Receptor and viral determinants of SARS-coronavirus adaptation to human ACE2

Abstract

Human angiotensin-converting enzyme 2 (ACE2) is a functional receptor for SARS coronavirus (SARS-CoV). Here we identify the SARS-CoV spike (S)-protein-binding site on ACE2. We also compare S proteins of SARS-CoV isolated during the 2002-2003 SARS outbreak and during the much less severe 2003-2004 outbreak, and from palm civets, a possible source of SARS-CoV found in humans. All three S proteins bound to and utilized palm-civet ACE2 efficiently, but the latter two S proteins utilized human ACE2 markedly less efficiently than did the S protein obtained during the earlier human outbreak. The lower affinity of these S proteins could be complemented by altering specific residues within the S-protein-binding site of human ACE2 to those of civet ACE2, or by altering S-protein residues 479 and 487 to residues conserved during the 2002-2003 outbreak. Collectively, these data describe molecular interactions important to the adaptation of SARS-CoV to human cells, and provide insight into the severity of the 2002-2003 SARS epidemic.

Figure 1

Introduction of rat ACE2 residues into the human ACE2 catalytic domain interferes with S-protein association. (A) HEK293T cells were transfected with plasmids encoding human or rat ACE2, or chimeras of these receptors in which residues 1–600, corresponding to the ACE2 catalytic domain, were exchanged. Transfected cells were metabolically labeled with [³⁵S]cysteine and [³⁵S]methionine, and lysed. Lysates were immunoprecipitated with Protein A–Sepharose together with either the S1 domain of the SARS-CoV (TOR2) S protein fused to the Fc domain of human IgG1 (S1-Ig), or with an antibody (1D4) recognizing a tag present at the carboxy-terminus of each of the ACE2 variants, and analyzed by SDS–PAGE. (B) HEK293T cells were transfected with plasmids encoding human ACE2, rat ACE2, or human ACE2 variants in which residues corresponding to those of rat ACE2 were introduced at the indicated position. Transfected cells were analyzed as in (A) and precipitated ACE2 was quantified by phosphorimaging. Values indicate the ratio of protein precipitated by S1-Ig to that precipitated by 1D4. Error bars indicate the range of two or more experiments.

Figure 2

Introduction of human ACE2 residues into rat ACE2 converts rat ACE2 to an efficient SARS-CoV receptor. (A) HEK293T cells transfected with plasmids encoding the indicated human and rat ACE2 variants or with vector alone were analyzed as in Figure 1 (light gray), or by flow cytometry (dark gray). Flow cytometry values indicate the ratio of mean fluorescence intensity (m.f.i.) observed using S1-Ig to that using the 9E10 antibody, which recognizes a tag present at the amino-terminus of each ACE2 variant. Error bars indicate the range of two or more experiments. (B) Representative example of an immunoprecipitation experiment used in (A). (C) Murine leukemia viruses (MLV) expressing green fluorescent protein (GFP), lacking its endogenous envelope glycoprotein (MLV-GFP), and pseudotyped with the S protein of SARS-CoV (TOR2 isolate) were incubated with HEK293T cells transfected with plasmids encoding the indicated human or rat ACE2 variants. Amount of ACE2-expressing plasmid was adjusted to maintain comparable receptor expression levels, as indicated, by flow cytometry using the antibody 9E10 that recognizes an amino-terminal myc tag on these receptors. GFP expression in cells was quantified by flow cytometry to measure infection of cells by pseudotyped viruses. Error bars indicate range of two experiments.

Figure 3

The S-protein-binding site of human ACE2. (A) Experiments identical to those of Figure 1 except that the indicated solvent-accessible residues common to human and rat ACE2 were modified in human ACE2 to either alanine or aspartic acid. (B) Representation of the crystal structure of human ACE2, with the collectrin domain oriented downward and viewed from the side of the cleft bearing the enzymatic active site. Residues of rat ACE2 whose alteration to the corresponding human residues converted rat ACE2 to an efficient SARS-CoV receptor are shown in red. Human ACE2 residues whose alteration substantially decreased S1-Ig association are shown in orange. Residues whose alteration did not affect S1-Ig association are shown in green. Low-resolution electron density associated with the collectrin domain is represented by a small β-sheet and α-helix at the base of the figure. (C) A view identical to that in (B) except that the molecule has been rotated 90° about the vertical axis. (D) A view identical to that in (C) except that the molecule has been rotated 90° about the horizontal axis.

