Biochemical and structural evidence demonstrates Omicron mutations are better adapted to mouse ACE2 than to human ACE2

By Neha MathurReviewed by Danielle Ellis, B.Sc.Oct 22 2022

In a recent study published in PNAS, researchers demonstrated the structural basis of how the receptor-binding domain (RBD) nested Omicron mutations have adapted to mouse angiotensin-converting enzyme 2 (ACE2) rather than human ACE2.

Background

Speculations about the source of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant of concern (VOC) are abundant, yet the experimental evidence for the same has been scarce. Its sudden emergence and rapid spread have raised questions about its animal reservoir.

A few amino acid residues differentiate the prototypic RBD from the RBD of bat coronaviruses. The Omicron BA.2 RBD differs from the prototypic RBD by 16 residues, with seven nested inside the receptor-binding motif (RBM) directly contacting ACE2.

About the study

In the present study, researchers recovered the evolutionary traces of Omicron RBM mutations. They investigated ACE2 recognition of the Omicron’s RBD, focusing on Q493R, Q498R, N501Y, and Y505H mutations, surrounding two mutational hotspots, hotspot-31 or hotspot-353.

The researchers deployed site-directed mutagenesis to synthesize the gene encoding the SARS-CoV-2 prototypic S, hACE2, and mACE2. Next, they used a surface plasmon resonance (SPR) assay to measure the binding interactions between RBDs and ACE2 molecules. To confirm the SPR data, the team also performed an Omicron pseudovirus entry assay. They packaged Omicron pseudoviruses with four reverse mutations (Q493R, Q498R, N501Y, and Y505H) before infecting mACE2-expressing cells.

Finally, the team determined the crystal structure of Omicron RBD complexed with mouse ACE2 at 2.84 Å.

Study findings

Although the prototypic SARS-CoV-2 did not infect mice efficiently, other earlier SARS-CoV-2 VOCs from humans and other animal species had evolved the N501Y mutation to facilitate the usage of the mACE2 receptor by SARS-CoV-2. Also, only mice have asparagine (Asn31) and histidine(His353) in their ACE2 sequence, suggesting that Omicron evolved in mice.

SPR assay showed that the prototypic RBD did not bind mACE2, while the Omicron RBD bound mACE2 with good affinity. Introducing R493Q, R498Q, Y501N, and H505Y reverse mutations to the Omicron's RBD, only slightly reduced mACE2 binding. Further, the study identified Q493R, Q498R, and Y505H RBM mutations, specifically structurally adapted to mACE2, suggesting that these mutations were the evolutionary traces left behind by Omicron.

It is what likely happened during SARS-CoV-2 evolution: a SARS-CoV-2 variant containing the N501Y mutation spread from humans or another animal species into mice. Later, as this variant spread in mice, mouse-specific RBM mutations (e.g., Q493R, Q498R, and Y505N) evolved, contributing to the emergence of the Omicron VOC. The ACE2 sequences of some rat species also contain Asn31 or His353. Besides humans, Omicron might have also been transmitted to other species whose ACE2 contained virus binding motifs (VBM) residues compatible with the Omicron RBD.

The complex of the chimeric Omicron RBD and chimeric mACE2 revealed the extensive interactions between the Omicron RBM and mACE2 virus binding motifs (VBMs). Hotspot-31 stabilizes the core of the RBM/VBMs interface, where lysine31 and glutamic acid35 VBM residues form a hydrogen bond with glutamine493. In mACE2, residue 31 is an asparagine, replacing Lys31 in hACE2. Thus, at the interface between the Omicron RBM and mouse VBMs, Arg493 in RBM forms two bifurcated hydrogen bonds with Asn31 VBM, stabilizing the RBM/VBMs interface and enhancing Omicron RBD’s affinity for mACE2. Overall, the Omicron mutation Q493R around hotspot-31 structurally adapted to Asn31 in mACE2.

Conclusions

The current study data revealed that the Omicron RBD was well adapted to mouse ACE2 before it even began infecting humans. The researchers used biochemical and structural evidence to show that mice facilitated the evolution of the Omicron VOC, providing much-needed insights into the evolutionary origin of SARS-CoV-2. These findings would also facilitate epidemiological surveillance of SARS-CoV-2 in animals, such as mice and rats, to clarify the SARS-CoV-2 evolutionary trajectory and prevent future coronavirus pandemics.