 5.43%), respectively. (B and C) For both SARS-CoV-2 and SARS-CoV, the respective cell–cell and pseudotype assay percentages for each ACE2 protein (relative to human ACE2) were plotted on an XY scatter graph, the Pearson correlation calculated and a linear line of regression fitted together with 95% confidence intervals. The data underlying this figure may be found in S1 Data . ACE2, angiotensin-converting enzyme 2; SARS-CoV, SARS Coronavirus; SARS-CoV-2, SARS Coronavirus 2.
    Human Ace2 Expression Plasmid, supplied by Addgene inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/human ace2 expression plasmid/product/Addgene inc
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    human ace2 expression plasmid - by Bioz Stars, 2021-06
    86/100 stars
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    N/A
    CRISPR Cas9 KO Plasmids consists of ACE2 specific 20 nt guide RNA sequences derived from the GeCKO v2 library For CRISPR gene knockout gRNA sequences direct the Cas9 protein to
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    N/A
    Gene Silencers generally consist of pools of three to five target specific 19 25 nucleotide sequences in length For independent verification of ACE2 gene silencing results individual duplex components or
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    N/A
    Standard format Plasmid sent in bacteria as agar stab
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    N/A
    Standard format Plasmid sent in bacteria as agar stab
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    Image Search Results


    Inhibitory effects of peptides on the SARS-CoV S protein and ACE2 interaction by competitive biotinylated ELISA. Biotin-labeled S protein (1 nmol) was mixed with 10 nmol of synthetic peptides or BSA and incubated at 37 °C with shaking. After a 2-h incubation, the mixtures were added to wells, which were coated with 1 ng of ACE2 and incubated at 37 °C for 1 h. Following three washes, peroxidase-conjugated avidin and chromatic substrate were sequentially added. The absorbance was read at 405 nm in an ELISA plate reader. The results are expressed as inhibition described in Section 2 . Values are mean ± standard error of six independent assays. ** p

    Journal: Antiviral Research

    Article Title: Design and biological activities of novel inhibitory peptides for SARS-CoV spike protein and angiotensin-converting enzyme 2 interaction

    doi: 10.1016/j.antiviral.2005.10.005

    Figure Lengend Snippet: Inhibitory effects of peptides on the SARS-CoV S protein and ACE2 interaction by competitive biotinylated ELISA. Biotin-labeled S protein (1 nmol) was mixed with 10 nmol of synthetic peptides or BSA and incubated at 37 °C with shaking. After a 2-h incubation, the mixtures were added to wells, which were coated with 1 ng of ACE2 and incubated at 37 °C for 1 h. Following three washes, peroxidase-conjugated avidin and chromatic substrate were sequentially added. The absorbance was read at 405 nm in an ELISA plate reader. The results are expressed as inhibition described in Section 2 . Values are mean ± standard error of six independent assays. ** p

    Article Snippet: By analyzing a series of peptides derived from S protein, we identified that peptides spanning residues 192–203, 483–494, and 660–683 of S protein efficiently blocked the binding of S protein to ACE2.

    Techniques: Enzyme-linked Immunosorbent Assay, Labeling, Incubation, Avidin-Biotin Assay, Inhibition

    Inhibitory effect of SP-10 on the SARS-CoV S-protein-pseudotyped retrovirus infectivity. S-protein-pseudotyped retroviruses were mixed with various amounts of SP-10 and incubated at 37 °C with shaking. After a 2-h incubation, the mixtures were inoculated with Vero E6 cells transfected with the plasmid encoding human ACE2. The luciferase activity of cell lysate was assayed 2 days postinfection. Relative infectivity is presented as comparison with the RLU relative to untreated cells. Values are mean ± standard error of four independent assays.

