ace2 open reading frame  (Addgene inc)

Journal: Cell Host & Microbe

Article Title: SARS-CoV-2 spike L452R variant evades cellular immunity and increases infectivity

doi: 10.1016/j.chom.2021.06.006

Figure Lengend Snippet: Increase in the binding affinity to ACE2, viral infectivity, viral fusogenicity, and viral replication capacity by the L452 mutation (A) Location of the NF9 peptide (residues 448–456) in the cocrystal structure of the SARS-CoV-2 S and human ACE2 proteins (PDB: 6M17 ) ( Yan et al., 2020 ). An overview (left), an enlarged view of the boxed area in the left panel (middle), and a view of the middle panel rotated 180° on the y axis (right) are shown. Residues 448–456 of SARS-CoV-2 S (corresponding to the NF9 peptide) are shown in black. (B–D) Binding affinity of SARS-CoV-2 S RBD to ACE2 by yeast surface display. The percentage of the binding of the SARS-CoV-2 S RBD expressed on yeast to soluble ACE2 (B) and the K D values (C) are shown. Assays were performed in quadruplicate. (D) The level of stable expression of the SARS-CoV-2 RBD on yeast (x axis) and the binding affinity toward ACE2 (y axis) compared to the parental RBD. In (B), the fitting curve of parental RBD is shown as black lines in all panels. (E and F) Pseudovirus assay. The HIV-1-based reporter virus pseudotyped with the parental SARS-CoV-2 S or its derivatives (E, L452R, Y453F, and N501Y; F, D614G, B.1.429 [S13I/W152C/L452R/D614G], and B.1.1.298 [HV69-70del/Y453F/D614G]) was inoculated into HEK293 cells transiently expressing human ACE2 and TMPRSS2 at four different doses (1, 3, 5, and 10 ng p24 antigens). Percentages of infectivity compared to the virus pseudotyped with parental S (10 ng p24 antigen) are shown. (G) Gain of electrostatic complementarity by the L452R substitution. Left: the surface structure of SARS-CoV-2 S and ACE2 (PDB: 6M17 ) ( Yan et al., 2020 ). Residue 452 of SARS-CoV-2 S and the negatively charged patch on ACE2 (residues E35, E37, and D38) are indicated in black and red. The boxed area is enlarged in the upper right panel. Right: Coulombic surface coloring of the structures of SARS-CoV-2 S and ACE2 (PDB: 6M17 ) ( Yan et al., 2020 ) (top) and a model of the L452R substitution (bottom). The black line indicates the border between SARS-CoV-2 S and ACE2. (H) Chromatograms of the mutated regions of SARS-CoV-2 viruses artificially generated by reverse genetics. Chromatograms of nucleotide positions 22,913–22,924 (left) and 23,060–23,068 (right) of parental SARS-CoV-2 (strain WK-521; GISAID ID: EPI_ISL_408667) and the L452R (T22917G in nucleotide), Y453F (A22920T in nucleotide), and N501Y (A23063T in nucleotide) mutants are shown. (I–L) Growth kinetics of parental SARS-CoV-2 and SARS-CoV-2 mutants. Parental SARS-CoV-2 and the L452R, Y453F, and N501Y mutants (100 plaque-forming units [PFU]) were inoculated into HEK293-ACE2 cells (I and J), A549-ACE2 cells (K), and VeroE6/TMPRSS2 cells (L), and the copy number of viral RNA in the culture supernatant was quantified by real-time PCR. (I) Representative bright-field images of HEK293-ACE2 cells uninfected or infected with the viruses indicated at 24, 48, or 72 h post-infection are also shown. Bars, 200 μm. (J–L) Left: the growth curve of the viruses inoculated. The result for the parental virus is shown in all panels as a black line. Right: the amount of viral RNA in the culture supernatant at 72 h post-infection. Assays were performed in triplicate (J) or quadruplicate (K and L). (M) Competition assay. Parental virus and the L452R mutant were mixed at a 1:1 ratio based on PFU, and the mixture was inoculated into HEK293-ACE2 cells. The percentage of L452R mutant at each time point was analyzed as described in . The data are shown as the average of four biological replicates. (N) SARS-CoV-2 S-based fusion assay. Effector cells (S-expressing cells) and target cells (ACE2-expressing cells) were prepared, and the fusion activity was measured as described in . Assays were performed in quadruplicate, and fusion activity (arbitrary unit) is shown. In (C), statistically significant differences ( ∗ p < 0.05) versus parental S are determined by the Mann-Whitney U test. In (E and F), statistically significant differences ( ∗ p < 0.05) versus parental S (E) and the D614G mutant (F) at the same dose were determined by Student’s t test. In (J–L and N), statistically significant differences ( ∗ p < 0.05) versus parental virus (J–L) or parental S (N) were determined by Student’s t test. In (M), values at respective time points were compared with those at the last time point using a two-tailed, paired Student’s t test, and an asterisk denotes familywise error rate <0.05 using the Holm test. See also Figure S1 .