Figure 4

ACE2 conformational changes induced by an enzymatic inhibitor do not alter S1-Ig association or S-protein-mediated entry. (A) Soluble ACE2 was incubated with a peptide substrate (methoxycoumarin- YVADAPK(dinitrophenyl)-OH) that fluoresces following cleavage, together with the indicated concentrations of MLN-4760, an inhibitor of ACE2 activity. Fluorescence was measured at 4 min intervals with a fluorescent plate reader. Values indicate average of duplicates. (B) S1-Ig was used to immunoprecipitate ACE2 from lysates of metabolically labeled HEK293T cells transfected with plasmid encoding human ACE2 in the presence of the indicated concentrations of MLN-4760. (C) MLV-GFP virions pseudotyped with the SARS-CoV S protein (diamonds) or with an MLV envelope glycoprotein (squares) were incubated as in Figure 2C with HEK293T cells expressing human ACE2 in the presence of the indicated concentrations of MLN-4760 or NH₄Cl. Infection as indicated by GFP fluorescence was quantified by flow cytometry. Figures are representative of two experiments with similar results.

Figure 5

Differential association of three S proteins with human and palm-civet ACE2. (A) HEK293T cells were transfected with plasmids encoding human or palm-civet ACE2, radiolabeled with [³⁵S]cysteine and [³⁵S]methionine, and lysed. ACE2 proteins were immunoprecipitated with 1D4, which recognizes a tag present at the carboxy-terminus of each receptor, or with S1-Ig variants containing the S1 domains of TOR2, GD, or SZ3 S proteins, and analyzed by SDS–PAGE. TOR2 SARS-CoV was isolated from humans infected during the 2002–2003 outbreak; GD, during the 2003–2004 outbreak; SZ3, from palm civets. (B) Experiment similar to that in (A) except that S-protein residues 318–510, comprising the RBDs of the indicated S proteins fused to the Fc domain of human IgG1 (RBD-Ig), were used to immunoprecipitate human or palm-civet ACE2. The bottom panel shows a Coomassie-stained SDS–PAGE gel of the individual RBDs used in this experiment.

Figure 6

S-protein RBD determinants of efficient association with human ACE2. (A) Table listing amino-acid differences among the RBDs of the S proteins of the indicated isolates. Residues critical to the differential association of these RBDs with palm-civet and human ACE2 are shown in gray. (B) Experiment similar to that shown in Figure 5B except that individual residues within the TOR2 RBD have been altered to the corresponding residues in the SZ3 RBD. (C) HIV-1-luciferase pseudotyped with S protein of the TOR2, GD, or SZ3 viruses, or with the indicated SZ3 or TOR2 variant, was incubated with HEK293T cells transfected with plasmid encoding human ACE2 or with palm-civet ACE2. Infection, measured as luciferase activity of cell lysates, was assayed 2 days postinfection. The figure shows the mean and range of two experiments.

Figure 7

Species-specific ACE2 determinants of differential S-protein association. (A) HEK293T cells transfected with plasmid encoding human or palm-civet ACE2, with human ACE2 bearing the indicated palm-civet residues, or with vector alone were analyzed by flow cytometry using S1-Ig variants of the TOR2, GD, and SZ3 isolates. Error bars indicate the range of two or more experiments. (B) HEK293T cells transfected with plasmid encoding the ACE2 variants used in (A) were incubated with HIV-1-luciferase virus pseudotyped with the S proteins of TOR2, GD, or SZ3 viruses. Infection was assayed as in Figure 6C. (C) HEK293T cells transfected with plasmid encoding human ACE2, human ACE2 variants bearing the indicated palm-civet residues, palm-civet ACE2, or the palm-civet ACE2 variant D354G were metabolically labeled and lysed. Cell lysates were immunoprecipitated with an anti-tag antibody recognizing an amino-terminal tag on these ACE2 variants (α-myc), or with RBD-Ig of TOR2 or SZ3, or with their variants with the indicated alterations of residues 479 and 487. The experiment is representative of at least two with similar results. (D) Amino-acid content of critical regions of ACE2 from human, palm civet, and rat. Orange indicates human-ACE2 residues whose alteration interferes with TOR2 S-protein association. Red indicates rat-ACE2 residues whose alteration to their human counterparts converts rat ACE2 to an efficient SARS-CoV receptor. Yellow indicates residues of palm-civet ACE2 that accommodate S-protein lysine 479 of SARS-CoV isolated from palm civets. Cyan indicates additional residues of palm-civet ACE2 that, when introduced into human ACE2, result in more efficient association with all S proteins assayed. This effect may be due to the loss of glycosylation at asparagine 90 of human ACE2, shown in green. (E) Ribbon diagram of human ACE2 from the top of the protein. Brown on the ribbon identifies regions shown in (D). Residues highlighted in (D) are shown in the same colors. (F) Surface diagram of human ACE2, from the same orientation as in (E), and colored consistently with (D, E).