    Journal: Antiviral Research

    Article Title: Design and biological activities of novel inhibitory peptides for SARS-CoV spike protein and angiotensin-converting enzyme 2 interaction

    doi: 10.1016/j.antiviral.2005.10.005

    Figure Lengend Snippet: Inhibitory effect of SP-10 on the SARS-CoV S-protein-pseudotyped retrovirus infectivity. S-protein-pseudotyped retroviruses were mixed with various amounts of SP-10 and incubated at 37 °C with shaking. After a 2-h incubation, the mixtures were inoculated with Vero E6 cells transfected with the plasmid encoding human ACE2. The luciferase activity of cell lysate was assayed 2 days postinfection. Relative infectivity is presented as comparison with the RLU relative to untreated cells. Values are mean ± standard error of four independent assays.

    Article Snippet: By analyzing a series of peptides derived from S protein, we identified that peptides spanning residues 192–203, 483–494, and 660–683 of S protein efficiently blocked the binding of S protein to ACE2.

    Techniques: Infection, Incubation, Transfection, Plasmid Preparation, Luciferase, Activity Assay

    Inhibitory effects of SP-4, SP-8, and SP-10 on the SARS-CoV S protein and ACE2 interaction by competitive biotinylated ELISA. Biotin-labeled S protein (1 nmol) was mixed with various amounts of SP-4 (A), SP-8 (B) or SP-10 (C), and incubated at 37 °C with shaking. After a 2-h incubation, the mixtures were added to wells, which were coated with 1 ng of ACE2, and incubated at 37 °C for 1 h. Following three washes, peroxidase-conjugated avidin and chromatic substrate were sequentially added. The absorbance was read at 405 nm in an ELISA plate reader. The results are expressed as inhibition described in Section 2 . Values are mean ± standard error of six independent assays.

    Journal: Antiviral Research

    Article Title: Design and biological activities of novel inhibitory peptides for SARS-CoV spike protein and angiotensin-converting enzyme 2 interaction

    doi: 10.1016/j.antiviral.2005.10.005

    Figure Lengend Snippet: Inhibitory effects of SP-4, SP-8, and SP-10 on the SARS-CoV S protein and ACE2 interaction by competitive biotinylated ELISA. Biotin-labeled S protein (1 nmol) was mixed with various amounts of SP-4 (A), SP-8 (B) or SP-10 (C), and incubated at 37 °C with shaking. After a 2-h incubation, the mixtures were added to wells, which were coated with 1 ng of ACE2, and incubated at 37 °C for 1 h. Following three washes, peroxidase-conjugated avidin and chromatic substrate were sequentially added. The absorbance was read at 405 nm in an ELISA plate reader. The results are expressed as inhibition described in Section 2 . Values are mean ± standard error of six independent assays.

    Article Snippet: By analyzing a series of peptides derived from S protein, we identified that peptides spanning residues 192–203, 483–494, and 660–683 of S protein efficiently blocked the binding of S protein to ACE2.

    Techniques: Enzyme-linked Immunosorbent Assay, Labeling, Incubation, Avidin-Biotin Assay, Inhibition

    Analysis of SARS-CoV S protein and ACE2 interaction by biotinylated ELISA. The wells were coated with 1 ng of ACE2 and challenged with various amounts of biotin-labeled S protein (●), biotin-labeled BSA (○) or biotin (▴). Following three washes, peroxidase-conjugated avidin and chromatic substrate were sequentially added. The absorbance was read at 405 nm in an ELISA plate reader. Values are mean ± standard error of four independent assays.

    Journal: Antiviral Research

    Article Title: Design and biological activities of novel inhibitory peptides for SARS-CoV spike protein and angiotensin-converting enzyme 2 interaction

    doi: 10.1016/j.antiviral.2005.10.005

    Figure Lengend Snippet: Analysis of SARS-CoV S protein and ACE2 interaction by biotinylated ELISA. The wells were coated with 1 ng of ACE2 and challenged with various amounts of biotin-labeled S protein (●), biotin-labeled BSA (○) or biotin (▴). Following three washes, peroxidase-conjugated avidin and chromatic substrate were sequentially added. The absorbance was read at 405 nm in an ELISA plate reader. Values are mean ± standard error of four independent assays.

