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: The Journal of Biological Chemistry

Article Title: Tumor Necrosis Factor-α Convertase (ADAM17) Mediates Regulated Ectodomain Shedding of the Severe-acute Respiratory Syndrome-Coronavirus (SARS-CoV) Receptor, Angiotensin-converting Enzyme-2 (ACE2) *

doi: 10.1074/jbc.M505111200

Figure Lengend Snippet: Sequences of sense strands of double-stranded RNA used to ablate specific protein expression in HEK-ACE2 and Huh7 cells

Article Snippet: Plasmid Construction— cDNA encoding full-length human ACE2 (GenBank™ accession number AB046569) was amplified from a human kidney cDNA library (Clontech) using the primer pair 5′-ACGTCCATGTCAAGCTCTTCCTGGCTCCTTCTC-3′ (forward) and 5′-CTAGGCTAAAAGGAGGTCTGAACATCATCAGTGTTT-3′ (reverse), digested, and ligated into the XbaI and XhoI sites of the pCI-Neo expression vector (Promega).

Techniques: Expressing, Sequencing

Journal: The Journal of Biological Chemistry

Article Title: Tumor Necrosis Factor-α Convertase (ADAM17) Mediates Regulated Ectodomain Shedding of the Severe-acute Respiratory Syndrome-Coronavirus (SARS-CoV) Receptor, Angiotensin-converting Enzyme-2 (ACE2) *

doi: 10.1074/jbc.M505111200

Figure Lengend Snippet: Shedding of ACE2 involves loss of its cytoplasmic tail. HEK293 cells were stably transfected with an expression vector encoding full-length ACE2 as described under “Materials and Methods.” OptiMEM containing 0.1 μ m PMA or an equal volume of Me 2 SO carrier was added to exponentially growing cells and collected after 1 h. Following sedimentation of cells, the media were concentrated 10-fold, and 40 μg of media proteins ( M ) were separated by SDS-PAGE (6% v/v) alongside 20 μg of corresponding detergent cell extract ( C ) and immunoblotted with an antibody raised to the ectodomain of ACE2 ( left panel , ectodomain) or the cytosolic tail of ACE2 ( right panel , cytosolic). Immunoreactive bands were visualized with enhanced chemiluminescence as described under “Materials and Methods.”

Article Snippet: Plasmid Construction— cDNA encoding full-length human ACE2 (GenBank™ accession number AB046569) was amplified from a human kidney cDNA library (Clontech) using the primer pair 5′-ACGTCCATGTCAAGCTCTTCCTGGCTCCTTCTC-3′ (forward) and 5′-CTAGGCTAAAAGGAGGTCTGAACATCATCAGTGTTT-3′ (reverse), digested, and ligated into the XbaI and XhoI sites of the pCI-Neo expression vector (Promega).

Techniques: Stable Transfection, Transfection, Expressing, Plasmid Preparation, Sedimentation, SDS Page

Journal: The Journal of Biological Chemistry

Article Title: Tumor Necrosis Factor-α Convertase (ADAM17) Mediates Regulated Ectodomain Shedding of the Severe-acute Respiratory Syndrome-Coronavirus (SARS-CoV) Receptor, Angiotensin-converting Enzyme-2 (ACE2) *

doi: 10.1074/jbc.M505111200

Figure Lengend Snippet: Shed ACE2 occurs as two distinct glycoforms. HEK-ACE2 cells were incubated in OptiMEM containing 0.1 μ m PMA or an equal volume of Me 2 SO carrier for 1 h, and the media were collected and concentrated as described. Media proteins ( M ; 40 μg) or detergent cell extracts ( C ; 20 μg) were incubated at 37 °C for 16 h in the presence or absence of endoglycosidase H ( Endo H ) or PNGase F and subsequently separated by SDS-PAGE. Following electrotransfer, immunoblotting was carried out using an antibody to the ectodomain of ACE2 as described under “Materials and Methods.”

Article Snippet: Plasmid Construction— cDNA encoding full-length human ACE2 (GenBank™ accession number AB046569) was amplified from a human kidney cDNA library (Clontech) using the primer pair 5′-ACGTCCATGTCAAGCTCTTCCTGGCTCCTTCTC-3′ (forward) and 5′-CTAGGCTAAAAGGAGGTCTGAACATCATCAGTGTTT-3′ (reverse), digested, and ligated into the XbaI and XhoI sites of the pCI-Neo expression vector (Promega).

Techniques: Incubation, SDS Page, Electrotransfer, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Tumor Necrosis Factor-α Convertase (ADAM17) Mediates Regulated Ectodomain Shedding of the Severe-acute Respiratory Syndrome-Coronavirus (SARS-CoV) Receptor, Angiotensin-converting Enzyme-2 (ACE2) *

doi: 10.1074/jbc.M505111200

Figure Lengend Snippet: Shedding of ACE2 is inhibited by broad spectrum hydroxamate-based metalloprotease inhibitors. HEK-ACE2 cells were incubated in OptiMEM containing various concentrations of the secretase inhibitors TAPI-1 or GM6001 or an equal volume of carrier (Me 2 SO). After 15 min, PMA (0.1 μ m ) or an equal volume of Me 2 SO was added, and incubation was continued for 1 h. The medium was subsequently harvested and concentrated as described; cells were pelleted and detergent extracts were collected as described under “Materials and Methods.” A , media proteins ( upper panel , 40 μg) and cell lysates ( lower panel , 20 μg) were separated by SDS-PAGE and immunoblotted for ACE2. Immunoreactive bands were visualized by enhanced chemiluminescence. B , graphical representation of results of densitometric analysis of three such experiments, ± S.E. Black shading , –PMA; gray shading , +PMA.

Article Snippet: Plasmid Construction— cDNA encoding full-length human ACE2 (GenBank™ accession number AB046569) was amplified from a human kidney cDNA library (Clontech) using the primer pair 5′-ACGTCCATGTCAAGCTCTTCCTGGCTCCTTCTC-3′ (forward) and 5′-CTAGGCTAAAAGGAGGTCTGAACATCATCAGTGTTT-3′ (reverse), digested, and ligated into the XbaI and XhoI sites of the pCI-Neo expression vector (Promega).

