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.

Severe acute respiratory syndrome coronavirus (SARS-CoV) infection inhibition using spike protein heptad repeat-derived peptides

“…Although cleavage does not occur in many coronaviruses, including SARS-CoV (our unpublished data and ref. 30), its inhibition in viruses carrying a cleaveable spike protein, by using a furin inhibitor, appeared not to affect virus infectivity, as we demonstrated for MHV (31). Moreover, in these latter coronaviruses, the cleavage does not expose a hydrophobic sequence in the N-terminal domain of the membrane-anchored subunit.…”

Severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice

“…We did not detect putative S1 and S2 cleavage products, as found for group 2 but not group 1 CoV S proteins (28). Xiao et al (13) expressed full-length S by transfection and detected low amounts of several smaller than full-length S fragments, which they suggested might include specific cleavage products, although no evidence to support this hypothesis was presented. Our characterization studies strongly suggested that MVA expressed a properly folded form of S, leading us to determine whether it would elicit neutralizing antibodies.…”

“…The SARS-CoV S is quite divergent from those of other CoVs, exhibiting only 20-27% overall amino acid identity (5). Recent studies indicated that the SARS-CoV S is expressed as a noncleaved glycoprotein with an apparent mass of 180-200 kDa that interacts with a functional receptor identified as angiotensin-converting enzyme 2 (12,13).…”

SARS Vaccine Development

“…These researchers also found that all of these MAbs specifically bound to RBD and blocked interaction between RBD and ACE2. These findings suggest that RBD contains the major neutralizing epitopes in the S protein and is an ideal SARS vaccine candidate because RBD contains the receptor-binding site, which is critical for virus attachment to the target cell for infection (15,(17)(18)(19). Antibodies specific for RBD are expected to block binding of virus to the target cell.…”

“…The S1 subunit is responsible for virus binding to the receptor, angiotensin-converting enzyme 2 (ACE2) (15,16). A fragment located in the middle region of the S1 subunit (aa 318-510) is the receptor-binding domain (RBD) for ACE2 (17)(18)(19). SARS-CoV may also bind to cells through the alternative receptors DC-SIGN or L-SIGN (20,21), but the binding sites for these alternative receptors have not been defined.…”