Fig 1.
Expressions and transduction of bat ACE2s by RaTG13 S pseudovirons.
(A) Western blot analysis of expression of different bat ACE2s in HEK293 cells. HEK293 cells were transfected with plasmids encoding FLAG-tagged bat ACE2s by PEI and lysed at 40 hrs post-transfection. Proteins were detected using anti-FLAG M2 antibody. The β-actin was used as a loading control. (B) Detection of the S proteins of RaTG13 and SARS-CoV-2 in celllysates and pseudovirions by western blot. HEK293T cells transfected with either empty vector or plasmids encoding the indicated CoV S proteins were harvested at 40 h post-transfection. The S proteins in cell lysate and pseudovirions were subjected to western blot analysis using an anti-FLAG M2 antibody. β-Actin and gag-p24 served as loading controls. The full-length S protein is about 180 kD, while the cleaved S protein is about 90 kD. Experiments were done three times and the representative was shown. (C) Entry mediated by the S protein of RaTG13 into cells expressing different bat and human ACE2s. HEK-293 cells transiently expressing bat and human ACE2s were transduced with RaTG13 S pseudovirions and the transduction efficiency was detected 40 hrs later and manifested as luciferase activities. Experiments were done in triplicate and repeated at least three times. One representative is shown with error bars indicating SEM. Statistical significance is set as * p<0.05 and ** p<0.01 and calculated by T-test.
Table 1.
Accession number for 18 different bat species.
Fig 2.
RaACE2 polymorphism confers different susceptibility to RaTG13.
(A) Phylogenetic tree of RaACE2 variants. The maximum-likelihood tree (left panel) was produced using MEGA X software, based on the alignment of ACE2 amino acid sequences of R. affinis. The branch lengths are scaled according to the number of amino acid substitutions per site, indicated by the scale bar. (B) The table of non-synonymous mutations present in the RaACE2 variants. The three of eight key residues which are involved in interacting with the RaTG13 and SARS-CoV-2 spike are shaded with a yellow background. The corresponding eight residues of human ACE2 are indicated at the bottom. (C) Analysis of RaACE2 proteins on the cell surface by cell surface protein biotinylation assay. HEK293 cells transiently overexpressing different RaACE2 and human ACE2 proteins were labeled with EZ-link Sulfo-NHS-LCLC-biotin on ice, and lysed with RIPA buffer. Biotinylated proteins were enriched with NeutrAvidin beads and detected by western blot using mouse monoclonal anti-FLAG M2 antibody. WCL, whole cell lysate. Entry mediated by RaTG13 S pseudovirions (D) and SARS-CoV-2 S pseudovirions (E) into cells expressing different RaACE2 variants. HEK-293 cells transiently expressing different RaACE2 variants were transduced with RaTG13 S pseudovirions or SARS-CoV-2 S pseudovirions and transduction efficiency was measured 40 hrs post-transduction. Experiments were done in triplicate and repeated at least three times. One representative is shown with error bars indicating SEM. Statistical significance is set as * p<0.05 and ** p<0.01 and calculated by t-test.
Fig 3.
Effect of RaACE2 polymorphism on RBD binding and cell-cell fusion mediated by RaTG13 and SARS-CoV-2 S protein.
HEK293 cells transiently expressing RaACE2 variants and human ACE2 were incubated with either SARS-CoV-2 (A) or RaTG13 (B) RBDs, followed by rabbit anti-Strep-tag II antibody incubation and then Alexa Fluor 488-conjugated goat anti-mouse IgG (1:500). Cells were fixed with 1% paraformaldehyde and then analyzed by flow cytometry. The results of the percentage of positive cells from hACE2 binding were set to 100%, the rest was calculated as a percentage of hACE2 binding according to results in flow cytometry analysis. Data are shown as the mean ± SEM. Cell-cell fusion mediated by SARS-CoV-2 (C) and RaTG13 (D) S proteins. HEK293T cells transiently expressing eGFP and S proteins of either SARS-CoV-2 or RaTG13 were detached with trypsin, and overlaid on different ACE2 expressing HEK293 cells. After 4 hrs of incubation, images were taken. The total number of nuclei and the number of nuclei in fused cells for each image were counted. The fusion efficiency was calculated as the number of nuclei in syncytia/total number of nuclei ×100. The scale bar indicates 250 μm. Statistical significance is set as * p<0.05 and ** p<0.01 and calculated by T-test.
Fig 4.
H34 and D38 in RaACE2 are critical for interaction with RaTG13 S protein.