Techniques: Incubation, SDS Page

Journal: The Journal of Biological Chemistry

Article Title: Tumor Necrosis Factor-α Convertase (ADAM17) Mediates Regulated Ectodomain Shedding of the Severe-acute Respiratory Syndrome-Coronavirus (SARS-CoV) Receptor, Angiotensin-converting Enzyme-2 (ACE2) *

doi: 10.1074/jbc.M505111200

Figure Lengend Snippet: PMA-stimulated ACE2 shedding is sensitive to ADAM17 inhibition. HEK-ACE2 cells were incubated for 15 min in the presence of the ADAM10 inhibitor GI254023X or the mixed ADAM10/ADAM17 inhibitor GW280264X or an equal volume of Me 2 SO. Subsequently, incubation was continued in the presence of PMA (0.1 μ m ). Media were harvested and concentrated as described under “Materials and Methods.” A , media proteins (40 μg) were separated by SDS-PAGE and immunoblotted for ACE2. B , graphical representation of results of densitometric analysis of three such experiments, ± S.E.

Article Snippet: Plasmid Construction— cDNA encoding full-length human ACE2 (GenBank™ accession number AB046569) was amplified from a human kidney cDNA library (Clontech) using the primer pair 5′-ACGTCCATGTCAAGCTCTTCCTGGCTCCTTCTC-3′ (forward) and 5′-CTAGGCTAAAAGGAGGTCTGAACATCATCAGTGTTT-3′ (reverse), digested, and ligated into the XbaI and XhoI sites of the pCI-Neo expression vector (Promega).

Techniques: Inhibition, Incubation, SDS Page

Journal: The Journal of Biological Chemistry

Article Title: Tumor Necrosis Factor-α Convertase (ADAM17) Mediates Regulated Ectodomain Shedding of the Severe-acute Respiratory Syndrome-Coronavirus (SARS-CoV) Receptor, Angiotensin-converting Enzyme-2 (ACE2) *

doi: 10.1074/jbc.M505111200

Figure Lengend Snippet: Stimulated ACE2 shedding is inhibited by NTIMP3 but not by NTIMP1. HEK-ACE2 cells were incubated for 15 min in the presence of NTIMP1, NTIMP3, or an equal volume of phosphate-buffered saline. Subsequently, incubation was continued in the presence of PMA (0.1 μ m ). Media were harvested and concentrated as described. A , media proteins (40 μg) were separated by SDS-PAGE and immunoblotted for ACE2. B , graphical representation of results of densitometric analysis of the immunoblots of three such experiments, ± S.E.

Article Snippet: Plasmid Construction— cDNA encoding full-length human ACE2 (GenBank™ accession number AB046569) was amplified from a human kidney cDNA library (Clontech) using the primer pair 5′-ACGTCCATGTCAAGCTCTTCCTGGCTCCTTCTC-3′ (forward) and 5′-CTAGGCTAAAAGGAGGTCTGAACATCATCAGTGTTT-3′ (reverse), digested, and ligated into the XbaI and XhoI sites of the pCI-Neo expression vector (Promega).

Techniques: Incubation, SDS Page, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Tumor Necrosis Factor-α Convertase (ADAM17) Mediates Regulated Ectodomain Shedding of the Severe-acute Respiratory Syndrome-Coronavirus (SARS-CoV) Receptor, Angiotensin-converting Enzyme-2 (ACE2) *

doi: 10.1074/jbc.M505111200

Figure Lengend Snippet: Ablation of ADAM17 expression by siRNA reduces stimulated ACE2 shedding. HEK-ACE2 cells were transiently transfected with a mixture of double-stranded RNA derived from the coding sequence of ADAM10, ADAM17, or the control sequence GL2 as described under “Materials and Methods.” Twenty-four hours after transfection, cells were incubated in OptiMEM containing 0.1 μ m PMA for 1 h. Media were concentrated as described, and cell lysates were prepared. A , media proteins (40 μg) and cell lysates (50 μg) were separated by SDS-PAGE and immunoblotted for ACE2, ADAM10, and ADAM17, as indicated. Mock , mock transfection. B , graphical representation of densitometric analysis of immunoblots of media ACE2 from three such experiments, ± S.E.

Article Snippet: Plasmid Construction— cDNA encoding full-length human ACE2 (GenBank™ accession number AB046569) was amplified from a human kidney cDNA library (Clontech) using the primer pair 5′-ACGTCCATGTCAAGCTCTTCCTGGCTCCTTCTC-3′ (forward) and 5′-CTAGGCTAAAAGGAGGTCTGAACATCATCAGTGTTT-3′ (reverse), digested, and ligated into the XbaI and XhoI sites of the pCI-Neo expression vector (Promega).

Techniques: Expressing, Transfection, Derivative Assay, Sequencing, Incubation, SDS Page, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Tumor Necrosis Factor-α Convertase (ADAM17) Mediates Regulated Ectodomain Shedding of the Severe-acute Respiratory Syndrome-Coronavirus (SARS-CoV) Receptor, Angiotensin-converting Enzyme-2 (ACE2) *

doi: 10.1074/jbc.M505111200

Figure Lengend Snippet: Overexpression of ADAM17 increases PMA-stimulated ACE2 shedding. HEK-ACE2 cells were transiently transfected with an expression vector encoding ADAM9, ADAM10, or ADAM17, as described under “Materials and Methods.” Thirty-six hours after transfection, cells were incubated in OptiMEM containing 0.1 μ m PMA for 1 h. Media were concentrated as described, and detergent cell extracts were harvested. A , media proteins (40 μg) and detergent cell extracts (50 μg) were separated by SDS-PAGE and immunoblotted for ACE2 ( upper panel , media), ADAM9, ADAM10, or ADAM17 ( lower panels , lysates). Membranes were stripped and reprobed for β-actin as a loading control. B , graphical representation of densitometric analysis of immunoblots of media ACE2 from three such experiments, ± S.E.

Article Snippet: Plasmid Construction— cDNA encoding full-length human ACE2 (GenBank™ accession number AB046569) was amplified from a human kidney cDNA library (Clontech) using the primer pair 5′-ACGTCCATGTCAAGCTCTTCCTGGCTCCTTCTC-3′ (forward) and 5′-CTAGGCTAAAAGGAGGTCTGAACATCATCAGTGTTT-3′ (reverse), digested, and ligated into the XbaI and XhoI sites of the pCI-Neo expression vector (Promega).

