Fig 1.
The mutation Y453F in SARS-CoV-2 spike protein is a potential genetic adaptation in minks.
(A) Schematic of amino acid changes in the spike protein of miSARS-CoV-2 variant “Cluster 5”. The four genetic changes in the S protein are marked in red. NTD, N-terminal domain; FP, fusion peptide; HR1, heptad repeat 1; HR2, heptad repeat 2; TM, transmembrane region; IC, intracellular domain. (B) Alignment of the residues of human (Homo sapiens; NCBI Reference Sequence: NM_001371415.1), mink (Neovison vison; NCBI Reference Sequence: MW269526.1), ferret (Mustela putorius furo; NCBI Reference Sequence: NM_001310190.1) and stoat (Mustela erminea; NCBI Reference Sequence: XM_032331786.1) ACE2 at the interface of ACE2 with the SARS-CoV-2 spike protein. The position of Y453 in the SARS-CoV-2 RBD is colored in red. The Y34 of mink, ferret and stoat ACE2 are highlighted in red. (C) Structural features of the SARS-CoV-2 RBD and ACE2. Contacting residues are shown as sticks at the hACE2-SARS-CoV-2 RBD interfaces (PDB ID: 6M0J). The key residues Y453 of the RBD (a and b) and H34 of human ACE2 (a and c) are labeled. The key residues F453 of the RBD (c and d) and Y34 substitution in human ACE2 (b and d) is labeled. SARS-CoV-2 RBD is shown in cyan, ACE2 in green. The dashed lines indicate hydrogen bonds.
Fig 2.
Increased binding of Y453F RBD protein to mink ACE2.
(A) Schematic of testing the efficiency of ACE2 variants binding WT or Y453F viral spikes. (B) HeLa cells were transduced with human and mink ACE2 or their mutants as indicated. The transduced cells were incubated with the WT or Y453F S1 domain of SARS-CoV-2 C-terminally fused with a His tag and then stained anti-His-PE for flow cytometry analysis. Values are expressed as the percent of cells positive for S1-Fc among the ACE2-expressing cells (zsGreen1+ cells) and shown as the means ± SD from 3 biological replicates. These experiments were independently performed three times with similar results. (C) Representative immunoblots of HeLa cells transduced with lentiviruses expressing FLAG-tagged ACE2. Actin used as the loading control. These experiments were independently performed twice with similar results. (D) Cell surface localization of human and mink ACE2 and their mutants. HeLa cells were transduced with lentivirus expressing human or mink ACE2 and their mutants. Cells were incubated with rabbit polyclonal antibody against ACE2 and then stained with goat anti-rabbit IgG (H+L) conjugated with Alexa Fluor 568 and DAPI (1μg/ml). The cell images were captured with a Zeiss LSM 880 Confocal Microscope. This experiment was independently repeated twice with similar results and the representative images are shown. (E) Table summarizing biochemical results for ACE2 variants bound to WT or Y453F RBD. These experiments were independently performed three times with similar results.
Fig 3.
Structural comparison of Y453F RBD/mink ACE2 and WT RBD/human ACE2 complexes.
(A)The overall structure is shown as ribbons. The Y453F-RBD is shown in cyan and mink ACE2 in green. The WT-RBD and human ACE2 are shown in magenta and orange, respectively. (B) Interaction residues around position 34 of human and mink ACE2. Left: complex of Y453F RBD/mink ACE2. Right: complex of WT RBD/human ACE2. RBD is shown in cyan, ACE2 in green. Contact residues are shown as sticks which were defined using a distance cutoff of 4Å. The PDB code for WT-RBD/human ACE2 complex is 6M0J[21].
Table 1.
Data collection and refinement statistics.
Fig 4.
Increased binding of Y453F RBD protein to Mustelidae ACE2 orthologs.
