Mutations from bat ACE2 orthologs markedly enhance ACE2-Fc neutralization of SARS-CoV-2

Abstract

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein mediates infection of cells expressing angiotensin-converting enzyme 2 (ACE2). ACE2 is also the viral receptor of SARS-CoV (SARS-CoV-1), a related coronavirus that emerged in 2002-2003. Horseshoe bats (genus Rhinolophus ) are presumed to be the original reservoir of both viruses, and a SARS-like coronavirus, RaTG13, closely related SARS-CoV-2, has been isolated from one horseshoe-bat species. Here we characterize the ability of S-protein receptor-binding domains (RBDs) of SARS-CoV-1, SARS-CoV-2, and RaTG13 to bind a range of ACE2 orthologs. We observed that the SARS-CoV-2 RBD bound human, pangolin, and horseshoe bat ( R. macrotis) ACE2 more efficiently than the SARS-CoV-1 or RaTG13 RBD. Only the RaTG13 RBD bound rodent ACE2 orthologs efficiently. Five mutations drawn from ACE2 orthologs of nine Rhinolophus species enhanced human ACE2 binding to the SARS-CoV-2 RBD and neutralization of SARS-CoV-2 by an immunoadhesin form of human ACE2 (ACE2-Fc). Two of these mutations impaired neutralization of SARS-CoV-1. An ACE2-Fc variant bearing all five mutations neutralized SARS-CoV-2 five-fold more efficiently than human ACE2-Fc. These data narrow the potential SARS-CoV-2 reservoir, suggest that SARS-CoV-1 and -2 originate from distinct bat species, and identify a more potently neutralizing form of ACE2-Fc.

Figure 1.. The receptor-binding domains (RBD) of SARS-CoV-1, SARS-CoV-2, and RaTG13 S proteins bind differentially to ACE2 orthologs.

HEK239T cells transfected to express the ACE2 orthologs of the indicate species, human DPP4 (the receptor of MERS-CoV), or with vector alone (mock) were assayed by flow cytometry for their ability to bind SARS1-RBD-Fc, SARS2-RBD-Fc, RaTG13-RBD-Fc, or MERS-RBD-Fc. Histograms display a representative experiment, and bars display the results of two experiments normalized to ACE2 expressing levels shown in Figure S1B and determined by an antibody recognizing an amino-terminal myc-tag.

Figure 2.. Quantitative comparisons of binding of RBD-Fc variants to soluble monomeric ACE2 orthologs.

(A) Biacore surface plasmon resonsance (SPR) studies were performed the indicated RBD-Fc (yellow and blue) captured on the sensor chip. The soluble monomeric forms of the indicated ACE2 variants (green) were injected for analysis as represented in the figure. Red indicates a four-amino acid at tag at the ACE2 C-termini. (B) Biacore X100 sensorgrams representing results from studies in which the indicated RBD-Fc variants were captured by an anti-human Fcγ antibody immobilized on a CM5 chip after instantaneous background depletion. Monomeric forms of human ACE2, human ACE2 lacking its active-site histidines (ACE2-NN), pangolin ACE2, or bat (R. macrotis) ACE2 were injected at five concentrations, serially diluted by two-fold from the highest concentration (100 nM for human ACE2 variants, 500 nM for non-human orthologs). The experiment is representative of two with similar results. (C) The association rate constant (k_on), dissociation rate constant (k_off), and equilibrium dissociation constant (K_d) were calculated and plotted from the study shown in (A). The negative of log₁₀ (K_d) is represented as the sum of log₁₀ (k_on), shown in red, and the negative of log₁₀ (k_off), shown in blue. Figure at bottom indicates that the RBD-Fc is immobilized and the monomeric soluble ACE2 is injected in this study.

Figure 3.. SARS1-RBD-Fc, SARS2-RBD-Fc, and human ACE2-Fc inhibit S-protein-mediated infection.