Techniques: Over Expression, Transfection, Expressing, Plasmid Preparation, Incubation, SDS Page, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Tumor Necrosis Factor-α Convertase (ADAM17) Mediates Regulated Ectodomain Shedding of the Severe-acute Respiratory Syndrome-Coronavirus (SARS-CoV) Receptor, Angiotensin-converting Enzyme-2 (ACE2) *

doi: 10.1074/jbc.M505111200

Figure Lengend Snippet: PMA-stimulated endogenous ACE2 shedding is sensitive to ADAM17 inhibition. A , OptiMEM containing 0.1 μ m PMA or an equal volume of Me 2 SO carrier was added to exponentially growing Huh7 cells and collected after 6 h. Following sedimentation of cells, the media were concentrated, and 100 μg of media proteins ( M ) was separated by SDS-PAGE (4–12% v/v) alongside 50 μg of corresponding detergent cell extract ( C ) and immunoblotted with an antibody raised to the ectodomain of ACE2 ( left panel , ectodomain) or the cytosolic tail of ACE2 ( right panel , cytosolic). Immunoreactive bands were visualized with enhanced chemiluminescence as described under “Materials and Methods.” B , Huh7 cells were incubated for 15 min in the presence of 50 μ m GM6001 or 1 μ m the ADAM10 inhibitor GI254023X or the mixed ADAM10/ADAM17 inhibitor GW280264X or an equal volume of Me 2 SO. Subsequently, incubation was continued for 4 h in the presence or absence of PMA (0.1 μ m ). Media were harvested and concentrated as described under “Materials and Methods.” Concentrated media samples (20 μg) were assayed for their ability to cleave an ACE2-specific fluorogenic substrate, as described under “Materials and Methods.” Black shading , –PMA; gray shading , +PMA.

Article Snippet: Plasmid Construction— cDNA encoding full-length human ACE2 (GenBank™ accession number AB046569) was amplified from a human kidney cDNA library (Clontech) using the primer pair 5′-ACGTCCATGTCAAGCTCTTCCTGGCTCCTTCTC-3′ (forward) and 5′-CTAGGCTAAAAGGAGGTCTGAACATCATCAGTGTTT-3′ (reverse), digested, and ligated into the XbaI and XhoI sites of the pCI-Neo expression vector (Promega).

Techniques: Inhibition, Sedimentation, SDS Page, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Tumor Necrosis Factor-α Convertase (ADAM17) Mediates Regulated Ectodomain Shedding of the Severe-acute Respiratory Syndrome-Coronavirus (SARS-CoV) Receptor, Angiotensin-converting Enzyme-2 (ACE2) *

doi: 10.1074/jbc.M505111200

Figure Lengend Snippet: Ablation of ADAM17 expression by siRNA reduces regulated endogenous ACE2 shedding. Huh7 cells (50% confluent) were transfected with double-stranded RNA oligomers (as described under “Materials and Methods”) to ADAM10, ADAM17, or a negative control sequence. After 40 h, the media were aspirated and replaced with OptiMEM containing 100 n m PMA or an equal volume of Me 2 SO. After a further 5-h incubation, the media were concentrated, and cell lysates were prepared as described under “Materials and Methods.” Cell lysates (50 μg) were separated by SDS-PAGE and immunoblotted for ADAM10 ( A ) and ADAM17 ( B ). Concentrated media samples (20 μg) were assayed for their ability to cleave an ACE2-specific fluorogenic substrate as described under “Materials and Methods.”

Article Snippet: Plasmid Construction— cDNA encoding full-length human ACE2 (GenBank™ accession number AB046569) was amplified from a human kidney cDNA library (Clontech) using the primer pair 5′-ACGTCCATGTCAAGCTCTTCCTGGCTCCTTCTC-3′ (forward) and 5′-CTAGGCTAAAAGGAGGTCTGAACATCATCAGTGTTT-3′ (reverse), digested, and ligated into the XbaI and XhoI sites of the pCI-Neo expression vector (Promega).

Techniques: Expressing, Transfection, Negative Control, Sequencing, Incubation, SDS Page

Journal: The Journal of Biological Chemistry

Article Title: Tumor Necrosis Factor-α Convertase (ADAM17) Mediates Regulated Ectodomain Shedding of the Severe-acute Respiratory Syndrome-Coronavirus (SARS-CoV) Receptor, Angiotensin-converting Enzyme-2 (ACE2) *

doi: 10.1074/jbc.M505111200

Figure Lengend Snippet: Overexpression of ADAM17 increases PMA-stimulated endogenous ACE2 shedding. HEK-ACE2 cells were transiently transfected with an expression vector encoding ADAM9, ADAM10, or ADAM17, as described under “Materials and Methods.” Thirty-six hours after transfection, cells were incubated in OptiMEM containing 0.1 μ m PMA for 4 h. Media were concentrated as described, and detergent cell extracts were harvested. A , detergent cell extracts (50 μg) were separated by SDS-PAGE and immunoblotted for ADAM9, ADAM10, or ADAM17. Membranes were stripped and reprobed for β-actin as a loading control. B , concentrated media samples (20 μg) were assayed for their ability to cleave an ACE2-specific fluorogenic substrate as described under “Materials and Methods.”

Article Snippet: Plasmid Construction— cDNA encoding full-length human ACE2 (GenBank™ accession number AB046569) was amplified from a human kidney cDNA library (Clontech) using the primer pair 5′-ACGTCCATGTCAAGCTCTTCCTGGCTCCTTCTC-3′ (forward) and 5′-CTAGGCTAAAAGGAGGTCTGAACATCATCAGTGTTT-3′ (reverse), digested, and ligated into the XbaI and XhoI sites of the pCI-Neo expression vector (Promega).

Techniques: Over Expression, Transfection, Expressing, Plasmid Preparation, Incubation, SDS Page

Journal: eLife

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

doi: 10.7554/eLife.61552

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

Article Snippet: Recombinant DNA reagent , ACE2 [v2] , This paper , Derived from RRID: Addgene_1786 , Plasmid to express full length human ACE2.

Techniques: Liquid Chromatography with Mass Spectroscopy, Glycoproteomics

Journal: eLife

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

doi: 10.7554/eLife.61552

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

Article Snippet: Recombinant DNA reagent , ACE2 [v2] , This paper , Derived from RRID: Addgene_1786 , Plasmid to express full length human ACE2.

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

Journal: eLife

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

doi: 10.7554/eLife.61552

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

Article Snippet: Recombinant DNA reagent , ACE2 [v2] , This paper , Derived from RRID: Addgene_1786 , Plasmid to express full length human ACE2.

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

Journal: eLife

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

doi: 10.7554/eLife.61552

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

Article Snippet: Recombinant DNA reagent , ACE2 [v2] , This paper , Derived from RRID: Addgene_1786 , Plasmid to express full length human ACE2.

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

Journal: eLife

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

doi: 10.7554/eLife.61552

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

Article Snippet: Recombinant DNA reagent , ACE2 [v2] , This paper , Derived from RRID: Addgene_1786 , Plasmid to express full length human ACE2.

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

Journal: eLife

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

doi: 10.7554/eLife.61552

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

Article Snippet: Recombinant DNA reagent , ACE2 [v2] , This paper , Derived from RRID: Addgene_1786 , Plasmid to express full length human ACE2.

