Cross-species recognition and molecular basis of SARS-CoV-2 and SARS-CoV binding to ACE2s of marine animals

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has an extremely broad host range that includes hippopotami, which are phylogenetically closely related to whales. The cellular ACE2 receptor is one of the key determinants of the host range. Here, we found that ACE2s from several marine mammals and hippopotami could efficiently bind to the receptor-binding domain (RBD) of both SARS-CoV and SARS-CoV-2 and facilitate the transduction of SARS-CoV and SARS-CoV-2 pseudoviruses into ACE2-expressing cells. We further resolved the cryo-electron microscopy complex structures of the minke whale ACE2 and sea lion ACE2, respectively, bound to the RBDs, revealing that they have similar binding modes to human ACE2 when it comes to the SARS-CoV-2 RBD and SARS-CoV RBD. Our results indicate that marine mammals could potentially be new victims or virus carriers of SARS-CoV-2, which deserves further careful investigation and study. It will provide an early warning for the prospective monitoring of marine mammals.

Keywords: SARS-CoV-2; cross-species recognition; cryo-EM structure; marine animals.

Figure 1.

Binding between ACE2s and SARS-CoV-2 or SARS-CoV RBD, and the transduction of pseudotyped SARS-CoV-2 or pseudotyped SARS-CoV into BHK-21 cells expressing the relevant ACE2s. (A) His-tagged SARS-CoV-2 RBD, SARS-CoV RBD or SARS-CoV-2 N-terminal domain (NTD) proteins were incubated with BHK-21 cells expressing EGFP-tagged ACE2s, respectively. Anti-His/APC antibodies were used to detect the His-tagged protein binding to the cells. Cells stained with the SARS-CoV-2 RBD, the SARS-CoV RBD and the SARS-CoV-2 NTD proteins are shown in bright blue, pink and brown, respectively. The mean fluorescence values of APC are presented. The SARS-CoV-2 NTD was used as the negative control. (B) The mFc-tagged ACE2s in the supernatants were captured by anti-mIgG Fc antibodies immobilized on the CM5 chip, and their binding was sequentially tested with serially diluted SARS-CoV-2 RBD or SARS-CoV RBD. The raw and fitted curves are displayed in dotted and solid lines, respectively. (C) The binding affinities between ACE2s and SARS-CoV-2 RBD or SARS-CoV RBD are shown as the means ± SD of three independent experiments. (D and E) Transduction of the pseudotyped SARS-CoV-2 and SARS-CoV on BHK-21 cells expressing the respective mammal ACE2 or hACE2. Error bars represent the SD from six replicates. P values were analyzed using the student's t test (*** P < 0.001, **** P < 0.0001).

Figure 2.

Overall architectures of the MW-ACE2/SARS-CoV-2 RBD, MW-ACE2/SARS-CoV RBD, SL-ACE2/SARS-CoV-2 RBD and SL-ACE2/SARS-CoV RBD complexes. Overall structure of the (A) MW-ACE2/SARS-CoV-2 RBD, (B) MW-ACE2/SARS-CoV RBD, (C) SL-ACE2/SARS-CoV-2 RBD and (D) SL-ACE2/SARS-CoV RBD complexes. Boxes indicate the interaction patches. The hydrogen bonds network of Patch 1 and Patch 2 are shown. A cartoon representation of the complex structure is shown, and residues participating in hydrogen bond formation are shown as sticks.

Figure 3.

Interface comparison among RBDs of SARS-CoV-2 and SARS-CoV with ACE2 orthologs. Binding interface of (A) hACE2/SARS-CoV-2 RBD, (B) hACE2/SARS-CoV RBD, (C) MW-ACE2/SARS-CoV-2 RBD, (D) MW-ACE2/SARS-CoV RBD, (E) SL-ACE2/SARS-CoV-2 RBD and (F) SL-ACE2/SARS-CoV RBD. Venn diagrams of key residues on (G) SARS-CoV-2 RBD and (I) SARS-CoV RBD that are involved in the interaction with the three ACE2s. Key residues on hACE2, MW-ACE2 and SL-ACE2 participate in the interaction with (H) SARS-CoV-2 RBD and (J) SARS-CoV RBD. In panels C–F, residues in blue indicate that they are only involved in RBD binding by hACE2 but not in the referred ACE2 ortholog. Residues in red indicate that they are only involved in the RBD interaction of the referred ACE2 ortholog but not in hACE2. Residues in yellow indicate that a substitution was observed on the ACE2 interface compared with hACE2.

Figure 4.

Structural details of MW-ACE2/SARS-CoV-2 RBD, MW-ACE2/SARS-CoV RBD, SL-ACE2/SARS-CoV-2 RBD and SL-ACE2/SARS-CoV RBD. (A and B) Structural alignment of hACE2/SARS-CoV-2 RBD (wheat) with MW-ACE2/SARS-CoV-2 RBD (purple, A), or with SL-ACE2/SARS-CoV-2 RBD (cyan, B). Substitutions of hACE2 with MW-ACE2 or SL-ACE2 are labeled above the figure. Residues involved in the interaction are presented as sticks, and polar interactions are presented by red dashes. (C and D) Structural alignment of hACE2/SARS-CoV RBD (yellow) with MW-ACE2/SARS-CoV-2 RBD (purple, C) or with SL-ACE2/SARS-CoV-2 RBD (cyan, D). Substitutions of hACE2 with MW-ACE2 or SL-ACE2 are labeled above the figure. Residues involved in the interaction are presented as sticks and polar interactions are presented by red dashes.