    Article Snippet: By analyzing a series of peptides derived from S protein, we identified that peptides spanning residues 192–203, 483–494, and 660–683 of S protein efficiently blocked the binding of S protein to ACE2.

    Techniques: Enzyme-linked Immunosorbent Assay, Labeling, Avidin-Biotin Assay

    Increased transduction of SARS-CoV-2 Spike-pseudotyped lentivirus in human cells that constitutively overexpress the human ACE2 receptor. ( a ) Percent of EGFP+ cells at 6 days post-transduction with 100 μL of supernatant SARS-CoV-2 Spike (D614) pseudotyped lentivirus and unpseudotyped lentivirus in human liver Huh7.5 with and without ACE2 overexpression. ( b ) Flow cytometry gating for GFP quantification. Cells were first gated by the forward and side scatter area to exclude debris and then doublets were excluded by gating on forward scatter area and width.

    Journal: eLife

    Article Title: The Spike D614G mutation increases SARS-CoV-2 infection of multiple human cell types

    doi: 10.7554/eLife.65365

    Figure Lengend Snippet: Increased transduction of SARS-CoV-2 Spike-pseudotyped lentivirus in human cells that constitutively overexpress the human ACE2 receptor. ( a ) Percent of EGFP+ cells at 6 days post-transduction with 100 μL of supernatant SARS-CoV-2 Spike (D614) pseudotyped lentivirus and unpseudotyped lentivirus in human liver Huh7.5 with and without ACE2 overexpression. ( b ) Flow cytometry gating for GFP quantification. Cells were first gated by the forward and side scatter area to exclude debris and then doublets were excluded by gating on forward scatter area and width.

    Article Snippet: ACE2 lentiviral cloning and ACE2 stable cell line overexpression To generate pLenti-ACE2-Hygro (Addgene 161758), we amplified human ACE2 (hACE2) from pcDNA3.1-ACE2 (Addgene 1786) and cloned it into a lentiviral transfer pLEX vector carrying the hygromycin resistance gene using Gibson Assembly Master Mix (NEB E2611L).

    Techniques: Transduction, Over Expression, Flow Cytometry

    Receptor screening using surrogate entry assays identifies SARS-CoV-2 Spike as a pan-tropic viral attachment protein. (A) A heatmap illustrating the receptor usage profile of SARS-CoV-2 and SARS-CoV in pseudotype entry and cell–cell fusion assays with various mammalian and bird ACE2s. The data in each row are normalised to the signal seen for human ACE2 (top), with results representing the mean percentage calculated from 3 separate experiments performed on different days. A vector-only control (pDISPLAY) was added to demonstrate specificity. Mammalian and bird ACE2s are organised, top to bottom, based on their phylogenetic relationship (rectangular cladogram, left). The inter-experimental standard error of the mean for the pseudotype and cell–cell fusion assays ranged from 0.01% to 47.92% (median 10.73%) and 0.12% to 32.97% (median 5.43%), respectively. (B and C) For both SARS-CoV-2 and SARS-CoV, the respective cell–cell and pseudotype assay percentages for each ACE2 protein (relative to human ACE2) were plotted on an XY scatter graph, the Pearson correlation calculated and a linear line of regression fitted together with 95% confidence intervals. The data underlying this figure may be found in S1 Data . ACE2, angiotensin-converting enzyme 2; SARS-CoV, SARS Coronavirus; SARS-CoV-2, SARS Coronavirus 2.

