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 β-sheets, β5 and β6. 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 β6 strand of S-RBM. In the present study, we demonstrate that patient sera predominantly recognized 2 linear epitopes outside the β6 fragment, while the mouse antisera, induced by immunization of BALB/c mice with recombinant S450–650, mainly recognized the β6 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 β6 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 β6 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.