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

Journal: eLife

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

doi: 10.7554/eLife.61552

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

Article Snippet: Recombinant DNA reagent , ACE2 [v2] , This paper , Derived from RRID: Addgene_1786 , Plasmid to express full length human ACE2.

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

Journal: eLife

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

doi: 10.7554/eLife.61552

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

Article Snippet: Recombinant DNA reagent , ACE2 [v2] , This paper , Derived from RRID: Addgene_1786 , Plasmid to express full length human ACE2.

Techniques: Binding Assay, Expressing, Generated, Virus

Journal: eLife

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

doi: 10.7554/eLife.61552

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , ACE2 [v2] , This paper , Derived from RRID: Addgene_1786 , Plasmid to express full length human ACE2.

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

Journal: Nature Chemical Biology

Article Title: Engineered ACE2 decoy mitigates lung injury and death induced by SARS-CoV-2 variants

doi: 10.1038/s41589-021-00965-6

Figure Lengend Snippet: a – c , Based on simulations of RBD-bound ACE2, new polar interactions were identified between the ACE2.v2.4 (orange) and RBD (yellow) loops (red) ( a ). ACE2 mutations are presented as sticks colored cyan with nearby RBD residues as sticks colored yellow. New polar interactions between ACE2.v2.4 and RBD loops 1 ( b ) and 2 ( c ) are indicated by the dotted blue lines. d , MSM-weighted distance distributions of newly formed hydrogen bonds. For each interacting pair, the ACE2.v2.4 residue is listed first and the RBD residue is listed second. e , Probabilities for stable hydrogen bond interactions using a 4-Å distance criterion between accepter and donor. f , Root mean square fluctuation (RMSF) of RBD residues when bound to WT (cyan) or v2.4 (orange) ACE2 receptors. The RBD regions that interface with ACE2 are shaded gray. The absolute difference in RMSF between WT and v2.4 proteins is shown in black. Distance distributions, hydrogen bond probabilities and RMSF calculations were based on 40,000 frames from the simulations. Frames were selected based on the MSM stationary probability to represent the entire conformational ensemble. The error bars represent the 95% confidence intervals calculated from 20 bootstrapped samples.

Article Snippet: For immunoblot analysis of protein degradation, samples were prepared in reducing (for the ACE2 blots) or nonreducing (for the human IgG1 blots) SDS load dye and separated by PAGE, transferred to polyvinylidene fluoride membrane, blocked with 5% skimmed milk and stained with 1:2,000 horseradish peroxidase (HRP)-conjugated antihuman ACE2 (OTI1D2 clone; Origene) or 1:5,000 HRP-conjugated donkey antihuman IgG (Jackson ImmunoResearch) in Tris-buffered saline containing 0.1% Tween 20 and 1% skimmed milk.

Techniques:

Journal: Nature Chemical Biology

Article Title: Engineered ACE2 decoy mitigates lung injury and death induced by SARS-CoV-2 variants

doi: 10.1038/s41589-021-00965-6

Figure Lengend Snippet: a , mRNA expression of ACE2 (NM_001371415.1) and TMPRSS2 (NM_001135099) in human lung epithelial A549 cells. A549 cells stably expressing hACE2 and hLMVECs were analyzed by one-step RT–PCR (top) and real-time PCR (bottom). Relative expression was normalized to glyceraldehyde 3-phosphate dehydrogenase expression. b , Cultured hACE2-A549 cells, A549 cells and hLMVECs were preincubated with sACE2 2 -IgG1 or sACE2 2 .v2.4-IgG1 at 5 µg ml −1 or 25 µg ml −1 for 1 h. SARS-CoV-2 pseudovirus (MOI = 0.1) was added to the cells, which were collected at 24 h. Virus entry was evaluated by luciferase activity. n = 4 replicates. c , A dose of 10 mg kg −1 sACE2 2 -IgG1, sACE2 2 .v2.4-IgG1 or buffer (PBS + 0.2% BSA) was administrated intravenously into K18-hACE2 transgenic mice for 30 min before SARS-CoV-2 pseudovirus (10 6 PFU) intraperitoneal injection. Tissue lysates were prepared at 24 h and virus entry in the selected organs was evaluated by luciferase activity. Buffer was applied as the control group, n = 4. d – g , K18-hACE2 transgenic mice were inoculated with the SARS-CoV-2 isolate WA-1/2020 at 1 × 10 4 PFU. Mice received control PBS or sACE2 2 .v2.4-IgG1 10 mg kg −1 via intravenous injection 12 h before inoculation. Mice were observed for survival ( d ) and body weight ( e ), n = 5. EBA as a marker of pulmonary endothelial permeability ( f ) and lung wet/dry ratio ( g ) as a measure of lung edema were quantified. Data are presented as the mean ± s.e.m., n = 4. b , c , f , g , P values were calculated by one-way ANOVA with Tukey post hoc test.

Article Snippet: For immunoblot analysis of protein degradation, samples were prepared in reducing (for the ACE2 blots) or nonreducing (for the human IgG1 blots) SDS load dye and separated by PAGE, transferred to polyvinylidene fluoride membrane, blocked with 5% skimmed milk and stained with 1:2,000 horseradish peroxidase (HRP)-conjugated antihuman ACE2 (OTI1D2 clone; Origene) or 1:5,000 HRP-conjugated donkey antihuman IgG (Jackson ImmunoResearch) in Tris-buffered saline containing 0.1% Tween 20 and 1% skimmed milk.

Techniques: Expressing, Stable Transfection, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Cell Culture, Luciferase, Activity Assay, Transgenic Assay, Injection, Marker, Permeability

Journal: Nature Chemical Biology

Article Title: Engineered ACE2 decoy mitigates lung injury and death induced by SARS-CoV-2 variants

doi: 10.1038/s41589-021-00965-6

Figure Lengend Snippet: a – d , sACE2 carrying the v2.4 mutations has increased S binding compared with WT sACE2. Human Expi293F cells expressing myc-tagged S from 4 SARS-CoV-2 variants (Wuhan ( a ), B.1.1.7/Alpha ( b ), B1.351/Beta ( c ) and P.1/Gamma ( d )) were incubated with monomeric sACE2-8 h (black) or dimeric sACE2 2 -IgG1 (gray); bound protein was detected by flow cytometry. WT ACE2 proteins are shown as broken lines, v2.4 proteins are shown as solid lines. n = 3 independent replicates; data are shown as the mean ± s.e.m. e – i , Binding of sACE2 2 .v2.4-IgG1 is comparable to clinically effective mAbs. Binding of mAbs versus sACE2 2 .v2.4-IgG1 to the S proteins of SARS-CoV-2 VOCs (B.1.1.7/Alpha ( e ), B1.351/Beta ( f ), B.1.617.2/Delta ( g ) and P.1/Gamma ( h )) and S protein of SARS-CoV-1 ( i ), as measured by flow cytometry. n = 3 independent replicates; data are shown as the mean ± s.e.m.