    Journal: PLoS Biology

    Article Title: The SARS-CoV-2 Spike protein has a broad tropism for mammalian ACE2 proteins

    doi: 10.1371/journal.pbio.3001016

    Figure Lengend Snippet: Receptor screening using surrogate entry assays identifies SARS-CoV-2 Spike as a pan-tropic viral attachment protein. (A) A heatmap illustrating the receptor usage profile of SARS-CoV-2 and SARS-CoV in pseudotype entry and cell–cell fusion assays with various mammalian and bird ACE2s. The data in each row are normalised to the signal seen for human ACE2 (top), with results representing the mean percentage calculated from 3 separate experiments performed on different days. A vector-only control (pDISPLAY) was added to demonstrate specificity. Mammalian and bird ACE2s are organised, top to bottom, based on their phylogenetic relationship (rectangular cladogram, left). The inter-experimental standard error of the mean for the pseudotype and cell–cell fusion assays ranged from 0.01% to 47.92% (median 10.73%) and 0.12% to 32.97% (median 5.43%), respectively. (B and C) For both SARS-CoV-2 and SARS-CoV, the respective cell–cell and pseudotype assay percentages for each ACE2 protein (relative to human ACE2) were plotted on an XY scatter graph, the Pearson correlation calculated and a linear line of regression fitted together with 95% confidence intervals. The data underlying this figure may be found in S1 Data . ACE2, angiotensin-converting enzyme 2; SARS-CoV, SARS Coronavirus; SARS-CoV-2, SARS Coronavirus 2.

    Article Snippet: For the initial optimisation of pseudoparticle activity, 3 conditions were tested: (1) ACE2 expression only: HEK293T target cells transfected with 500 ng of a human ACE2 expression plasmid (Addgene, USA) and were seeded at 2 × 104 in 100 μL DMEM-10% in a white-bottomed 96-well plate (Corning, USA) 1 day prior to infection; (2) Spike activation with TMPRSS2: HEK293T target cells were transfected with 25 ng TMPRSS2 alongside human ACE2 as above; (3) Spike activation by trypsin treatment: viral pseudoparticles were treated with 2.5 mg/mL trypsin for 1 h at 37°C before addition to target cells overexpressing human ACE2.

    Techniques: Plasmid Preparation

    Substitutions at the interface between SARS-CoV-2 RBD and mammalian ACE2 proteins impact receptor utilisation. (A) Residues of mammalian ACE2 sequences used in this study that are predicted to interact with the RBD SARS-CoV and SARS-CoV-2, based on the structures of human ACE2 in complex with SARS-CoV [ 19 ] and SARS-CoV-2 [ 6 ]. Differences between closely related species that may impact RBD binding are highlighted. (B) Interface between human ACE2 (green) and SARS-CoV-2 RBD (yellow). Insets 1 to 5 show molecular interactions discussed in the main text. Bonds that may be disrupted are shown as grey lines, with bond distances in grey text, and hydrophobic interactions that may be disrupted are marked with asterisks. ACE2, angiotensin-converting enzyme 2; RBD, receptor binding domain; SARS-CoV, SARS Coronavirus; SARS-CoV-2, SARS Coronavirus 2.

    Journal: PLoS Biology

    Article Title: The SARS-CoV-2 Spike protein has a broad tropism for mammalian ACE2 proteins

    doi: 10.1371/journal.pbio.3001016

    Figure Lengend Snippet: Substitutions at the interface between SARS-CoV-2 RBD and mammalian ACE2 proteins impact receptor utilisation. (A) Residues of mammalian ACE2 sequences used in this study that are predicted to interact with the RBD SARS-CoV and SARS-CoV-2, based on the structures of human ACE2 in complex with SARS-CoV [ 19 ] and SARS-CoV-2 [ 6 ]. Differences between closely related species that may impact RBD binding are highlighted. (B) Interface between human ACE2 (green) and SARS-CoV-2 RBD (yellow). Insets 1 to 5 show molecular interactions discussed in the main text. Bonds that may be disrupted are shown as grey lines, with bond distances in grey text, and hydrophobic interactions that may be disrupted are marked with asterisks. ACE2, angiotensin-converting enzyme 2; RBD, receptor binding domain; SARS-CoV, SARS Coronavirus; SARS-CoV-2, SARS Coronavirus 2.

