A study on antigenicity and receptor-binding ability of fragment 450-650 of the spike protein of SARS coronavirus

Abstract

The spike (S) protein of SARS coronavirus (SARS-CoV) is responsible for viral binding with ACE2 molecules. Its receptor-binding motif (S-RBM) is located between residues 424 and 494, which folds into 2 anti-parallel beta-sheets, beta5 and beta6. We have previously demonstrated that fragment 450-650 of the S protein (S450-650) is predominantly recognized by convalescent sera of SARS patients. The N-terminal 60 residues (450-510) of the S450-650 fragment covers the entire beta6 strand of S-RBM. In the present study, we demonstrate that patient sera predominantly recognized 2 linear epitopes outside the beta6 fragment, while the mouse antisera, induced by immunization of BALB/c mice with recombinant S450-650, mainly recognized the beta6 strand-containing region. Unlike patient sera, however, the mouse antisera were unable to inhibit the infectivity of S protein-expressing (SARS-CoV-S) pseudovirus. Fusion protein between green fluorescence protein (GFP) and S450-650 (S450-650-GFP) was able to stain Vero E6 cells and deletion of the beta6 fragment rendered the fusion product (S511-650-GFP) unable to do so. Similarly, recombinant S450-650, but not S511-650, was able to block the infection of Vero E6 cells by the SARS-CoV-S pseudovirus. Co-precipitation experiments confirmed that S450-650 was able to specifically bind with ACE2 molecules in lysate of Vero E6 cells. However, the ability of S450-510, either alone or in fusion with GFP, to bind with ACE2 was significantly poorer compared with S450-650. Our data suggest a possibility that, although the beta6 strand alone is able to bind with ACE2 with relatively high affinity, residues outside the S-RBM could also assist the receptor binding of SARS-CoV-S protein.

Fig. 1

Relative locations of the S-RBD, S-RBM and recombinant polypeptides prepared in this study. (A) Recombinant S450–650 and truncated polypeptides covering its sequence, including S-I (450–510), S-II (511–560), S-III (546–595), S-IV (581–630), S-V (601–650) and S511–650 were expressed in E. coli. Recombinant GFP and its fusion proteins with S protein fragments were also prepared. (B) The purifications product of GFP (lane 1) and its fusion proteins with S-I (S450–510-GFP, lane 2), S511–650 (S511–650-GFP, lane 3) or S450–650 (S450–650-GFP, lane 4) were analyzed by SDS-PAGE electrophoresis and visualized by Commassie blue staining. Molecular weight markers were loaded in the left-hand-side lane.

Fig. 2

Possible B cell epitopes in S450–650. Recombinant constructs S-I (450–510), S-II (511–560), S-III (546–595), S-IV (581–630) and S-V (601–650) were run on SDS-PAGE 12% gels (0.5 μg in each lane) and visualized by Commassie blue staining (top row). The protein bands were transferred onto nitrocellulose membranes for WB with convalescent patient sera (PT31, PT41, PT42, PT53, PT58) or mouse anti-S450–650 antiserum (MAS) as first Abs. HRP-conjugated goat anti-human, or goat-anti-mouse, IgG Abs were employed, respectively, as detection Abs.

Fig. 3

Comparison for neutralization ability of patient sera and mouse antiserum. (A) Infectivity of the SARS-CoV-S pseudovirus. Preparations of SARS-CoV-S pseudovirus, or VSV-G pseudovirus, or mock transfection supernatant (Control) were added to triplicate wells on 96-well plates containing monolayer of Vero E6 cells (8 × 10³ cells/well) and incubated at 37 °C for 1 h. The incubation continued for 48 h after fresh medium replacement and the cells were then lysed and luciferase activity in the lysate determined. The results are expressed as luciferase activity (log₁₀). (B and C) Neutralization assay. Serial diluted serum samples from convalescent patients (PT31 and PT53), or healthy blood donors (HDS), or S450–650-immunized mice (MAS), or naïve BALB/c mice (NMS) were mixed with medium containing SARS-CoV-S, or VSV-G, pseudovirus, and incubated for 30 min at 37 °C. The mixtures were then distributed into triplicate wells in 96-well plates containing monolayer of Vero E6 cells. Luciferase activity in the infected cells was determined 48 h later and the results expressed as percent infection compared with the control group (Vero E6 cells only treated with pseudovirus preparations).

