The SARS-CoV S glycoprotein: expression and functional characterization

Abstract

We have cloned, expressed, and characterized the full-length and various soluble fragments of the SARS-CoV (Tor2 isolate) S glycoprotein. Cells expressing S fused with receptor-expressing cells at neutral pH suggesting that the recombinant glycoprotein is functional, its membrane fusogenic activity does not require other viral proteins, and that low pH is not required for triggering membrane fusion; fusion was not observed at low receptor concentrations. S and its soluble ectodomain, S(e), were not cleaved to any significant degree. They ran at about 180-200kDa in SDS gels suggesting post-translational modifications as predicted by previous computer analysis and observed for other coronaviruses. Fragments containing the N-terminal amino acid residues 17-537 and 272-537 but not 17-276 bound specifically to Vero E6 cells and purified soluble receptor, ACE2, recently identified by M. Farzan and co-workers [Nature 426 (2003) 450-454]. Together with data for inhibition of binding by antibodies developed against peptides from S, these findings suggest that the receptor-binding domain is located between amino acid residues 303 and 537. These results also confirm that ACE2 is a functional receptor for the SARS virus and may help in the elucidation of the mechanisms of SARS-CoV entry and in the development of vaccine immunogens and entry inhibitors.

Fig. 1

Schematic diagram of a monomer of expressed full-length SARS-CoV S glycoprotein and various soluble fragments after removal of the signal sequence (residues 1–16). TM denotes the transmembrane segment and the arrow indicates possible cleavage site at amino acid residues 758–761 (RNTR). RBD indicates the potential receptor-binding domain that is within S272–537 likely between a residue downstream from 303 and a residue upstream of 537 as suggested by this work.

Fig. 2

Expression of soluble S fragments. (A) Supernatants of 293 and Vero E6 cells transfected with plasmids encoding S fragments (S276, S537, and S756) in the absence or presence of T7 polymerase expressed by recombinant vaccinia virus (VTF7.3) were transferred to nitrocellulose membranes (dot blots) and detected with anti-c-Myc epitope antibody. PC is the positive control for this antibody provided by the manufacturer and NC is a negative control of cells transfected with empty vector. (B) Supernatants from transfected cells as described above (A) were incubated with Ni–NTA agarose beads, washed, and subjected to Western blotting with the same anti-c-Myc epitope antibody as in A. (C) Detection of S fragments by two rabbit polyclonal antibodies raised against peptides corresponding to sequences starting at residues 24 (D24) and 540 (P540), respectively (right two panels). The left panel shows for comparison Western blot where S537 and S756 were detected by the anti-c-Myc epitope antibody.

Fig. 3

Surface expression of full-length membrane-associated S protein demonstrated by flow cytometry using the rabbit polyclonal antibody P540. Full-length S glycoprotein was used to transfect 293 cells, which were then infected with VTF7.3. Cells were collected and incubated with P540 polyclonal antibody plus anti-rabbit secondary antibody conjugated with FITC, washed, and subjected to flow cytometry analysis. The same plasmid used to express S but without the gene for S was used to transfect cells in a control experiment denoted as negative control (NC); the full-length S glycoprotein is denoted as S.

Fig. 4

Characterization of the S756, S_e, and S glycoproteins. Close to background level cleavage of S and S_e. Western blots of supernatants from transfected 293 cells expressing S756, S_e, and cell lysate of 293 cells expressing the S glycoproteins using the P540 antibody are shown. The left panel shows Western blots of samples kept for three days at 4 °C before analysis to monitor the effect of nonspecific protease activity on the cleavage pattern, whereas the right one—blots with samples used immediately after preparation.

Fig. 5

Cell fusion mediated by the S glycoprotein. (A) Syncytium formation between 293T cells transfetced with pSecTag2B-S and pCDNA3-ACE2, respectively (right panel). There was no syncytium formation between 293T cells transfected with pSecTag2B-S and pCDNA3-ACE2-Ecto (left panel). (B) Cell fusion measured by a reporter gene-based assay. S glycoprotein expressed in both pCDNA3 and pSecTag2B vectors can be used in a β-gal reporter gene-based cell–cell fusion assay. A pCDNA3-based plasmid without S insert was used as plasmid control, and fusion between S-expressing cells with ACE2-ecto expressing cells was used as negative control.

Fig. 6

Identification of the S glycoprotein receptor-binding domain (RBD). (A) Comparison of the binding of two different S soluble fragments (S537 and S756) to 293 and Vero E6 cells. (B) Binding of various S fragments to Vero E6 cells. The background OD405 measured for the negative control was subtracted from the OD405s of each S fragment. The resulting OD405 for each fragment was then presented as a percentage of the OD405 for S537. (C) Interaction of S fragments with purified soluble ACE2 as measured by ELISA. In all experiments, the negative control (NC) represents sample processed exactly the same way as the others except that the plasmid used for transfection did not encode any protein. Data shown here represent at least three independent experiments. OD405 for all samples is presented as percentages of that for S537.