title: 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.
authors.affiliation: Laboratory of Experimental and Computational Biology, CCR, NCI-Frederick, NIH, Frederick, MD 21702-1201, USA.
authors.forename: Xiaodong
authors.initials: X
authors.lastname: Xiao
authors.forename: Samitabh
authors.initials: S
authors.lastname: Chakraborti
authors.forename: Anthony S
authors.initials: AS
authors.lastname: Dimitrov
authors.forename: Kosi
authors.initials: K
authors.lastname: Gramatikoff
authors.forename: Dimiter S
authors.initials: DS
authors.lastname: Dimitrov
mesh_terms: D000818:Animals; D001665:Binding Sites; D002448:Cell Adhesion / Q000502:physiology; D002522:Chlorocebus aethiops; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D045473:Severe acute respiratory syndrome-related coronavirus / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D013045:Species Specificity; D064370:Spike Glycoprotein, Coronavirus; D014709:Vero Cells; D014759:Viral Envelope Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D014760:Viral Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins; D014760:Viral Fusion Proteins