Structural basis of increased binding affinities of spikes from SARS-CoV-2 Omicron variants to rabbit and hare ACE2s reveals the expanding host tendency

Abstract

The potential host range of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been expanding alongside its evolution during the pandemic, with rabbits and hares being considered important potential hosts, supported by a report of rabbit sero-prevalence in nature. We measured the binding affinities of rabbit and hare angiotensin-converting enzyme 2 (ACE2) with receptor-binding domains (RBDs) from SARS-CoV, SARS-CoV-2, and its variants and found that rabbit and hare ACE2s had broad variant tropism, with significantly enhanced affinities to Omicron BA.4/5 and its subsequent-emerged sub-variants (>10 fold). The structures of rabbit ACE2 complexed with either SARS-CoV-2 prototype (PT) or Omicron BA.4/5 spike (S) proteins were determined, thereby unveiling the importance of rabbit ACE2 Q34 in RBD-interaction and elucidating the molecular basis of the enhanced binding with Omicron BA.4/5 RBD. These results address the highly enhanced risk of rabbits infecting SARS-CoV-2 Omicron sub-variants and the importance of constant surveillance.IMPORTANCEThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has swept the globe and caused immense health and economic damage. SARS-CoV-2 has demonstrated a broad host range, indicating a high risk of interspecies transmission and adaptive mutation. Therefore, constant monitoring for potential hosts is of immense importance. In this study, we found that Omicron BA.4/5 and subsequent-emerged sub-variants exhibited enhanced binding to both rabbit and hare angiotensin-converting enzyme 2 (ACE2), and we elucidated the structural mechanism of their recognition. From the structure, we found that Q34, a unique residue of rabbit ACE2 compared to other ACE2 orthologs, plays an important role in ACE2 recognition. These results address the probability of rabbits/hares being potential hosts of SARS-CoV-2 and broaden our knowledge regarding the molecular mechanism of SARS-CoV-2 interspecies transmission.

Keywords: ACE2; RBD; SARS-CoV; SARS-CoV-2; cryo-EM structure; hare; rabbit; spike (S) proteins.

FIG 1

Rabbit and hare ACE2s broadly bind to RBDs of SARS-CoV, SARS-CoV-2, and SARS-CoV-2 variants. (A and B) The SPR assay of rabbit (A) and hare (B) ACE2s binding to RBDs of SARS-CoV, SARS-CoV-2 PT, and SARS-CoV-2 variants. (C) Comparison of rabbit ACE2 (upper) and rabbit ACE2 Q34H (lower) binding to SARS-CoV-2 PT and variants. Dissociation constants (K_D) were calculated from three independent experiments and presented as mean ± standard deviation. The actual and fitted curves were colored in black and red, respectively.

FIG 2

Structures of SARS-CoV-2 PT and omicron BA.4/5 S proteins in complex with rabbit ACE2. Cryo-EM density maps of the PT (A) and BA.4/5 (C) S proteins complexed with rabbit ACE2. The local refinement was performed at the binding interface of the RBD and rabbit ACE2. The density maps on the binding interface are shown as mesh. The overall structures of PT RBD/rabbit ACE2 (B) and Omicron BA.4/5 RBD/rabbit ACE2 (D) are shown as cartoons. The residues that participate in the H-bond networks of Patch 1 and Patch 2 are shown as sticks. The PT RBD and BA.4/5 RBD are colored in green and pink, respectively, and rabbit ACE2 in blue.

FIG 3

Rabbit ACE2-induced RBD erection in SARS-CoV S/rabbit ACE2 complex. (A-C) Cryo-EM density maps of SARS-CoV S/rabbit ACE2 complexes. The molar ratio of mixed S protein and ACE2 during sample preparation is labeled above the complexes. Three protomers of SARS-CoV S are colored in purple, red, and pale purple, respectively, and rabbit ACE2 in blue. (D) Cryo-EM density map of the SARS-CoV RBD/rabbit ACE2 complex and its binding interface. The main chains of interacting residues are presented as cartoons and side chains as sticks. The density of residues is presented as mesh. (E) Molecular model of SARS-CoV RBD/rabbit ACE2 complex and H-bond network. Residues involved in the H-bond network are shown as sticks.

FIG 4

Comparison of the binding interface among PT and omicron BA.4/5 RBDs with human ACE2 or rabbit ACE2. (A-D) Binding interfaces of PT RBD/rabbit ACE2 (A), PT RBD/human ACE2 (B), Omicron BA.4/5 RBD/rabbit ACE2 (C), and Omicron BA.4/5 RBD/human ACE2 (D). Outline of the binding interface between rabbit ACE2 and designated RBDs is colored in blue and cyan, respectively. (E and F) Venn diagrams of key residues on the binding interface. PT RBD (E) and Omicron BA.4/5 RBD (F) residues involved in the interaction with human ACE2 and rabbit ACE2 are presented.

FIG 5

The role of mutated residues of variant RBDs in receptor binding. (A) Crucial ACE2 residues interacting with the PT RBD are listed. The highlighted residues indicate the distinctive residues among ACE2 orthologs. (B-D) Structural comparison of designated key ACE2 residues (labeled above) affecting RBD binding. The key residues are shown as sticks. The PT and Omicron BA.4/5 RBDs are colored in green and pink, respectively. The human ACE2 and rabbit ACE2 are in orange and blue, respectively. (E and F) Structural comparison of S477N (E) and Q498R (F) substitutions affects rabbit ACE2 binding.