Article Snippet: For immunoblot analysis of protein degradation, samples were prepared in reducing (for the ACE2 blots) or nonreducing (for the human IgG1 blots) SDS load dye and separated by PAGE, transferred to polyvinylidene fluoride membrane, blocked with 5% skimmed milk and stained with 1:2,000 horseradish peroxidase (HRP)-conjugated antihuman ACE2 (OTI1D2 clone; Origene) or 1:5,000 HRP-conjugated donkey antihuman IgG (Jackson ImmunoResearch) in Tris-buffered saline containing 0.1% Tween 20 and 1% skimmed milk.

Techniques: Binding Assay, Expressing, Incubation, Flow Cytometry

Journal: PLoS Pathogens

Article Title: Phosphatidylserine receptors enhance SARS-CoV-2 infection

doi: 10.1371/journal.ppat.1009743

Figure Lengend Snippet: A) Cells transfected with PS receptor expression plasmids, AXL or TIM-1, with or without 50 ng of ACE2 and infected 48 hours later with SARS-CoV-2 (MOI = 0.5). Resulting cellular infection was determined by viral loads 24 hours after initial infection using RT-qPCR. B-C) PS receptors, TIM-1 (B) and AXL (C) , enhance rVSV/Spike infection at low concentrations of transfected ACE2. D) Virus binding of cells transfected with PS receptor plasmids with or without 50 ng of ACE2. rVSV/Spike was bound to transfected cells at 48 h following transfection and bound virus was measured via RT-qPCR . E) PS receptors mediate internalization of rVSV/Spike. Virion internalization was measured at 24 h after transfection with 1 μg of the indicated plasmids. FITC-labeled rVSV/Spike was bound for 1-hour, unbound virions washed away, and cells shifted to 37°C for 30 minutes. Non-internalized virus was then cleaved from cell surface by trypsin. Cells were washed, and FITC retention quantified by flow cytometry. F) HEK 293T cells transfected with PS receptor plasmids, TYRO3 or TIM-4, with or without 250 ng of ACE2 and infected 48 hours later with VSV/Spike. Viral loads were determined 24 hours following infection. Data shown are pooled from at least 3 independent experiments ( A , B , C , D , E , F ). Data represented as means ± SEM. Student’s t-test (A, E) and multiple t-test (B, C) , One-Way ANOVA with multiple comparisons (D, F) ; asterisks represent p < 0.05.

Article Snippet: Specific primary antibodies used as follows: goat anti-ACE2 (R&D; AF933), goat anti-AXL (R&D; 154), goat anti-TIM-1 (R&D; 1750), goat anti- Tyro3 (R&D; AF859), goat anti-TIM-4 (R&D; 2929), goat anti-MerTK (R&D; AF891), rabbit anti-TMPRSS2 (ABCAM; ab92323).

Techniques: Transfection, Expressing, Infection, Quantitative RT-PCR, Binding Assay, Labeling, Flow Cytometry

Journal: PLoS Pathogens

Article Title: Phosphatidylserine receptors enhance SARS-CoV-2 infection

doi: 10.1371/journal.ppat.1009743

Figure Lengend Snippet: A) PS is readily detectable on UV irradiated SARS-CoV-2 virions and rVSV/Spike. Indicated quantities of viral particles (determined by protein content) were coated in ELISA plates, and PS was detected using bavituximab followed by secondary antisera . B-C) PS liposomes interfere with rVSV/Spike infection. HEK 293T cells transfected with 50 ng of ACE2 plasmid and 1 μg of TIM-1 ( B ) or AXL ( C ) plasmid. Cells were infected with rVSV/Spike in the presence of increasing concentrations of PS or PC liposomes and assessed for nanoluciferase activity 24 hours later. D) HEK 293T cells were transfected with 1 μg of plasmid expressing WT or PS binding pocket mutant TIM-1 (ND115DN) with or without 250 ng of ACE2 plasmid and infected 48 hours later with rVSV/Spike. Luminescence fold change were compared to Mock transfected lysates that were set to a value of 1. E) Surface expressed AXL is unable to directly interact with purified SARS-CoV-2 spike/Fc proteins. HEK 293T cells transfected with AXL or ACE2 were incubated with soluble Spike protein-Fc, S1 RBD-Fc or S1 NTD-Fc and subsequently incubated with an Alexa 647 secondary against Fc. Transfected cells bound to spike constructs were detected by flow cytometry. F) Purified AXL does not bind to the NTD of SARS-CoV-2 spike. Biolayer interferometry association curves show that immobilized AXL-Fc fails to interact with purified NTD of spike. Data are pooled from at least 3 independent experiments ( B , C ) or are representative of at least 3 experiments ( A , D , E , F ). Data represented as means (or individual datapoints) ± SEM. Multiple t-test ( B , C ), One-way ANOVA with multiple comparisons ( A , D ); asterisks represent p < 0.05.

Article Snippet: Specific primary antibodies used as follows: goat anti-ACE2 (R&D; AF933), goat anti-AXL (R&D; 154), goat anti-TIM-1 (R&D; 1750), goat anti- Tyro3 (R&D; AF859), goat anti-TIM-4 (R&D; 2929), goat anti-MerTK (R&D; AF891), rabbit anti-TMPRSS2 (ABCAM; ab92323).

Techniques: Irradiation, Enzyme-linked Immunosorbent Assay, Infection, Transfection, Plasmid Preparation, Activity Assay, Expressing, Binding Assay, Mutagenesis, Purification, Incubation, Construct, Flow Cytometry