    Article Snippet: For the initial optimisation of pseudoparticle activity, 3 conditions were tested: (1) ACE2 expression only: HEK293T target cells transfected with 500 ng of a human ACE2 expression plasmid (Addgene, USA) and were seeded at 2 × 104 in 100 μL DMEM-10% in a white-bottomed 96-well plate (Corning, USA) 1 day prior to infection; (2) Spike activation with TMPRSS2: HEK293T target cells were transfected with 25 ng TMPRSS2 alongside human ACE2 as above; (3) Spike activation by trypsin treatment: viral pseudoparticles were treated with 2.5 mg/mL trypsin for 1 h at 37°C before addition to target cells overexpressing human ACE2.

    Techniques: Binding Assay

    Amino acid variation in SARS-CoV-2 and RaTG13 RBD impacts human ACE2 receptor utilisation. Structure of RaTG13 RBD (orange) [ 12 ] superposed onto the structure of human ACE2 (green) in complex with the SARS-CoV-2 RBD (yellow) [ 10 ]. Selected RBD residues that promote association with ACE2 are highlighted, as are the ACE2 “hotspot” residues K31 and K353 [ 13 ]. Insets 1 to 5 show molecular interactions discussed in the main text. Bonds that may be disrupted are shown as grey lines, with bond distances in grey text, and hydrophobic interactions that may be disrupted or potential steric clashes are marked with asterisks. ACE2, angiotensin-converting enzyme 2; RBD, receptor binding domain; SARS-CoV-2, SARS Coronavirus 2.

    Journal: PLoS Biology

    Article Title: The SARS-CoV-2 Spike protein has a broad tropism for mammalian ACE2 proteins

    doi: 10.1371/journal.pbio.3001016

    Figure Lengend Snippet: Amino acid variation in SARS-CoV-2 and RaTG13 RBD impacts human ACE2 receptor utilisation. Structure of RaTG13 RBD (orange) [ 12 ] superposed onto the structure of human ACE2 (green) in complex with the SARS-CoV-2 RBD (yellow) [ 10 ]. Selected RBD residues that promote association with ACE2 are highlighted, as are the ACE2 “hotspot” residues K31 and K353 [ 13 ]. Insets 1 to 5 show molecular interactions discussed in the main text. Bonds that may be disrupted are shown as grey lines, with bond distances in grey text, and hydrophobic interactions that may be disrupted or potential steric clashes are marked with asterisks. ACE2, angiotensin-converting enzyme 2; RBD, receptor binding domain; SARS-CoV-2, SARS Coronavirus 2.

    Article Snippet: For the initial optimisation of pseudoparticle activity, 3 conditions were tested: (1) ACE2 expression only: HEK293T target cells transfected with 500 ng of a human ACE2 expression plasmid (Addgene, USA) and were seeded at 2 × 104 in 100 μL DMEM-10% in a white-bottomed 96-well plate (Corning, USA) 1 day prior to infection; (2) Spike activation with TMPRSS2: HEK293T target cells were transfected with 25 ng TMPRSS2 alongside human ACE2 as above; (3) Spike activation by trypsin treatment: viral pseudoparticles were treated with 2.5 mg/mL trypsin for 1 h at 37°C before addition to target cells overexpressing human ACE2.

    Techniques: Binding Assay

    A cognate ACE2 receptor is required for SARS-CoV-2 infection. (A) Various cell lines derived from birds, dogs, rabbits, rodents, pigs, ruminants, and primates were experimentally infected with SARS-CoV-2 at a MOI of 0.001. At 72 h postinfection, the supernatants from cells were harvested and titred by TCID-50. For each cell line, RNA from uninfected cells was also extracted, and RT-qPCR was performed to detect ACE2 mRNA, with the value above each line indicating the cycle when PCR positivity was achieved (Ct). (B) Four of the same cell lines were infected again, this time at high MOI (1). (C) BHK-21 hamster cells were transiently transfected with ACE2 expression constructs (or a vector control [pDISPLAY]) before being infected with SARS-CoV-2 at high MOI (1). * p