Expression of WT and mutant RaACE2 variants, RA-01, RA-03, and mutants (A), RA-07 and mutants (C). HEK293 cells were transfected with plasmids encoding FLAG-tagged RaACE2s using PEI, and lysed at 40 hrs post-transfection. Proteins were detected using an anti-FLAG M2 antibody. The β-actin was used as a loading control. (B) (D) Entry mediated by the S protein of RaTG13 into cells expressing WT and mutant RaACE2 variants, RA-01, RA-03, and mutants (B), and RA-07 and mutants (D). Experiments were done three times, and one representative is shown with error bars indicating SEM. Statistical significance is set as * p<0.05 and ** p<0.01 and calculated by T-test.
Fig 5.
Identification of critical residues in RaTG13 and SARS-CoV-2 S protein for virus entry.
(A) Alignment of RaTG13 RBD and SARS-CoV-2 RBD. The RBM of RaTG13 RBD and SARS-CoV-2 RBD are highlighted in yellow background. The mutations chosen are indicated in red. (B) Detection of mutant S proteins of RaTG13 (B) and SARS-CoV-2 (C) in cell lysates and pseudovirions by western blot using anti-FLAG M2 antibody. Top panel, cell lysate; bottom panel, pseudovirions; β-actin and HIV p24 were used as loading controls. Entry mediated by mutant RaTG13 (D) and SARS-CoV-2 (E) S pseudovirions into HEK-293 cells expressing RaACE2 variants. HEK-293 cells transiently expressing RaACE2 variants were transduced with indicated S pseudovirions as well as mutants. Experiments were performed three times, and one representative is shown as the heat map.
Fig 6.
Effect of RaACE2 polymorphism on entry of other SC2r-CoVs.
(A) Alignment of amino acid sequences from aa490 to aa510 of RaTG13, BANAL-20-52, BANAL-20-236, Pangolin-GD, and SARS-CoV-2 S proteins. Residues 501 and 505 are labeled in red. (B) Expression and pseudovirion incorporation of RaTG13, BANAL-20-52, BANAL-20-236, Pangolin-GD and SARS-CoV-2 S proteins. Top panel, cell lysate; bottom panel, pseudovirions; β-actin and HIV p24 were used as loading controls. Entry of pseudovirions of BANAL-20-52 (C), BANAL-20-236 (D), Pangolin-GD (E) S proteins on 293/RaACE2s and 293/hACE2 cells.
Fig 7.
Effect of individual D501N and H505Y changes in RaTG13 S protein on entry of cells expressing bat, pangolin, and mouse ACE2s.
Transduction of D501N and H505Y individual mutant RaTG13 S pseudoviruses on cells expressing different bat ACEs (A), pACE2, and mACE2 (B). Effect of D501N and H505Y mutations in RaTG13 S on cell-cell fusion with cells expressing pACE2 and mACE2. The representative images were shown in (C), and the total number of nuclei and the number of nuclei in fused cells for each image were counted. The fusion efficiency was calculated as the number of nuclei in syncytia/total number of nuclei ×100. The scale bar indicates 250 μm. Statistical significance is set as * p<0.05 and ** p<0.01 and calculated by T-test.
Fig 8.
Effect of T372A mutation in RaTG13 S on entry on bat ACE2s and neutralization activities from BANAL-20-52 immunized mouse sera.
(A) Western blot analysis of RaTG13 and T372A S proteins in cell lysates and pseudovirions. Top panel, cell lysate; bottom panel, pseudovirions, β-actin, and HIV p24 were used as loading controls. The WT and T372A mutant RaTG13 S pseudovirions were transduced on cells expressing RaACE2 variants (B) and different bat ACE2s (C), and the transduction efficiencies were determined according to luciferase activities. (D) Effect of T372A substitution in RaTG13 on neutralization activities of mouse sera immunized with trimeric BANAL-20-52 S proteins. Mice were immunized with trimeric BANAL-20-52 S proteins twice at days 0 and 14, and sera were collected at day 28 post immunization. The sera from BANAL-20-52 immunized mice were serially diluted and incubated with pseudovirions for 1 hr at 37°C, and the mixture was then incubated with RA-07 expressing cells. After 12 hrs incubation at 37 °C, cells were fed with fresh medium. After another 24 hrs incubation, the transduction efficiencies were measured using Steady-Glo, and neutralization titers were calculated according to the dilution with 50% inhibition. Statistical significance is set as * p<0.05 and ** p<0.01, using an unpaired t-test (Wilcoxon rank test).
Fig 9.
In silico analysis of RaTG13 RBD (green) binding to RA-06/RA-07 (gray) showing key interactions on the binding interfaces.
Key interactions with H34/R34, D38/E38, and Y83/H83 are shown in the blue square (A), the orange square (B), and the purple square (C), respectively. Hydrogen bonding and salt bridge are displayed in yellow and red dash lines, respectively. The side chains of H34, D38, and Y83 are labeled in grey, and the side chains of R34, E38, and H83 are labeled in purple. (D) Key interactions with residues 501 and 505 in S protein. D501 and H505 are in green, and N501 and Y505 are in purple.