Journal: PLoS Pathogens

Article Title: Phosphatidylserine receptors enhance SARS-CoV-2 infection

doi: 10.1371/journal.ppat.1009743

Figure Lengend Snippet: A) PS liposomes interfere with SARS-CoV-2 pseudovirion entry. Vero E6 cells were treated with increasing concentrations of PS or PC liposomes and infected with VSV/Spike pseudovirions for 24 hours. Infection was detected by GFP fluorescence expressed from the VSV genome. B) PS liposomes disrupt SARS-CoV-2 binding. Vero E6 cells were incubated with SARS-CoV-2 (MOI = 5) at 10°C for 1 hour in the presence of indicated liposomes, washed extensively, and viral load assessed by RT-qPCR. C) AXL signaling inhibitor bemcentinib inhibits SARS-CoV-2 infection in Vero E6 cells. Cells were treated with bemcentinib and infected with SARS-CoV-2 (MOI = 0.01). Viral loads were measured 24 hpi by RT-qPCR. E) RNAseq studies in Vero E6 cells demonstrate bemcentinib inhibition. Cells were treated with 1 μM bemcentinib, infected with SARS-CoV-2 (MOI = 0.01) and mRNA harvested 18 hpi. mRNA was deep sequenced on an Illumina platform, and ‘Percent Viral Reads’ were calculated by alignment to the SARS-CoV-2 genome. E) Broad spectrum TAM inhibitor BMS-777607 inhibits SARS-CoV-2 infection in Vero E6 cells. Cells were treated with inhibitor at indicated concentrations prior to challenged (MOI = 0.01), and viral loads measured 24 hpi by RT-qPCR. F-G) Enhanced colocalization of AXL and ACE2 during SARS-CoV-2 infection. STED micrographs shows staining for ACE2 (red) and AXL (green) and merged in Vero E6 cells ( F ). Insets are enlarged images from regions highlighted by yellow rectangles. White arrows indicate shared vesicular structures between the two channels. Yellow arrowheads indicate objects that are only seen in one channel. Plot profiles are shown in S4F , representing signal intensity along the yellow lines in the merged panels. Pearson’s correlation coefficients of ACE2 and AXL were calculated for n = 20 mock and infected cells (ROI determined by cell borders) ( G ). Data are pooled from at least 3 independent experiments ( B , E ) or are representative of at least 3 experiments ( A , C , F , G ). Data are represented as means ± SEM. Multiple t-tests ( A ) Student’s t-test ( B , C , D , G ); asterisks represent p < 0.05.

Article Snippet: Specific primary antibodies used as follows: goat anti-ACE2 (R&D; AF933), goat anti-AXL (R&D; 154), goat anti-TIM-1 (R&D; 1750), goat anti- Tyro3 (R&D; AF859), goat anti-TIM-4 (R&D; 2929), goat anti-MerTK (R&D; AF891), rabbit anti-TMPRSS2 (ABCAM; ab92323).

Techniques: Infection, Fluorescence, Binding Assay, Incubation, Quantitative RT-PCR, Inhibition, Staining

Journal: PLoS Pathogens

Article Title: Phosphatidylserine receptors enhance SARS-CoV-2 infection

doi: 10.1371/journal.ppat.1009743

Figure Lengend Snippet: A-E) SARS-CoV-2 infection (MOI = 0.5) is reduced by AXL inhibition by bemcentinib or E64 in multiple human lung cell lines, including A549 ACE2 ( A ), H1650 ( B ), HCC1944 ( C ), HCC2302 ( D ). Inhibitors were added to cells 1 h prior to infection and maintained on the cells for the entire infection. At 24 hpi, viral load was determined. E) Infectious virus produced by HCC2302 cells was inhibited by bemcentinib. HCC2302 cells were treated with bemcentinib at the indicated concentrations and infected with SARS-CoV-2 (MOI = 0.5). Input virus was removed 6 hpi and media containing the appropriate bemcentinib concentration was added. Supernatant was collected at 24 and 48 hpi and titered by TCID 50 assays on Vero E6-TMPRSS2 cells. TCID 50 /mL was calculated by the Spearmann-Karber method. F) Timing of effect bemcentinib inhibition of SARS-CoV-2 infection. In time-of-addition studies, SARS-CoV-2 (MOI = 0.01) was added to infected H1650 cells (human lung cells) to initiate the experiment and 1 μM bemcentinib was added at the times noted. Cells were harvested at 24 hpi for viral load determinations. G) Calu-3 are insensitive to bemcentinib and E64. Studies were performed as described for panels A-E. H) A549 ACE2 were treated with bemcentinib, infected with SARS-CoV-2 (MOI = 0.5) and mRNA harvested 24 hpi. mRNA was deep sequenced and viral loads calculated by alignment to the SARS-CoV-2 genome. Data are representative of at least 3 experiments ( A , B , C , D , E , F , G ). Data represented as means ± SEM. Student’s t-test; asterisks represent p < 0.05.

Article Snippet: Specific primary antibodies used as follows: goat anti-ACE2 (R&D; AF933), goat anti-AXL (R&D; 154), goat anti-TIM-1 (R&D; 1750), goat anti- Tyro3 (R&D; AF859), goat anti-TIM-4 (R&D; 2929), goat anti-MerTK (R&D; AF891), rabbit anti-TMPRSS2 (ABCAM; ab92323).

Techniques: Infection, Inhibition, Produced, Concentration Assay

Journal: Vaccine

Article Title: SARS-COV-2 recombinant Receptor-Binding-Domain (RBD) induces neutralizing antibodies against variant strains of SARS-CoV-2 and SARS-CoV-1

doi: 10.1016/j.vaccine.2021.08.081

Figure Lengend Snippet: Vaccinated antisera blocks RBD-ACE-2 interaction. (A) 3-fold serial diluted antisera was added to micro-titer plates coated with recombinant RBD protein. After 30 min incubation, FLAG-tagged ACE-2 protein were added and detected with anti-FLAG antibody. Pooled pre-immune serum of each group was used as a control and theamount of bound ACE-2 determined. Each colored line represents serum of an indvidual mouse with pooled pre-immune serum in grey. (B) Reciprocal Inhibitory Dose 50 (IC50) was calculated for sera of each animals. Comparison of IC50 values between the two adjuvants is shown. (*) indicates p < 0.05 in unpaired T-test.

Article Snippet: Full length human ACE-2-MycDDK in pCMV-6 entry vector (Origene, cat #RC208442) was expressed in Expi293™ cells.

Techniques: Recombinant, Incubation

Journal: Vaccine

Article Title: SARS-COV-2 recombinant Receptor-Binding-Domain (RBD) induces neutralizing antibodies against variant strains of SARS-CoV-2 and SARS-CoV-1

doi: 10.1016/j.vaccine.2021.08.081

Figure Lengend Snippet: Vaccination-induced neutralizing antibodies (nAb) protects from SARS-CoV-2 infection. Pre-immune or post-vaccinated mice sera were evaluated for their ability to neutralize SARS-CoV-2 pseudoparticles (CoV2pp) (A) or infectious SARs-CoV-2 virions (B). (A) Neutralization CoV2pp was performed using pre- and post- vaccination sera (1:50) in 293 T ACE-2 cells. The group means (in triplicate) with SEM were plotted and virus/particle entry normalized to entry of CoV2pp in the presence of PBS as 100%. (B) Neutralization titer (N100) of vaccinated mouse sera was determined in Vero E6 cells using infectious SARS-CoV-2. Serially diluted sera were pre-incubated with 100 PFU of SARS-CoV-2 for 1 h at 37 °C followed by addition to Vero E6 cells. Three days post-infection, cells were formaldehyde fixed and stained with crystal violet. Neutralization titer was determined as the minimal dilution of each mouse serum required to prevent CPE. A representative of two independent experiments (performed in duplicate) is shown. (*) indicates p < 0.05 in Tukey’s multiple comparisons test. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Full length human ACE-2-MycDDK in pCMV-6 entry vector (Origene, cat #RC208442) was expressed in Expi293™ cells.