Article Snippet: The DNA fragment including the mink ACE2 open reading frame was obtained by RT-PCR using PrimeSTAR GXL DNA polymerase (Takara, cat# R050A) and the following primers: Mink ACE2 forward, 5′-ATG TTA GGC TCT TCC TGG CTC CTT-3′; and Mink ACE2 reverse, 5′-CTA AAA TGA CGT CTG AAC ATC ATC GAC-3′.

Techniques: Binding Assay, Infection, Mutagenesis, Expressing, Virus, Residue, Generated, Real-time Polymerase Chain Reaction, Competitive Binding Assay, Single Vesicle Fusion Assay, Activity Assay, MANN-WHITNEY, Two Tailed Test

Journal: Cell Host & Microbe

Article Title: SARS-CoV-2 spike L452R variant evades cellular immunity and increases infectivity

doi: 10.1016/j.chom.2021.06.006

Figure Lengend Snippet:

Article Snippet: The DNA fragment including the mink ACE2 open reading frame was obtained by RT-PCR using PrimeSTAR GXL DNA polymerase (Takara, cat# R050A) and the following primers: Mink ACE2 forward, 5′-ATG TTA GGC TCT TCC TGG CTC CTT-3′; and Mink ACE2 reverse, 5′-CTA AAA TGA CGT CTG AAC ATC ATC GAC-3′.

Techniques: Virus, Recombinant, Modification, Expressing, Reverse Transcription, Transfection, Luciferase, Mutagenesis, Gel Extraction, Cloning, Plasmid Preparation, Quantitative RT-PCR, Competitive Binding Assay, Sequencing, Random Hexamer, Software, Pore Size, Membrane

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: Vaccine

Article Title: Immunisation of ferrets and mice with recombinant SARS-CoV-2 spike protein formulated with Advax-SM adjuvant protects against COVID-19 infection

doi: 10.1016/j.vaccine.2021.07.087

Figure Lengend Snippet: Modelling and expression of SARS-CoV-2 spike ectodomain (ECD) vaccine antigen. (A) Diagram of the ECD construct of SARS-CoV-2 spike (S) protein. Honeybee melittin signal (HBMss) peptide was used for efficient secretion expression. Transmembrane domain and intracellular domain were removed and replaced with a TEV cleavage site followed by 6x Histidines (6xHis). (B) SARS-CoV-2 receptor binding domain (RBD) (green) binding human Angiotensin-Converting Enzyme 2 (ACE2) (red). (C) Molecular dynamic simulation (MDS) run for 100 ns showed the structure was stable. (D) Representative Coomassie Brilliant Blue stained SDS-PAGE of purified S-dTM. (E) Purified S-dTM was confirmed by Western blot using anti-SARS-CoV-2 spike mouse polyclonal antibody. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The human ACE2 open reading frame (Addgene# 1786) was cloned into a 3rd generation lentiviral expression vector pRRLSIN.cPPT.PGK-GFP.WPRE (Addgene# 122053), and clonal HEK 293 T cells stably expressing ACE2 were generated by lentiviral transductions as described previously , followed by single cell sorting into 50% HEK 293 T conditioned media (media conditioned from 50% confluent HEK 293 T cultures).

Techniques: Expressing, Construct, Binding Assay, Staining, SDS Page, Purification, Western Blot