Endocytosis of the receptor-binding domain of SARS-CoV spike protein together with virus receptor ACE2

Abstract

Cell entry of severe acute respiratory syndrome coronavirus (SARS-CoV) is mediated by the viral spike (S) protein. Amino acids 319-510 on the S protein have been mapped as the receptor-binding domain (RBD), which mediates binding to the SARS-CoV receptor angiotensin converting enzyme 2 (ACE2) on SARS-CoV susceptible cells. In this study, we expressed a fusion protein containing the human codon-optimized RBD of the SARS-CoV spike protein linked to the Fc portion of human IgG1 (named RBD-Fc) in HEK293 cells. The RBD-Fc protein was purified by affinity chromatography. The flow cytometry assay showed that the purified RBD-Fc protein could bind to ACE2. We demonstrated that the RBD spike protein alone could be internalized into SARS-CoV susceptible cells together with ACE2. We also showed that the removal of N-glycans from the RBD spike protein did not abolish this phenomenon. Our discoveries may have some implications for the development of the SARS vaccine.

Fig. 1

Expression and purification of the RBD spike protein of SARS-CoV. Western blot (WB) analysis of RBD-Fc protein expression using a human IgG-specific antibody. HEK293 cell culture supernatant (lane 1), the culture supernatant of HEK293 cells transiently transfected with Peak13-RBD-Fc (lane 2). Coomassie Brilliant Blue (CBB) staining of 10% SDS-PAGE analysis of the purified recombinant RBD-Fc protein.

Fig. 2

SARS-CoV RBD spike protein was internalized by the susceptible cells together with ACE2. (A) Decreased cell surface-bound RBD-Fc protein following incubation with VeroE6 cells at 37 °C compared to 4 °C. After incubation with VeroE6 cells for 3 h, cell surface-bound Fc or RBD-Fc proteins were detected using a FITC-conjugated human IgG-specific antibody. Representative FACS histograms are shown for three independent experiments. Blue and black histograms represent the results from the cells incubated with the RBD-Fc or Fc at 4 °C, respectively. The red histogram represents the results from the cells incubated with the RBD-Fc at 37 °C. (B) ACE2 internalization is the RBD spike protein-dependent. After ACE2-GFP-293 cells were incubated with the RBD-Fc or Fc protein at 37 °C for 3 h, the distribution of ACE2-GFP molecules in live cells was observed under a fluorescence microscope. (C) Co-localization of ACE2 and the RBD of SARS-CoV in ACE2-GFP-293 cells under a confocal laser-scanning microscope. After incubation with the RBD-Fc or Fc protein at 37 °C for 3 h, ACE2-GFP-293 cells were washed to remove excess RBD-Fc or Fc protein and then fixed with 4% paraformaldehyde. Immunostaining was performed using an Alexa Fluor 568-conjugated human IgG-specific antibody to detect the distribution of the Fc or RBD-Fc protein in the cells. (D) and (E) Statistical analysis of the differences between the RBD-Fc and Fc protein treatments (Chi-square test). **P < 0.001. “Live cells” means that the treated cells were directly observed under a fluorescence microscope. “Immunostaining cells” means that the treated cells were first fixed and immunostained, and then observed under a confocal laser-scanning microscope.

Fig. 3

Removal of N-glycans of RBD-Fc can still induce ACE2 internalization (A) the RBD-Fc protein was N-linked glycosylated in HEK293 cells. The purified RBD-Fc (lane 1), the purified RBD-Fc was deglycosylated with PNGase F in the presence of SDS (lane 2), the purified RBD-Fc was deglycosylated with PNGase F in the absence of SDS (lane 3). (B) The deglycosylated RBD-Fc under nondenaturing conditions (dg-RBD-Fc) can still induce ACE2 internalization as the glycosylated RBD-Fc. ACE2-GFP-293 cells were incubated with the Fc protein (a), the RBD-Fc protein (b), the mixture of PNGase F and RBD-Fc (c) and the dg-RBD-Fc (d) at 37 °C for 3 h. The distribution of ACE2-GFP molecules in live cells was observed under a fluorescence microscope. (C) and (D) Statistical analysis of the differences among treatments of the RBD-Fc, the dg-RBD-Fc or the mixture of PNGase F and the RBD-Fc using Chi-square test. P < 0.05 was considered to be significantly different.