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. 2004 May;78(9):4552-60.
doi: 10.1128/jvi.78.9.4552-4560.2004.

Amino acids 270 to 510 of the severe acute respiratory syndrome coronavirus spike protein are required for interaction with receptor

Gregory J Babcock  1 , Diana J Esshaki, William D Thomas Jr, Donna M Ambrosino
Affiliations

Amino acids 270 to 510 of the severe acute respiratory syndrome coronavirus spike protein are required for interaction with receptor

Gregory J Babcock et al. J Virol. 2004 May.

Abstract

A novel coronavirus, severe acute respiratory syndrome coronavirus (SARS-CoV), has recently been identified as the causative agent of severe acute respiratory syndrome (SARS). SARS-CoV appears similar to other coronaviruses in both virion structure and genome organization. It is known for other coronaviruses that the spike (S) glycoprotein is required for both viral attachment to permissive cells and for fusion of the viral envelope with the host cell membrane. Here we describe the construction and expression of a soluble codon-optimized SARS-CoV S glycoprotein comprising the first 1,190 amino acids of the native S glycoprotein (S(1190)). The codon-optimized and native S glycoproteins exhibit similar molecular weight as determined by Western blot analysis, indicating that synthetic S glycoprotein is modified correctly in a mammalian expression system. S(1190) binds to the surface of Vero E6 cells, a cell permissive to infection, as demonstrated by fluorescence-activated cell sorter analysis, suggesting that S(1190) maintains the biologic activity present in native S glycoprotein. This interaction is blocked with serum obtained from recovering SARS patients, indicating that the binding is specific. In an effort to map the ligand-binding domain of the SARS-CoV S glycoprotein, carboxy- and amino-terminal truncations of the S(1190) glycoprotein were constructed. Amino acids 270 to 510 were the minimal receptor-binding region of the SARS-CoV S glycoprotein as determined by flow cytometry. We speculate that amino acids 1 to 510 of the SARS-CoV S glycoprotein represent a unique domain containing the receptor-binding site (amino acids 270 to 510), analogous to the S1 subunit of other coronavirus S glycoproteins.

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Figures

FIG. 1.
FIG. 1.
Expression and purification of soluble S1190 glycoprotein. Codon-optimized S1190 glycoprotein was cloned into pcDNA3.1 Myc/His and expressed in HEK-293T/17 cells to a level of 5 mg/liter. Protein was purified using metal-affinity chromatography, dialyzed, and concentrated. Purified protein was analyzed by Coomassie staining (A) or Western blotting using the anti-c-myc antibody for detection (B).
FIG. 2.
FIG. 2.
The apparent molecular mass of the S1190 protein is similar to that of native S protein. S1190 protein (200 ng), Vero E6 cell extract, and SARS CoV-infected Vero E6 cell extract were resolved by SDS-PAGE. Proteins were transferred to a solid support, and Western blotting was performed using either human SARS convalescent-phase serum (top panel) or mouse anti-synthetic S protein (bottom panel). The major species detected for S1190 and SARS-infected extract in both cases was of an apparent molecular mass of approximately 170 kDa. No signal was observed in the lane containing the Vero E6 cell extract.
FIG. 3.
FIG. 3.
S1190 specifically binds to the surface of Vero E6 cells. (A) Vero E6 cells (diamonds) or HEK-293T/17 cells (squares) were incubated with 1 to 100 nM S1190 protein to determine the ability to bind to cell surfaces. S1190 binding was detected by anti-c-myc antibody followed by anti-mouse IgG-PE. Samples were analyzed by flow cytometry, and mean fluorescence intensity was plotted. (B) Vero E6 cells were incubated with 30 nM S1190 glycoprotein in the presence of increasing concentrations of normal (circles) or SARS convalescent-phase (squares) human serum. Serum concentration was maintained at 10% by the addition of fetal bovine serum. Binding was measured by flow cytometry, and the results were plotted as a percentage of the signal observed in a sample containing no test serum.
FIG. 4.
FIG. 4.
Soluble S glycoproteins. Shown is a schematic of the native SARS CoV S glycoprotein as well as the various S glycoproteins synthesized. Landmarks include the leader peptide (black; amino acids 1 to 14), the predicted ectodomain (white; amino acids 15 to 1190), the transmembrane domain (dark gray; amino acids 1191 to 1227), and the cytoplasmic tail (light gray; amino acids 1228 to 1255). All soluble constructs were C-terminally truncated, and the relative sizes are shown.
FIG. 5.
FIG. 5.
Expression and binding of C-terminally truncated S proteins. (A) DNA encoding C-terminally truncated S proteins (S350, S490, S590, S690, and S790) was synthesized via PCR using the vector encoding the S1190 glycoprotein as template. PCR products were cloned into pcDNA3.1 Myc/His and expressed in 293T cells. Western blotting was performed using the anti-c-myc antibody for detection. (B) Vero E6 (black bars) or HEK-293T/17 (gray bars) cells were incubated with a 100 nM concentration of each soluble S glycoprotein fragment and detected with antibody directed against the c-myc tag. FACS analysis was performed, and mean fluorescence intensity was plotted.
FIG. 6.
FIG. 6.
Amino acids 1 to 510 represent the ligand-binding domain of the SARS CoV S protein. Constructs encoding C-terminally truncated proteins covering the gap between amino acids 490 and 590 (S510, S520, S540, S550, S560, S570, and S580) were synthesized using PCR and cloned into pcDNA3.1 Myc/His. All constructs were expressed in HEK-293T/17 cells and purified by metal-affinity chromatography. (A) Proteins were resolved by SDS-PAGE and Western blot analysis performed in conjunction with detection with the anti-c-myc antibody. (B) All purified S glycoproteins were incubated with Vero E6 cells at a concentration of 100 nM. Binding was measured using the anti-c-myc antibody and FACS analysis. Mean fluorescence intensity was plotted for each construct and compared.
FIG. 7.
FIG. 7.
S510 and S1190 have comparable binding profiles for Vero E6 cell surfaces. Vero E6 cells were incubated with various concentrations of S1190 (squares), S510 (triangles), and S350 (circles) glycoproteins. Binding was detected with the anti-c-myc antibody, and FACS analysis was performed. Concentration versus mean fluorescence intensity was plotted, and the results for each protein were compared.
FIG. 8.
FIG. 8.
S270-510 binds to Vero E6 cells. (A) Schematic representation of the S510 and S270-510 glycoproteins. The black box (amino acids 1 to 14) represents the S glycoprotein leader sequence. (B) Vero E6 cells or HEK-293T/17 cells were incubated with S350, S590, or S270-510 at concentrations ranging from 0.1 nM to 1 μM. Binding of S glycoproteins was detected using the anti-c-myc antibody followed by anti-mouse IgG conjugated to PE. FACS analysis was performed, and mean fluorescence was plotted as a function of S glycoprotein concentration.
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