title: A study on antigenicity and receptor-binding ability of fragment 450-650 of the spike protein of SARS coronavirus.
abstract: The spike (S) protein of SARS coronavirus (SARS-CoV) is responsible for viral binding with ACE2 molecules. Its receptor-binding motif (S-RBM) is located between residues 424 and 494, which folds into 2 anti-parallel beta-sheets, beta5 and beta6. We have previously demonstrated that fragment 450-650 of the S protein (S450-650) is predominantly recognized by convalescent sera of SARS patients. The N-terminal 60 residues (450-510) of the S450-650 fragment covers the entire beta6 strand of S-RBM. In the present study, we demonstrate that patient sera predominantly recognized 2 linear epitopes outside the beta6 fragment, while the mouse antisera, induced by immunization of BALB/c mice with recombinant S450-650, mainly recognized the beta6 strand-containing region. Unlike patient sera, however, the mouse antisera were unable to inhibit the infectivity of S protein-expressing (SARS-CoV-S) pseudovirus. Fusion protein between green fluorescence protein (GFP) and S450-650 (S450-650-GFP) was able to stain Vero E6 cells and deletion of the beta6 fragment rendered the fusion product (S511-650-GFP) unable to do so. Similarly, recombinant S450-650, but not S511-650, was able to block the infection of Vero E6 cells by the SARS-CoV-S pseudovirus. Co-precipitation experiments confirmed that S450-650 was able to specifically bind with ACE2 molecules in lysate of Vero E6 cells. However, the ability of S450-510, either alone or in fusion with GFP, to bind with ACE2 was significantly poorer compared with S450-650. Our data suggest a possibility that, although the beta6 strand alone is able to bind with ACE2 with relatively high affinity, residues outside the S-RBM could also assist the receptor binding of SARS-CoV-S protein.
authors.affiliation: Department of Immunology, Peking University Health Science Center, Peking University, 38 Xueyuan Road, Beijing 100083, China.
authors.forename: Jincun
authors.initials: J
authors.lastname: Zhao
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.forename: Zhihong
authors.initials: Z
authors.lastname: Yuan
authors.forename: Rujing
authors.initials: R
authors.lastname: Jia
authors.forename: Zhendong
authors.initials: Z
authors.lastname: Zhao
authors.forename: Xiaojun
authors.initials: X
authors.lastname: Xu
authors.forename: Ping
authors.initials: P
authors.lastname: Lv
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhang
authors.forename: Chengyu
authors.initials: C
authors.lastname: Jiang
authors.forename: Xiao-Ming
authors.initials: XM
authors.lastname: Gao
mesh_terms: D000085962:Angiotensin-Converting Enzyme 2; D000818:Animals; D000914:Antibodies, Viral; D000915:Antibody Affinity; D002460:Cell Line; D002522:Chlorocebus aethiops; D018985:Epitopes, B-Lymphocyte; D005260:Female; D015967:Gene Expression Regulation, Viral; D006801:Humans; D008562:Membrane Glycoproteins / Q000276:immunology* / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008958:Models, Molecular; D007703:Peptidyl-Dipeptidase A / Q000378:metabolism; D011487:Protein Conformation; D011991:Receptors, Virus / Q000378:metabolism*; D045473:Severe acute respiratory syndrome-related coronavirus / Q000276:immunology / Q000378:metabolism*; D064370:Spike Glycoprotein, Coronavirus; D014709:Vero Cells; D014759:Viral Envelope Proteins / Q000276:immunology* / Q000378:metabolism*
chemical_list: D000914:Antibodies, Viral; D018985:Epitopes, B-Lymphocyte; D008562:Membrane Glycoproteins; D011991:Receptors, Virus; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins; C578557:spike glycoprotein, SARS-CoV; C578553:spike protein, mouse hepatitis virus; D007703:Peptidyl-Dipeptidase A; C000705307:ACE2 protein, human; C000705328:Ace2 protein, mouse; D000085962:Angiotensin-Converting Enzyme 2