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. 2003 Feb;77(4):2530-8.
doi: 10.1128/jvi.77.4.2530-2538.2003.

Identification of a receptor-binding domain of the spike glycoprotein of human coronavirus HCoV-229E

Aurelio Bonavia  1 , Bruce D Zelus, David E Wentworth, Pierre J Talbot, Kathryn V Holmes
Affiliations

Identification of a receptor-binding domain of the spike glycoprotein of human coronavirus HCoV-229E

Aurelio Bonavia et al. J Virol. 2003 Feb.

Abstract

Human coronavirus HCoV-229E uses human aminopeptidase N (hAPN) as its receptor (C. L. Yeager et al., Nature 357:420-422, 1992). To identify the receptor-binding domain of the viral spike glycoprotein (S), we expressed soluble truncated histidine-tagged S glycoproteins by using baculovirus expression vectors. Truncated S proteins purified by nickel affinity chromatography were shown to be glycosylated and to react with polyclonal anti-HCoV-229E antibodies and monoclonal antibodies to the viral S protein. A truncated protein (S(547)) that contains the N-terminal 547 amino acids bound to 3T3 mouse cells that express hAPN but not to mouse 3T3 cells transfected with empty vector. Binding of S(547) to hAPN was blocked by an anti-hAPN monoclonal antibody that inhibits binding of virus to hAPN and blocks virus infection of human cells and was also blocked by polyclonal anti-HCoV-229E antibody. S proteins that contain the N-terminal 268 or 417 amino acids did not bind to hAPN-3T3 cells. Antibody to the region from amino acid 417 to the C terminus of S blocked binding of S(547) to hAPN-3T3 cells. Thus, the data suggest that the domain of the spike protein between amino acids 417 and 547 is required for the binding of HCoV-229E to its hAPN receptor.

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Figures

FIG. 1.
FIG. 1.
Diagram of the baculovirus expression constructs for expression of soluble, six-histidine-tagged (6xH) truncated HCoV-229E spike glycoprotein (S). The number next to the S indicates the amino acid at which the construct was truncated. Each construct contains the HCoV-229E signal sequence. The region corresponding to the TGEV receptor-binding domain was identified by Godet et al. (14).
FIG. 2.
FIG. 2.
Characterization of the purified, soluble, truncated HCoV-229E spike proteins. (A) Immunoblot of 1.2 μg of purified S547 (lane 1), S417 (lane 2), and S268 (lane 3) with polyclonal goat anti-HCoV-229E antiserum. (B) Immunoblot as for that in panel A of proteins treated with PNGase F.
FIG. 3.
FIG. 3.
Binding of anti-HCoV-229E MAbs to the three truncated HCoV-229E spike glycoproteins. ELISA detection of 350 ng of S547, S417, and S268 by using anti-HCoV-229E MAbs followed by HRP-conjugated anti-mouse Igs. The diagram below shows the probable locations of MAb-binding epitopes in the linear map of the N-terminal domain of the HCoV-229E spike protein. OD (450), optical density at 450 nm.
FIG. 4.
FIG. 4.
Binding of truncated HCoV-229E S glycoproteins to hAPN on cell membranes. (A) Detection of S547, S417, and S268 binding to 3T3 cells or hAPN-3T3 cells by using normal goat serum (C) or polyclonal goat anti-HCoV-229E antiserum (E). (B) Detection of S547 binding to hAPN-3T3 cells with preimmune goat serum (C) or polyclonal goat anti-HCoV-229E antiserum (E) after the cells were preincubated with isotype control (graph a) or anti-hAPN MAb-BB1 that blocks HCoV-229E infection of human cells or hAPN-expressing cells (graph b). The y axis indicates cell numbers, and the x axis indicates fluorescence intensity.
FIG. 5.
FIG. 5.
Binding of S547 to hAPN-3T3 cells is blocked by preincubation of S547 with neutralizing polyclonal goat anti-HCoV-229E antiserum. Detection of S547 by flow cytometry with anti-HCoV-229E-S MAb5-11.H6 (E) and isotype control (C). The amount (in micrograms) of polyclonal goat anti-HCoV-229E antiserum used in the preincubation step is shown above each panel. The y axis indicates cell numbers, and the x axis indicates fluorescence intensity.
FIG. 6.
FIG. 6.
(A) ELISA of binding to S547, S417, and S268 of polyclonal anti-HCoV-229E (G1), purified polyclonal (G1Ig), and anti-HCoV-229E G1 antiserum immunoadsorbed against S547 (G1/S547), S417 (G1/S417), and S268 (G1/S268). (B) Binding of 1.5 μg of S547 to 106 hAPN/3T3 cells after incubation of S547 with various amounts of G1 (a), G1Ig (b), G1/S547(c), G1/S417 (d), and G1/S268 (e) preimmune goat serum (PI) (f) detected with anti-HCoV-229E S MAb 5-11H.6 or isotype control (M). The y axis indicates cell numbers, and the x axis indicates fluorescence intensity.
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