(A) HeLa cells were transduced with human or Mustelidae ACE2 orthologs as indicated. Transduced cells were incubated with WT or Y453F S1 domain of SARS-CoV-2 C-terminally fused with His tag and then stained with anti-His-PE for flow cytometry analysis. Values are expressed as the percent of cells positive for S1-Fc among the ACE2-expressing cells (zsGreen1+ cells) and shown as the means ± SD from 3 biological replicates. These experiments were independently performed three times with similar results. (B) Cell surface localization of human, ferret and stoat ACE2. HeLa cells were transduced with lentivirus expressing ACE2 orthologs as indicated. Cells were incubated with rabbit polyclonal antibody against ACE2 and then stained with goat anti-rabbit IgG (H+L) conjugated with Alexa Fluor 568 and DAPI (1μg/ml). The cell images were captured with a Zeiss LSM 880 Confocal Microscope. This experiment was independently repeated twice with similar results and the representative images are shown. (C) The binding kinetics of ACE2 proteins (ferret or stoat) with recombinant WT or Y453F SARS-CoV-2 RBD were obtained using the BIAcore. ACE2 proteins were captured on the chip, and serial dilutions of RBD were then injected over the chip surface. Experiments were performed three times with similar result, and one set of representative data is displayed.
Fig 5.
Enhanced entry of Y453F spike pseudotyped virion by utilization of mink ACE2.
(A) pTG-MLV-Fluc, pTG-MLV-Gag-pol, and pcDNA3.1 expressing WT or mutant SARS-CoV-2 spike genes as indicated were co-transfected into HEK293T cells. After 48h, the cell lysates (left panel) and the cell culture medium (right panel) were collected for Western blotting analysis of abundance of spike proteins and their proteolytic processing. Mink W/O Y453F: del69-70/I692V/M1229I; Mink: del69-70/Y453F/I692V/M1229I. Experiments were performed three times with similar result, and one set of representative blotting is displayed. (B) A549 cells transduced with mouse ACE2, human ACE2, or mink ACE2 were infected with indicated SARS-CoV-2 pseudoparticles. Two days after infection, cells were lysed and luciferase activity determined. All infections were performed in triplicate, and the data are representative of two independent experiments (mean ± SD). ns, no significance,**, P < 0.01, ***, P < 0.001. Significance assessed by one-way ANOVA.
Fig 6.
Y453F mutation in spike protein promote SARS-CoV-2 GFP/ΔN trVLP infection of Caco-2ACE2KO cells expressing exogenous mink ACE2.
(A) Schematic representation of the experiment. SARS-CoV-2 GFP/ΔN trVLP (WT or Y453F) were produced as previously described[26]. The trVLP were used to infect cells as indicated. (B-C) Endogenous ACE2 knockout Caco-2 cells expressing SARS-CoV-2 N protein were infected with SARS-CoV-2 GFP/ΔN (MOI = 0.1). After 36 hours, GFP expression was observed using immunofluorescence microscopy and GFP-positive cells were quantified by flow cytometry. All infections were performed in triplicate, and the data are pooled from two independent experiments (mean ± SD).
Fig 7.
Sensitivity of miSARS-CoV-2 “Cluster Five” spike pseudotyped virus to neutralization by convalescent sera of patients or soluble ACE2.
(A) Convalescent serum samples from donors were serially diluted and incubated with WT or “Cluster Five” spike-pseudotyped viruses. The serum dilution factor leading to 50% reduction of pseudotyped virion entry was calculated as the NT50 (neutralizing titer 50). Identical plasma samples are connected with lines. These analyses were repeated twice with similar results. Statistical analysis of the difference between neutralization of the WT and “Cluster Five” was performed using two-tailed Wilcoxon matched-pairs signed-rank test. (B) Recombinant ACE2-Ig was diluted at the indicated concentrations. Viral entry was determined by assessing Luc activity 48 hours post infection. The dilution factors leading to 50% reduction of pseudotyped virion entry was calculated as the IC50. Data shown are representative of three independent experiments with similar results, and data points represent mean ± SD in triplicate.