    Journal: PLoS Biology

    Article Title: The SARS-CoV-2 Spike protein has a broad tropism for mammalian ACE2 proteins

    doi: 10.1371/journal.pbio.3001016

    Figure Lengend Snippet: A cognate ACE2 receptor is required for SARS-CoV-2 infection. (A) Various cell lines derived from birds, dogs, rabbits, rodents, pigs, ruminants, and primates were experimentally infected with SARS-CoV-2 at a MOI of 0.001. At 72 h postinfection, the supernatants from cells were harvested and titred by TCID-50. For each cell line, RNA from uninfected cells was also extracted, and RT-qPCR was performed to detect ACE2 mRNA, with the value above each line indicating the cycle when PCR positivity was achieved (Ct). (B) Four of the same cell lines were infected again, this time at high MOI (1). (C) BHK-21 hamster cells were transiently transfected with ACE2 expression constructs (or a vector control [pDISPLAY]) before being infected with SARS-CoV-2 at high MOI (1). * p

    Article Snippet: For the initial optimisation of pseudoparticle activity, 3 conditions were tested: (1) ACE2 expression only: HEK293T target cells transfected with 500 ng of a human ACE2 expression plasmid (Addgene, USA) and were seeded at 2 × 104 in 100 μL DMEM-10% in a white-bottomed 96-well plate (Corning, USA) 1 day prior to infection; (2) Spike activation with TMPRSS2: HEK293T target cells were transfected with 25 ng TMPRSS2 alongside human ACE2 as above; (3) Spike activation by trypsin treatment: viral pseudoparticles were treated with 2.5 mg/mL trypsin for 1 h at 37°C before addition to target cells overexpressing human ACE2.

    Techniques: Infection, Derivative Assay, Quantitative RT-PCR, Polymerase Chain Reaction, Transfection, Expressing, Construct, Plasmid Preparation

    The SARS-CoV-2 binding site on ACE2 is highly variable. (A) A phylogenetic tree of ACE2 proteins assembled using the neighbor-joining method [ 51 ] conducted in MEGA7 (Temple University, USA) [ 52 ] with ambiguous positions removed. The tree is drawn to scale, and support was provided with 500 bootstraps. (B) Structure of human ACE2 ectodomain (green) in complex with the RBD of SARS-CoV-2 [ 10 ]. (C) Conservation of mammalian ACE2 amino acid residues, estimated from site-specific evolutionary rates [ 50 ], mapped onto the surface of the ACE2 ectodomain [ 10 ], and coloured from blue (divergent) to purple (conserved) and presented in 2 orientations. Inset depicts the SARS-CoV-2 binding region of ACE2 (outlined), with residues that contact the SARS-CoV-2 RBD highlighted [ 6 ]. (D) WebLogo (University of California, Berkeley, USA) [ 53 ] plots summarising the amino acid divergence within the mammalian and bird ACE2 sequences characterised in this study. The single letter amino acid (aa) code is used with the vertical height of the amino acid representing its prevalence at each position in the polypeptide (aa 18–46, 78–91, 324–358, and 392–394 are indicated). The aa sites bound by SARS-CoV and SARS-CoV-2 Spike [ 11 ] are indicated by red arrows. *SARS-CoV-specific interactions. (E) ACE2 sequences were cloned into the pDISPLAY expression construct in frame with an N-terminal signal peptide (the murine Ig κ-chain leader sequence) and HA-tag. (F) Expression of individual mammal or bird ACE2 proteins was confirmed at a whole cell level by western blot. (G) Flow cytometry was performed to examine surface expression of each ACE2 protein on non-permeabilised cells. For gated cells, the percentage positivity and MFI are plotted. The data underlying this figure may be found in S1 Data and S1 Raw Images . aa, amino acid; ACE2, angiotensin-converting enzyme 2; MFI, mean fluorescence intensity; RBD, receptor binding domain; SARS-CoV, SARS Coronavirus; SARS-CoV-2, SARS Coronavirus 2.