Techniques: Infection, Neutralization, Incubation, Staining

Journal: Vaccine

Article Title: SARS-COV-2 recombinant Receptor-Binding-Domain (RBD) induces neutralizing antibodies against variant strains of SARS-CoV-2 and SARS-CoV-1

doi: 10.1016/j.vaccine.2021.08.081

Figure Lengend Snippet: Antisera exhibits similar neutralization activity against SARS-CoV-2 pp containing variant mutations N501Y/E484K/K417N. Neutralization activity of mouse sera were tested at 1:50 to 1:109,350 in 3-fold dilution against lentivirus particles pseudotyped with either spike of SARS-CoV-2(WT) or S encoding triple mutations N501Y/E484K/K417N. Pre- or Post-vaccination sera were pre-incubated with pseudoparticles (PP) encoding different surface proteins for 1 h followed by addition to 293 T ACE-2 cells. 48 h post-transduction, entry of PP were quantitated by luciferase activity andresults (in duplicate) normalized to entry of PP in the presence of PBS. IC50 of neutralization activity was calculated and compared between groups. (A) Mean and standard error of IC50 values is shown. (*) indicates p < 0.05 in Tukey’s multiple comparisons test. (B) Change in neutralization (IC50) between WT and variant (501/484/417) PP. A representative of two independent experiments (performed in duplicate) is shown with fold change of the mean IC50 value compared to WT.

Article Snippet: Full length human ACE-2-MycDDK in pCMV-6 entry vector (Origene, cat #RC208442) was expressed in Expi293™ cells.

Techniques: Neutralization, Activity Assay, Variant Assay, Incubation, Transduction, Luciferase

Journal: Vaccine

Article Title: SARS-COV-2 recombinant Receptor-Binding-Domain (RBD) induces neutralizing antibodies against variant strains of SARS-CoV-2 and SARS-CoV-1

doi: 10.1016/j.vaccine.2021.08.081

Figure Lengend Snippet: Antisera from RBD SARS-CoV-2 vaccinated mice exhibits cross-neutralization against SARS-CoV-1. RBD is either formulated with Alum + MPLA (left) or with AddaS03 (right). Neutralization activity of mouse sera were tested at 1:100 against pseudotyped virus particles (PP) expressing either SARS-CoV-2 spike (CoV-2), SARS-CoV-1 spike (CoV-1) or the glycoprotein of VSV (VSV). Pre- or post-vaccination sera were pre-incubated with PP and added to 293 T ACE-2 cells and assessed for luciferase activity according to the Materials and Methods. Triplicate samples were normalized to entry of Pre-immune sera and the mean with SEM plotted. (*) indicates p < 0.05 in Tukey's multiple comparison test. A representative of two independent experiments done in triplicates is shown.

Article Snippet: Full length human ACE-2-MycDDK in pCMV-6 entry vector (Origene, cat #RC208442) was expressed in Expi293™ cells.

Techniques: Neutralization, Activity Assay, Expressing, Incubation, Luciferase

Journal: Signal Transduction and Targeted Therapy

Article Title: ACE2-independent infection of T lymphocytes by SARS-CoV-2

doi: 10.1038/s41392-022-00919-x

Figure Lengend Snippet: SARS-CoV-2 infection of T cell is spike-ACE2/TMPRSS2-independent. a The ACE2 expression level of Scramble or ACE2-knockdown Caco2 or Jurkat cells was analyzed by qPCR or WB. b ACE2 stably knockdown Caco2 or activated Jurkat cells were infected with SARS-CoV-2 (MOI = 0.01). Viral RNA or viral NP in cells was analyzed by qPCR or WB at 24 h post infection. c The ACE2 expression level of control or ACE-knockout Caco2 or Jurkat cells were quantified by qPCR or detected by WB. d ACE2 stably knock-out Caco2 or activated Jurkat cells were infected with SARS-CoV-2 (MOI = 0.01). Viral RNA or viral NP was detected using qPCR or WB. e For ACE2 blocking, Caco2 or activated Jurkat cells were pre-incubated with anti-ACE2 Ab (3.33 ng/μl final) before infected with SARS-CoV-2. For virus blocking, ACE2-Fc protein (10 μg/μl final) or RD#4-anti-Spike Ab (160 ng/μl final) were incubated with SARS-CoV-2 at a volume of 1:1 at 37 °C for 30 min. Cells were infected at 0.01 MOI for 24 h before they were quantified for SARS-CoV-2 viral RNA or NP protein by qPCR or WB. f The TMPRSS2 expression level of Caco2, Jurkat and activated Jurkat cells was analyzed using qPCR. g Caco2 or activated Jurkat cells were pre-incubated with Camostat (2 μM or 20 μM) for 1 h and then infected with SARS-CoV-2 (MOI = 0.01). Viral RNA or NP proteins were quantified. The results were derived from three independent experiments. Statistical analyses were carried out using Student’s t test (* P < 0.05; ** P < 0.01; **** P < 0.0001; NS no significance)

Article Snippet: Human recombinant full-length ACE2-Fc protein (GenScript, Z03484), Anti-ACE2 Ab (R&D, AF933) and RD#4-anti-Spike Ab (house-made monoclonal antibodies) were used.