Fig. 4

Staining of Vero E6 cells with S450–650 or its GFP fusion proteins. For indirect staining (A, B), Vero E6 cells were treated with S450–650 (0.5 μM) in PBS at 4 °C for 1 h, followed by convalescent patient serum (PT31), or mouse anti-S450–650 antiserum (MAS), or serum sample from healthy blood donors (HDS), or normal mouse serum (NMS). FITC-conjugated goat anti-human, or goat-anti-mouse, IgG Abs were employed as secondary Abs. As controls, Vero E6 cells and 293T cells were treated with S-RBD-Fc, or human IgG (0.5 μM), at 4 °C for 1 h and followed by FITC-conjugated goat anti-human IgG (C, F). For direct staining, Vero E6 (D), or 293T (E), cells were treated with GFP, S450–510-GFP, S510–650-GFP or S450–650-GFP (0.5 μM) at 4 °C for 30 min. After washes, the cells were subjected to flow cytometric analysis.

Fig. 5

Pseudovirus infection blocking assay. SARS-CoV-S pseudovirus preparations were mixed with, or without (reference group), RBD-Fc or human IgG and incubated together for 30 min at 37 °C. The mixtures were then distributed into triplicate wells containing monolayer of Vero E6 cells seeded on 96-well plates 18 h previously. Luciferase activity in the infected cells was determined 48 h later (A). In a parallel experiment (B), SARS-CoV-S pseudovirus preparations were mixed with, or without (reference group), recombinant GFP, S450–650, S450–510-GFP or S510–650 at 37 °C for 30 min and then used to infect Vero E6 cells. The results are expressed as percent infection (luciferase activity of experimental group compared with that of the reference group).

Fig. 6

Co-precipitation of recombinant S450–650 with ACE2 in the lysate of Vero E6 cells. Vero E6 cells (1 × 10⁶) were sequentially treated with goat anti-human ACE2 Abs, or goat IgG (2 μg), and FITC-conjugated rabbit anti-goat IgG. The stained cells were subjected to flow cytometric analysis for determination of ACE2 expression on the cell surface (A). For the co-precipitation experiment (B), Vero E6 cells (2.5 × 10⁷) were treated with, or without, recombinant S450–650 (15 μg/ml) at 4 °C for 1 h. After washes, the cells were lysed using lysis buffer and the lysate treated with PT31 serum followed by protein-A/G-agarose precipitation. The precipitated materials were run on SDS-PAGE 12% gels and the protein bands transferred to nitrocellulose membranes for WB assays using goat anti-human ACE2 Abs as the first Abs, the detection Abs were HRP-labeled rabbit anti-goat IgG. In a parallel experiment (C), lysate of the S450–650-treated, or untreated, Vero E6 cells were precipitated using goat anti-human ACE2 Abs and protein-A/G-agarose. The precipitates were assayed by WB using PT31 serum (first Ab) and HRP-labeled goat anti-human IgG (detection Ab).

Fig. 7

ACE2 interaction with the β5 and β6 strands of S-RBM. (A) Crystal structure of the RBD in complex of human receptor ACE2 as reported by Li and co-workers (Li et al., 2005). Details of the binding interface between S-RBM and the ACE2 molecule and calculation results of the contact area and binding energy are shown in panels B and C, respectively. The S protein-binding areas of the ACE2 molecule (purple) are colored in green and the β5 and β6 strands in gold. The binding energy here is the energy required to disassemble the RBD-ACE2 complex into separate parts. The figure is prepared using PYMOL and contact area and binding energy calculated by SPDBV. PDB file accession number is 2AJF.