(A) The indicated concentrations of the indicated RBD-Fc variants were incubated with retroviral pseudoviruses (PV) pseudotyped with the S proteins of SARS-CoV-1 (SARS1-PV), SARS-CoV-2 (SAR2-PV), or with the G protein of vesicular stomatitis virus (VSV-G-PV). Infection is normalized to that in the absence of inhibitors. (B) Experiments similar to those in (A) except that the indicated soluble monomeric ACE2 variants, or human ACE2-Fc were used to inhibit the indicated pseudoviruses. Error bars in (A) and (B) indicate standard error of the mean (S.E.M), and are representative at least two experiments with similar results.

Figure 4.. Horseshoe bat and pangolin-derived mutations characterized in Figures 5 and 6.

(A) Human ACE2 (grey) is shown bound to the SARS-CoV-2 RBD (blue), based on PDB accession number 6M0J (Lan et al., 2020). Yellow indicates the ACE2 surface that is within 5.5 Å of the RBD. Red indicates residues that vary between human and horseshoe bat species and were characterized in the subsequent Figures. Specific mutations are indicated in the rightmost figure. A glycosylation at asparagine 90, also characterized, is indicated in green. Figure shows successive 90 degree rotations around the vertical and horizontal axes, respectively. The RBD is removed from the rightmost figure for clarity. (B) Sequences of human ACE2 directly associating, or proximal to, the SARS-CoV-2 RBD are shown, aligned with the sequence of the pangolin ACE2 ortholog, and those of all available horseshoe-bat ACE2 orthologs. Yellow indicates a residue that directly contacts the RBD. Red indicates human residues that were altered to the indicated pangolin and bat residues shown in gray and characterized in the subsequent figures. Bold indicates a glycosylated asparagine.

Figure 5.. Residues from horseshoe-bat ACE2 orthologs improved human ACE2-Fc binding to SARS2-RBD and neutralization of SARS2-PV.

(A) SARS1-PV (blue), SARS2-PV (red), or VSV-G-PV (grey) were used to infect ACE2–239T cells in the presence of the indicated concentration of human ACE2-Fc (hACE2-Fc) or hACE2-Fc with the indicated mutations. Mean half-maximal inhibitory concentration (IC₅₀) shown each variant. Error bars indicate standard error of the mean (S.E.M) for replicates. Experiment is representative of at least two with similar results. (B) SARS2-PV IC₅₀ values for three studies similar to those shown in (A) are plotted. Error bars indicate the upper and lower IC₅₀ value observed for each hACE2-Fc variant. Asterisks indicate that the neutralization was incomplete and therefore no IC₅₀ was calculated. (C) Percentage of SARS2-PV infection in the presence of 16.67 μg/ml indicated hACE2-Fc variant. Error bars indicate standard error of the mean (S.E.M). (D) A diagram is shown illustrating that hACE2-Fc variants were captured and that soluble monomeric SARS2-RBD was injected for these studies. (E) SPR analyses showing the k_on, k_off, and K_d of SARS2-RBD to hACE2-Fc variants.

Figure 6.. A combination of bat-derived residues improves SARS-PV neutralization five-fold.

(A) Experiments similar to those shown Figure 5A except that the indicated ACE2-Fc variant (dark colors) is directly compared with wild-type human ACE2-Fc (light colors) for their ability to neutralize SARS1-PV (light and dark blue), SARS2-PV (light and dark red), or VSV-G-PV (light and dark grey). IC₅₀ values for SARS-CoV-2 of the indicated ACE2-Fc variant over two independent studies are provided in bottom left of each figure. Neutralization results were normalized to the virus infection without any ACE2-Fc. Error bars indicate standard error of the mean (S.E.M). Neutralization data are representative of at least two experiments. (B) SARS2-PV IC₅₀ values for two studies similar to those shown in (A) are plotted. Error bars indicate the upper and lower IC₅₀ value observed for each hACE2-Fc variant. Asterisks indicate that the neutralization was incomplete and therefore no IC₅₀ was calculated. (C) Sensorgrams of SPR analyses used to determine bind constants of the SARS2-RBD for wild-type hACE2-Fc (Wt) and hACE2-Fc with the five indicated mutations (5M). Diagram illlustrates that ACE2-Fc was captured in these studies while soluble monomeric RBD was injected. (D) k_on, k_off, and K_d of SARS2-RBD binding to wild-type hACE2-Fc or to hACE2-Fc 5M.