    Journal: PLoS Biology

    Article Title: The SARS-CoV-2 Spike protein has a broad tropism for mammalian ACE2 proteins

    doi: 10.1371/journal.pbio.3001016

    Figure Lengend Snippet: The SARS-CoV-2 binding site on ACE2 is highly variable. (A) A phylogenetic tree of ACE2 proteins assembled using the neighbor-joining method [ 51 ] conducted in MEGA7 (Temple University, USA) [ 52 ] with ambiguous positions removed. The tree is drawn to scale, and support was provided with 500 bootstraps. (B) Structure of human ACE2 ectodomain (green) in complex with the RBD of SARS-CoV-2 [ 10 ]. (C) Conservation of mammalian ACE2 amino acid residues, estimated from site-specific evolutionary rates [ 50 ], mapped onto the surface of the ACE2 ectodomain [ 10 ], and coloured from blue (divergent) to purple (conserved) and presented in 2 orientations. Inset depicts the SARS-CoV-2 binding region of ACE2 (outlined), with residues that contact the SARS-CoV-2 RBD highlighted [ 6 ]. (D) WebLogo (University of California, Berkeley, USA) [ 53 ] plots summarising the amino acid divergence within the mammalian and bird ACE2 sequences characterised in this study. The single letter amino acid (aa) code is used with the vertical height of the amino acid representing its prevalence at each position in the polypeptide (aa 18–46, 78–91, 324–358, and 392–394 are indicated). The aa sites bound by SARS-CoV and SARS-CoV-2 Spike [ 11 ] are indicated by red arrows. *SARS-CoV-specific interactions. (E) ACE2 sequences were cloned into the pDISPLAY expression construct in frame with an N-terminal signal peptide (the murine Ig κ-chain leader sequence) and HA-tag. (F) Expression of individual mammal or bird ACE2 proteins was confirmed at a whole cell level by western blot. (G) Flow cytometry was performed to examine surface expression of each ACE2 protein on non-permeabilised cells. For gated cells, the percentage positivity and MFI are plotted. The data underlying this figure may be found in S1 Data and S1 Raw Images . aa, amino acid; ACE2, angiotensin-converting enzyme 2; MFI, mean fluorescence intensity; RBD, receptor binding domain; SARS-CoV, SARS Coronavirus; SARS-CoV-2, SARS Coronavirus 2.

    Article Snippet: For the initial optimisation of pseudoparticle activity, 3 conditions were tested: (1) ACE2 expression only: HEK293T target cells transfected with 500 ng of a human ACE2 expression plasmid (Addgene, USA) and were seeded at 2 × 104 in 100 μL DMEM-10% in a white-bottomed 96-well plate (Corning, USA) 1 day prior to infection; (2) Spike activation with TMPRSS2: HEK293T target cells were transfected with 25 ng TMPRSS2 alongside human ACE2 as above; (3) Spike activation by trypsin treatment: viral pseudoparticles were treated with 2.5 mg/mL trypsin for 1 h at 37°C before addition to target cells overexpressing human ACE2.

    Techniques: Binding Assay, Clone Assay, Expressing, Construct, Sequencing, Western Blot, Flow Cytometry, Fluorescence

