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.

Long-lived memory B cells (BMem) provide an archive of historic Ab responses. By contrast, circulating Abs typically decline once the immunogen is cleared. Consequently, circulating Abs can underestimate the nature of cognate humoral immunity. On the other hand, the BMem pool should provide a comprehensive picture of Ab specificities that arise over the entire course of infection. To test this hypothesis, we compared circulating Ab and BMem from natural virus suppressors who control HIV-1 without therapy and maintain a relatively intact immune system. We found high frequencies of BMem specific for the conserved neutralizing CD4 induced or CD4 binding site epitopes of gp120, whereas low Ab titers to these determinants were detected in contemporaneous plasma. These data suggest that plasma Ab repertoires can underestimate the breadth of humoral immunity, and analyses of BMem should be included in studies correlating Ab specificity with protective immunity to HIV-1.CD4i antibody ͉ CD4bs antibody ͉ human monoclonal antibody ͉ natural viral suppressors ͉ elite controller