human ace2  (Addgene inc)

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 . " title="Increase in the binding affinity to ACE2, viral infectivity, viral fusogenicity, and viral replication capacity ..." property="contentUrl" width="100%" height="100%"/>
Figure Legend 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 .

Techniques Used: 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

Figure Legend Snippet:

Techniques Used: 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