Structural basis for raccoon dog receptor recognition by SARS-CoV-2

Abstract

Since the COVID-19 outbreak, raccoon dogs have been suggested as a potential intermediary in transmitting SARS-CoV-2 to humans. To understand their role in the COVID-19 pandemic and the species barrier for SARS-CoV-2 transmission to humans, we analyzed how their ACE2 protein interacts with SARS-CoV-2 spike protein. Biochemical data showed that raccoon dog ACE2 is an effective receptor for SARS-CoV-2 spike protein, though not as effective as human ACE2. Structural comparisons highlighted differences in the virus-binding residues of raccoon dog ACE2 compared to human ACE2 (L24Q, Y34H, E38D, T82M, R353K), explaining their varied effectiveness as receptors for SARS-CoV-2. These variations contribute to the species barrier that exists between raccoon dogs and humans regarding SARS-CoV-2 transmission. Identifying these barriers can help assess the susceptibility of other mammals to SARS-CoV-2. Our research underscores the potential of raccoon dogs as SARS-CoV-2 carriers and identifies molecular barriers that affect the virus's ability to jump between species.

Fig 1. Molecular characterization of ACE2 from different species.

(A) A phylogenetic tree based on the full-length ACE2 gene among different species was built using the maximum likelihood estimation (MLE) method. The Kimura 2-parameter (K2P) mode was selected under a discrete gamma distribution with bootstraps for 1,000 replications. (B) Sequence alignment of three virus-binding motifs (VBMs) among the ACE2 molecules from human, raccoon dog and domestic dog. Residues that directly contact SARS-CoV-2 RBD are labeled in blue. Among these RBD-contacting residues, those that differ between human ACE2 and raccoon dog ACE2 are labeled in red. Asterisks indicate positions that have a single, fully conserved residue. Colons indicate positions that have strongly conserved residues. Periods indicate positions that have weakly conserved residues. Hyphen indicates deletion at residue 21 in raccoon dog ACE2 and domestic dog ACE2. For the sake of clarity, the residue numbering in raccoon dog ACE2 and domestic dog ACE2 is based on the human ACE2 sequence, ignoring the deleted residue at position 21.

Fig 2. Binding interactions between SARS-CoV-2 RBD and the ACE2 molecules of different species.

(A) Flow cytometry between recombinant SARS-CoV-2 RBD and cell surface-expressed ACE2 molecules from human, raccoon dog and domestic dog. The mean fluorescence intensity (M.F.I.) is determined using PE anti-His tag antibodies, which target the His-tagged RBD protein bound to ACE2-positive cells. The mock group represents cells transfected with an empty vector. The data are presented as mean ± SEM (n = 4) (S1A Fig). A Student’s two-tailed t-test was performed to analyze the statistical difference between the indicated groups; the results are labeled on top of each bar. ***p < 0.001. n.s.: not significant. (B) Surface plasmon resonance (SPR) between recombinant SARS-CoV-2 RBD and recombinant ACE2 molecules from human, raccoon dog and domestic dog. The data are presented as mean ± SEM (n = 3) (S1 Table; S2 Fig). A Student’s two-tailed t-test was performed to analyze the statistical difference between the indicated groups; the results are labeled on top of each bar. ***p < 0.001. n.s.: not significant. (C) Flow cytometry between recombinant SARS-CoV-2 RBD and raccoon dog ACE2 (wild type or containing one of the indicated mutations). The data are presented as mean ± SEM (n = 3) (S1B Fig). A Student’s two-tailed t-test was performed to analyze the statistical difference between the indicated groups; the results are labeled on top of each bar. ***p < 0.001.

Fig 3. Crystal structure of chimeric SARS-CoV-2 RBD complexed with chimeric raccoon dog ACE2.

(A) Overall structure of the complex. The chimeric RBD contains the core structure (in cyan) from SARS-CoV-1 RBD and receptor-binding motif (RBM) (in magenta) from SARS-CoV-2 RBD. The chimeric raccoon dog ACE2 contains the core structure (in green) from human ACE2 and three virus-binding motifs (VBMs) (in orange) from raccoon dog ACE2. (B) Structural interface between SARS-CoV-2 RBM and raccoon dog VBMs. Three virus-binding hotspots are highlighted. The key residues that differ between human ACE2 and raccoon dog ACE2 are shown in sticks.

Fig 4. Structural details at hotspot-ridge.

(A) (C) Structural interface between SARS-CoV-2 RBM and human VBMs at the hotspot-ridge. PDB ID: 6VW1. (B) (D) Structural interface between SARS-CoV-2 RBM and raccoon dog VBMs at hotspot-ridge. The dotted line indicates a hydrogen bond.

Fig 5. Structural details at hotspot-31 and hotspot-353.

(A) Structural interface between SARS-CoV-2 RBM and human VBMs at the hotspot-31. PDB ID: 6VW1. (B) Structural interface between SARS-CoV-2 RBM and raccoon dog VBMs at the hotspot-31. (C) Structural interface between SARS-CoV-2 RBM and human VBMs at the hotspot-353. PDB ID: 6VW1. (D) Structural interface between SARS-CoV-2 RBM and raccoon dog VBMs at the hotspot-31. The dotted lines indicate hydrogen bonds or salt bridges.