Expression cloning of functional receptor used by SARS coronavirus

Abstract

We have expressed a series of truncated spike (S) glycoproteins of SARS-CoV and found that the N-terminus 14-502 residuals were sufficient to bind to SARS-CoV susceptible Vero E6 cells. With this soluble S protein fragment as an affinity ligand, we screened HeLa cells transduced with retroviral cDNA library from Vero E6 cells and obtained a HeLa cell clone which could bind with the S protein. This cell clone was susceptible to HIV/SARS pseudovirus infection and the presence of a functional receptor for S protein in this cell clone was confirmed by the cell-cell fusion assay. Further studies showed the susceptibility of this cell was due to the expression of endogenous angiotensin-converting enzyme 2 (ACE2) which was activated by inserted LTR from retroviral vector used for expression cloning. When human ACE2 cDNA was transduced into NIH3T3 cells, the ACE2 expressing NIH3T3 cells could be infected with HIV/SARS pseudovirus. These data clearly demonstrated that ACE2 was the functional receptor for SARS-CoV.

CD209L (L-SIGN) is a receptor for severe acute respiratory syndrome coronavirus

“…2B) and immunofluorescence (data not shown) showed that ACE2 was expressed only on Vero E6 cells and not on clone 2.27 cells or CHO cells. These observations show that binding of SARS-CoV S 590 glycoprotein to clone 2.27 cells was not due to increased expression of human ACE2, as in the experiments of Wang et al (10).…”

“…Angiotensin-converting enzyme 2 (ACE2) was found to be an efficient receptor for the S glycoprotein of SARS-CoV (9,10). Because the Vero line of rhesus monkey kidney cells is highly susceptible to infection with SARS-CoV, Li et al (9) used a codon-optimized soluble SARS-CoV spike glycoprotein (amino acids 12-672) fused to the Fc domain of human IgG1 to immunoprecipitate a putative receptor glycoprotein from Vero cell membranes and identified the simian ACE2 protein by mass spectrometry.…”

Comparison of vesicular stomatitis virus pseudotyped with the S proteins from a porcine and a human coronavirus

“…2(a) for the deletion mutant Sdel18, the S protein of SARS-CoV mediated infection of VeroE6 cells, whereas the two porcine cell lines, PK15 and LLC-PK1, as well as BHK21 cells were resistant to infection. This finding is consistent with previous reports that human and simian ACE2 can serve as receptors for SARS-CoV (Li et al, 2003;Wang et al, 2004b). The corresponding deletion mutant of the TGEV S protein was able to mediate infection of the two porcine cell lines, but not infection of VeroE6 or BHK21 cells.…”

“…In addition, it is the main target for neutralizing antibodies elicited in the infected host. SARS-CoV, the aetiological agent of severe acute respiratory syndrome, uses angiotensin-converting enzyme 2 (ACE2) as a receptor to infect cells (Li et al, 2003;Wang et al, 2004b). Porcine aminopeptidase N is known to serve as a receptor for transmissible gastroenteritis virus (TGEV), a porcine coronavirus (Delmas et al, 1992).…”

“…We and others have analysed the role of the S protein of SARSCoV in the initial stage of infection using VSV or lentiviral pseudotype systems (Wang et al, 2004b;Giroglou et al, 2004;Ren et al, 2006;Glende et al, 2008;Watanabe et al, 2008). …”