A 193-amino acid fragment of the SARS coronavirus S protein efficiently binds angiotensin-converting enzyme 2

Abstract

The coronavirus spike (S) protein mediates infection of receptor-expressing host cells and is a critical target for antiviral neutralizing antibodies. Angiotensin-converting enzyme 2 (ACE2) is a functional receptor for the coronavirus (severe acute respiratory syndrome (SARS)-CoV) that causes SARS. Here we demonstrate that a 193-amino acid fragment of the S protein (residues 318-510) bound ACE2 more efficiently than did the full S1 domain (residues 12-672). Smaller S protein fragments, expressing residues 327-510 or 318-490, did not detectably bind ACE2. A point mutation at aspartic acid 454 abolished association of the full S1 domain and of the 193-residue fragment with ACE2. The 193-residue fragment blocked S protein-mediated infection with an IC(50) of less than 10 nm, whereas the IC(50) of the S1 domain was approximately 50 nm. These data identify an independently folded receptor-binding domain of the SARS-CoV S protein.

Fig. 1

Residues 318–510 of the SARS-CoV S protein include the receptor-binding domain.A, S1-Ig, containing S protein residues 12–672 fused to the Fc region of human IgG1, or truncation variants of S1-Ig containing the indicated S protein residues, were purified from media of transfected 293T cells. S1-Ig and variants were normalized for expression, as shown by Coomassie staining (top panel), and used to precipitate soluble metabolically labeled ACE2 (bottom panel). Precipitates were analyzed by SDS-PAGE, and ACE2 was quantified by phosphorimaging. B, the indicated S1-Ig variants were incubated with ACE2-transfected 293T cells and analyzed by flow cytometry (black bars) or used, as in (A), to immunoprecipitate soluble ACE2 (gray bars). Bars indicate averages of two or more experiments normalized to results for S1-Ig. C, representation of truncation variants assayed in A and B. Dark gray indicates association with ACE2 greater than 25% of that observed for S1-Ig in both precipitation and flow-cytometry assays. Light gray indicates ACE2 association less than 10%, in both assays, of that for S1-Ig. The arrow indicates 318–510 variant, the smallest fragment observed to bind ACE2.

Fig. 2

An S1-Ig variant containing residues 318–510 associates with ACE2 and blocks S protein-mediated entry better than does S1-Ig.A, S1-Ig, or variants containing residues 318–510 or 12–327, were purified from transfected 293T cells and quantified. An aliquot of each variant diluted to the indicated concentrations was visualized by SDS-PAGE and Coomassie staining (top panel) and used to precipitate soluble metabolically labeled ACE2. Precipitates were analyzed by SDS-PAGE, and ACE2 was quantified by phosphorimaging. B, ACE2 precipitated by the indicated concentrations of S1-Ig and the indicated variants. An average of two experiments is shown. C, the indicated concentrations of S1-Ig, or of the 318–510 or 12–327 variants, were incubated with 293T cells expressing ACE2, together with an SIV modified to express green fluorescent protein (SIV-GFP) and pseudotyped with S protein of SARS-CoV or with VSV-G. Infection with pseudotyped virus was quantified by measuring green fluorescence by flow cytometry and shown here as mean fluorescent intensity (m.f.i.). D, fluorescent microscopic fields of 293T cells transfected with ACE2 and incubated with SIV-GFP pseudotyped with S protein in the presence of a 250 nm concentration of the 12–327 (right) or the 318–510 (left) variants. Many microscopic fields of cells incubated with the 318–510 variant lacked observable fluorescing cells.

Fig. 3

Analysis of point mutations of S1-Ig and the 318–510 variant.A, the 318–510 S1-Ig truncation variant, or variants thereof in which each of seven cysteines was altered individually to alanine, were analyzed as described in the legend to Fig. 1. Variants in which cysteine 366 or 419 was altered to alanine did not express and were not further analyzed. A variant containing alterations of both cysteines 323 and 378 was also analyzed (right panel). B, 318–510 variants (left panel) or S1-Ig variants (right panel) in which glutamic acid 452 or aspartic acids 454, 463, or 480 were altered individually to alanine were analyzed as in Fig. 1. C, representation of the S proteins of SARS-CoV, HCoV-229E, and MHV, aligned by their S2 domains. Dark gray indicates leader and transmembrane sequences. Light gray indicates receptor-binding domain. The receptor-binding domain of SARS-CoV is shown with N-glycosylation sites (small circles) and cysteines indicated. Residues that make a substantial contribution to ACE2 association (glutamic acid 452 and aspartic acid 454) are shown as white bars.