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. 2012 Mar 16;287(12):8904-11.
doi: 10.1074/jbc.M111.325803. Epub 2012 Jan 30.

Mechanisms of host receptor adaptation by severe acute respiratory syndrome coronavirus

Kailang Wu  1 , Guiqing Peng, Matthew Wilken, Robert J Geraghty, Fang Li
Affiliations

Mechanisms of host receptor adaptation by severe acute respiratory syndrome coronavirus

Kailang Wu et al. J Biol Chem. .

Abstract

The severe acute respiratory syndrome coronavirus (SARS-CoV) from palm civets has twice evolved the capacity to infect humans by gaining binding affinity for human receptor angiotensin-converting enzyme 2 (ACE2). Numerous mutations have been identified in the receptor-binding domain (RBD) of different SARS-CoV strains isolated from humans or civets. Why these mutations were naturally selected or how SARS-CoV evolved to adapt to different host receptors has been poorly understood, presenting evolutionary and epidemic conundrums. In this study, we investigated the impact of these mutations on receptor recognition, an important determinant of SARS-CoV infection and pathogenesis. Using a combination of biochemical, functional, and crystallographic approaches, we elucidated the molecular and structural mechanisms of each of these naturally selected RBD mutations. These mutations either strengthen favorable interactions or reduce unfavorable interactions with two virus-binding hot spots on ACE2, and by doing so, they enhance viral interactions with either human (hACE2) or civet (cACE2) ACE2. Therefore, these mutations were viral adaptations to either hACE2 or cACE2. To corroborate the above analysis, we designed and characterized two optimized RBDs. The human-optimized RBD contains all of the hACE2-adapted residues (Phe-442, Phe-472, Asn-479, Asp-480, and Thr-487) and possesses exceptionally high affinity for hACE2 but relative low affinity for cACE2. The civet-optimized RBD contains all of the cACE2-adapted residues (Tyr-442, Pro-472, Arg-479, Gly-480, and Thr-487) and possesses exceptionally high affinity for cACE2 and also substantial affinity for hACE2. These results not only illustrate the detailed mechanisms of host receptor adaptation by SARS-CoV but also provide a molecular and structural basis for tracking future SARS-CoV evolution in animals.

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Figures

FIGURE 1.
FIGURE 1.
Interface between SARS-CoV RBD and hACE2. A, list of mutations in the RBMs of various SARS-CoV strains. Five representative existent strains and two predicted future strains are defined in the Introduction. B, overall structure of the hTor02 RBD-hACE2 complex (Protein Data Bank code 2AJF). hACE2 is in green, and hTor02 RBD is in cyan (core) and red (RBM). RBM residues that underwent mutations are displayed. C, detailed structure of the hTor02 RBD/hACE2 interface. hACE2 residues are in green, SARS-CoV residues that underwent mutations are in magenta, and SARS-CoV residues that played significant roles in the mutations are in cyan.
FIGURE 2.
FIGURE 2.
Structures and functions of two virus-binding hot spots on hACE2. A, surface plasmon resonance Biacore analysis of the binding interactions between hTor02 RBD and wild-type or mutant hACE2. hTor02 RBD was immobilized, and wild-type or mutant ACE2 was flowed through. Each experiment was repeated six times at three different protein concentrations. The corresponding S.E. values are shown. B, pseudotyped viral infection assays of the interactions between hTor02 spike protein and wild-type or mutant hACE2. Retroviral murine leukemia viruses expressing β-galactosidase and pseudotyped with hTor02 spike protein were used to infect HEK293T cells expressing wild-type or mutant hACE2. Infection efficiency of pseudotyped viruses was measured by β-galactosidase assays and normalized against the infection efficiency in cells expressing wild-type hACE2. Each experiment was repeated six times. The corresponding S.E. values are shown. C, use of a human-civet chimeric ACE2 in crystallographic studies of RBD/cACE2 interactions. The chimeric ACE2 contains SARS-CoV-binding residues from cACE2 and other residues from hACE2. The chimeric ACE2 has the same receptor activities as cACE2 but the same crystallographic activities as hACE2 (27). SARS-CoV-binding residues that differ between hACE2 and cACE2 are shown. D, structure of the interface between hTor02 RBD and the chimeric ACE2.
FIGURE 3.
FIGURE 3.
Molecular mechanisms of RBM mutations in SARS-CoV. A, surface plasmon resonance Biacore analysis of the binding interactions between hACE2 and wild-type or mutant hTor02 RBD. hACE2 was immobilized, and wild-type or mutant hTor02 RBD was flowed through. B, surface plasmon resonance Biacore analysis of the binding interactions between cACE2 and wild-type or mutant hTor02 RBD. cACE2 was immobilized, and wild-type or mutant hTor02 RBD was flowed through. C, pseudotyped viral infection assays of the interactions between different spike proteins and hACE2. D, pseudotyped viral infection assays of the interactions between different spike proteins and cACE2.
FIGURE 4.
FIGURE 4.
Structural mechanisms of RBM mutations in SARS-CoV. A, structure of the interface between hACE2 and hOptimize RBD. B, structure of the interface between the chimeric ACE2 and cOptimize RBD. C, structure of the interface between hACE2 and cOptimize RBD. D, superimposed structures of the interfaces between the chimeric ACE2 and cOptimize RBD (colored) and between hACE2 and hTor02 (white).
FIGURE 5.
FIGURE 5.
Summary of host receptor adaptation by SARS-CoV. Listed are adaptations of RBM residues to hACE2 or cACE2. Arrows point from less well adapted residues to better adapted residues. Double arrows connect equally well adapted residues.
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