Techniques: Infection, Expressing, Stable Transfection, Knock-Out, Blocking Assay, Incubation, Derivative Assay

Journal: Signal Transduction and Targeted Therapy

Article Title: ACE2-independent infection of T lymphocytes by SARS-CoV-2

doi: 10.1038/s41392-022-00919-x

Figure Lengend Snippet: Exploration of potential receptors in T cells. a The expression of ACE2, TMPRSS2, and ITGB2 (LFA-1) in blood T cells from healthy donors and COVID-19 patients. The analysis is dependent on public single-cell NGS data. The expression of the three genes is indicated in SARS-CoV-2 viral RNA-positive T cells for patient penal. b BEAS-2B and activated Jurkat cells were incubated with different concentrations of AXL proteins (25, 50, or 100 μg/ml) at 37 °C for 30 min before infected by SARS-CoV-2 (MOI of 0.01). Intracellular viral RNA (RBD) at 24 h post infection was quantified using qPCR. c Knockdown or overexpression of AXL were performed on Jurkat cells and the expression level of AXL was detected using qPCR. Cell lines were infected by SARS-CoV-2 at an MOI of 0.01 for 24 h and viral RNA was quantified. d LFA-1 was stably overexpressed on ACE2 knockdown Caco2 (Caco2-ACE2-shRNA) or Jurkat cells, and the RNA level was quantified using qPCR. e Caco2, activated Jurkat and their respective LFA-1-overexpression cells were infected by SARS-CoV-2 at an MOI of 0.01 and harvested at 24 h post infection. Intracellular viral RNA was detected using qPCR. f , g Caco2, Caco2-ACE2-shRNA, and its LFA-1 overexpression cell line were infected by SARS-CoV-2 at an MOI = 5 for 8 h. The high content microscope was used to observe ( f ) and quantify ( g ) the viral NP-positive cells. h LFA-1 knockdown Jurkat cells were infected with SARS-CoV-2 (MOI = 0.01) for 24 h, and the expression of LFA-1 and intracellular viral RNA was analyzed using qPCR. i Activated Jurkat cells were pretreated with Lifitegrast, an inhibitor of LFA-1 at different concentrations (50, 100, or 200 nM) at 37 °C for 30 min before infection (MOI = 0.01). Intracellular viral RNA was quantified at 24 h post infection. The statistics was performed using Student’s t test (* P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; NS no significance)

Article Snippet: Human recombinant full-length ACE2-Fc protein (GenScript, Z03484), Anti-ACE2 Ab (R&D, AF933) and RD#4-anti-Spike Ab (house-made monoclonal antibodies) were used.

Techniques: Expressing, Incubation, Infection, Over Expression, Stable Transfection, shRNA, Microscopy

Journal: Journal of Biochemistry

Article Title: SARS-CoV-2 spike protein binding selectively accelerates substrate-specific catalytic activity of ACE2

doi: 10.1093/jb/mvab041

Figure Lengend Snippet: Binding of the SARS-CoV-2 spike protein increases the rate of ACE2 activity. ( A ) Kinetic curves showing the effect of full-length SARS-CoV-2 spike on ACE2 activity. ( B ) Kinetic curves showing the effect of SARS-CoV-1 and SARS-CoV-2 RBD spike on ACE2 activity. ( C ) Michaelis–Menten plot showing effect of SARS-CoV-1 and SARS-CoV-2 RBD on catalytic activity of ACE2. k obs , observed rate constant. ( D ) Catalytic rate ( k cat ) and K M of ACE2 in the absence or presence of SARS-CoV-1 and SARS-CoV-2 RBD spike protein [mean (95% confidence intervals)]. Pseudosubstrate concentration in (A) and (B) is 20 µM. Assays were conducted in two biological replicates.

Article Snippet: The extracellular catalytic domain of recombinant human ACE2, SARS-CoV-2 spike full-length and spike RBD, SARS-CoV-1 RBD proteins and ACE2/Caspase-1 quenched fluorogenic peptide pseudosubstrate were obtained from R&D systems (cat# 933-ZN, 10549-CV, 10500-CV-100, 10558-CV-100 and ES007, respectively, R&D Systems Inc., MN, USA).

Techniques: Binding Assay, Activity Assay, Concentration Assay

Journal: Journal of Biochemistry

Article Title: SARS-CoV-2 spike protein binding selectively accelerates substrate-specific catalytic activity of ACE2

doi: 10.1093/jb/mvab041

Figure Lengend Snippet: SARS-CoV-2 spike protein accelerates the activity of ACE2 in a substrate-dependent manner. Kinetic curves showing the effect of SARS-CoV-2 spike RBD binding on ACE2 activity in the presence of ( A ) pseudosubstrate MCA-YVADAPK(Dnp); ( B ) angiotensin II mimic [MCA-DRVYIHPK(Dnp)]; ( C ) apelin 13 mimic [MCA-QRPRLSHKGPMPK(Dnp)]; ( D ) des-Arg9-bradykinin mimic [MCA-RPPGFSPK(Dnp)] ( E ) angiotensin I mimic [MCA-DRVYIHPFK(Dnp)]; ( F ) dynorphin A mimic [MCA-YGGFLRRIRPKLK(Dnp)] substrates; ( G ) Michaelis–Menten plot showing effect of SARS-CoV-2 RBD on catalytic activity of ACE2 in the presence of des-Arg9-bradykinin mimic substrate. ( H ) Catalytic rate ( k cat ) and K M of ACE2 in the absence or presence of SARS-CoV-2 RBD spike protein and des-Arg9-bradykinin mimic substrate [mean (95% confidence intervals)]. Substrate concentration in (A)–(F) is 20 µM. Assays were conducted in two biological replicates.

Article Snippet: The extracellular catalytic domain of recombinant human ACE2, SARS-CoV-2 spike full-length and spike RBD, SARS-CoV-1 RBD proteins and ACE2/Caspase-1 quenched fluorogenic peptide pseudosubstrate were obtained from R&D systems (cat# 933-ZN, 10549-CV, 10500-CV-100, 10558-CV-100 and ES007, respectively, R&D Systems Inc., MN, USA).

Techniques: Activity Assay, Binding Assay, Concentration Assay

Journal: Journal of Biochemistry

Article Title: SARS-CoV-2 spike protein binding selectively accelerates substrate-specific catalytic activity of ACE2

doi: 10.1093/jb/mvab041

Figure Lengend Snippet: Binding of the heat-inactivated SARS-CoV-2 viral particles accelerates ACE2 catalytic activity. ( A – C ) Kinetic curves showing the effect of different concentrations of heat-inactivated SARS-CoV-2 on ACE2 activity in the presence of des-Arg9-bradykinin mimic [MCA-RPPGFSPK(Dnp)] substrate (A); MCA-YVADAPK(Dnp) substrate (B); and angiotensin II mimic [MCA-DRVYIHPK(Dnp)] substrate (C). Substrate concentration in (A)–(C) is 20 µM. Assays were conducted in two biological replicates.

Article Snippet: The extracellular catalytic domain of recombinant human ACE2, SARS-CoV-2 spike full-length and spike RBD, SARS-CoV-1 RBD proteins and ACE2/Caspase-1 quenched fluorogenic peptide pseudosubstrate were obtained from R&D systems (cat# 933-ZN, 10549-CV, 10500-CV-100, 10558-CV-100 and ES007, respectively, R&D Systems Inc., MN, USA).

Techniques: Binding Assay, Activity Assay, Concentration Assay