    Amino acid substitutions within SARS-CoV-2 Spike RBD may have contributed to zoonotic emergence. (A) Comparison of the RBD structures of SARS-CoV-2 and RaTG13 Spike proteins [ 6 , 12 ] identified a high degree of structural similarity. Nevertheless, a number of amino acid changes between RaTG13 and SARS-CoV-2 were identified at residues interacting directly with ACE2 (according to [ 6 ]). SARS-CoV-2 N439, which does not interact directly with ACE2, was included because of its functional stabilisation role in the 498–505 loop, its previous identification within the SARS-CoV Spike RBD–ACE2 interface [ 19 ], and the N439K substitution present in RaTG13. (B) An XY scatter plot demonstrating the receptor usage profile of RaTG13 Spike in pseudotype (X) and cell–cell fusion assays (Y). Each point represents the mean signal seen from 3 experiments performed on separate days, with the human ACE2 highlighted in blue. Human ACE2 utilisation by SARS-CoV-2 and SARS-CoV Spike is also plotted for reference. In this graph, values are not normalised to human ACE2 since RaTG13 is, to our knowledge, not a human-tropic virus. (C and D) Specific amino acids within the RBDs of SARS-CoV-2 and RaTG13 Spike, which directly interact with ACE2 yet vary between these 2 sequences, were mutated to generate chimaeric SARS-CoV-2-RaTG13 Spike proteins. The cell–cell fusion activity of individual point mutants, as well as a full chimaera containing all 8 mutations (chimaera), was then examined using human ACE2 expressing target cells with activity normalised to the fusion seen with the wildtype (WT) viral glycoprotein of (C) SARS-CoV-2 or (D) RaTG13. The data points shown are the mean cell–cell fusion results seen in 3 completely independent experiments (green, increased relative activity; red, decreased relative activity). * p

    Journal: PLoS Biology

    Article Title: The SARS-CoV-2 Spike protein has a broad tropism for mammalian ACE2 proteins

    doi: 10.1371/journal.pbio.3001016

    Figure Lengend Snippet: Amino acid substitutions within SARS-CoV-2 Spike RBD may have contributed to zoonotic emergence. (A) Comparison of the RBD structures of SARS-CoV-2 and RaTG13 Spike proteins [ 6 , 12 ] identified a high degree of structural similarity. Nevertheless, a number of amino acid changes between RaTG13 and SARS-CoV-2 were identified at residues interacting directly with ACE2 (according to [ 6 ]). SARS-CoV-2 N439, which does not interact directly with ACE2, was included because of its functional stabilisation role in the 498–505 loop, its previous identification within the SARS-CoV Spike RBD–ACE2 interface [ 19 ], and the N439K substitution present in RaTG13. (B) An XY scatter plot demonstrating the receptor usage profile of RaTG13 Spike in pseudotype (X) and cell–cell fusion assays (Y). Each point represents the mean signal seen from 3 experiments performed on separate days, with the human ACE2 highlighted in blue. Human ACE2 utilisation by SARS-CoV-2 and SARS-CoV Spike is also plotted for reference. In this graph, values are not normalised to human ACE2 since RaTG13 is, to our knowledge, not a human-tropic virus. (C and D) Specific amino acids within the RBDs of SARS-CoV-2 and RaTG13 Spike, which directly interact with ACE2 yet vary between these 2 sequences, were mutated to generate chimaeric SARS-CoV-2-RaTG13 Spike proteins. The cell–cell fusion activity of individual point mutants, as well as a full chimaera containing all 8 mutations (chimaera), was then examined using human ACE2 expressing target cells with activity normalised to the fusion seen with the wildtype (WT) viral glycoprotein of (C) SARS-CoV-2 or (D) RaTG13. The data points shown are the mean cell–cell fusion results seen in 3 completely independent experiments (green, increased relative activity; red, decreased relative activity). * p

    Article Snippet: For the initial optimisation of pseudoparticle activity, 3 conditions were tested: (1) ACE2 expression only: HEK293T target cells transfected with 500 ng of a human ACE2 expression plasmid (Addgene, USA) and were seeded at 2 × 104 in 100 μL DMEM-10% in a white-bottomed 96-well plate (Corning, USA) 1 day prior to infection; (2) Spike activation with TMPRSS2: HEK293T target cells were transfected with 25 ng TMPRSS2 alongside human ACE2 as above; (3) Spike activation by trypsin treatment: viral pseudoparticles were treated with 2.5 mg/mL trypsin for 1 h at 37°C before addition to target cells overexpressing human ACE2.

    Techniques: Functional Assay, Activity Assay, Expressing