The spike protein of severe acute respiratory syndrome (SARS) is cleaved in virus infected Vero-E6 cells

Abstract

Spike protein is one of the major structural proteins of severe acute respiratory syndrome-coronavirus. It is essential for the interaction of the virons with host cell receptors and subsequent fusion of the viral envelop with host cell membrane to allow infection. Some spike proteins of coronavirus, such as MHV, HCoV-OC43, AIBV and BcoV, are proteolytically cleaved into two subunits, S1 and S2. In contrast, TGV, FIPV and HCoV-229E are not. Many studies have shown that the cleavage of spike protein seriously affects its function. In order to investigate the maturation and proteolytic processing of the S protein of SARS CoV, we generated S1 and S2 subunit specific antibodies (Abs) as well as N, E and 3CL protein-specific Abs. Our results showed that the antibodies could efficiently and specifically bind to their corresponding proteins from E.coli expressed or lysate of SARS-CoV infected Vero-E6 cells by Western blot analysis. Furthermore, the anti-S1 and S2 Abs were proved to be capable of binding to SARS CoV under electron microscope observation. When S2 Ab was used to perform immune precipitation with lysate of SARS-CoV infected cells, a cleaved S2 fragment was detected with S2-specific mAb by Western blot analysis. The data demonstrated that the cleavage of S protein was observed in the lysate, indicating that proteolytic processing of S protein is present in host cells.

Figure 1

Recombinant SARS-CoV nucleocapsid (N), spike (S, S1 and S2), small envelope (E) protein and 3-CL proteinase were purified and size fractionated on 10% SDS-PAGE. The gel was stained by coomassie brilliant blue staining.

Figure 2

Antibodies binding to their corresponding proteins by western blot analysis. (A) anti-E antibody; (B) anti-3CL antibody; (C) anti-N antibody; (D) anti-S1 antibody; (E) anti-S2 antibody. The proteins were subjected to SDS-PAGE and transferred to Nitrocellulose Transfer Membrane. The blots were probed with the antisera (1:10000) and detected with HRP-conjugated goat anti-rabbits IgG.

Figure 3

Antibodies binding to relevant proteins from the lysates of SARS-CoV infected Vero E6 cell. (A) anti-S1 antibody; (B) anti-S2 antibody; (C) anti-N antibody. The E.coli expressed S or N protein and the lysates were subjected to SDS-PAGE and transferred to Nitrocellulose Transfer Membrane. The blots were probed with the antisera (1:10000) and detected with HRP-conjugated goat anti-rabbits IgG.

Figure 4

Electromicroscopy of SARS-CoV with human Serum of SARS patients and rabbits antisera of S1 and S2. (A) sera from SARS patient; (B) anti-S1 Ab; (C) anti-S2 Ab. After staining with sera, the SARS-CoVs were incubated with the complex of protein-A and golden-particles conjugate. Then the images were observed by electron microscopy.

Figure 5

The S2-specifc monoclonal antibody to detect the subunit of S2 fragment. The lysate with or without immunoprecipitation (IP) with S2 pAb and then the target proteins were determined by S2 mAb using western blot (WB) analysis. The lysate was prepared from SARS-CoV infected Vero E6 cells.