Development of a safe neutralization assay for SARS-CoV and characterization of S-glycoprotein.
The etiological agent of severe acute respiratory syndrome (SARS) has been identified as a novel coronavirus SARS-CoV. Similar to other coronaviruses, spike (S)-glycoprotein of the virus interacts with a cellular receptor and mediates membrane fusion to allow viral entry into susceptible target cells. Accordingly, S-protein plays an important role in virus infection cycle and is the primary target of neutralizing antibodies. To begin to understand its biochemical and immunological properties, we expressed both full-length and ectodomain of the protein in various primate cells. Our results show that the protein has an electrophoretic mobility of about 160-170 kDa. The protein is glycosylated with high mannose and/or hybrid oligosaccharides, which account for approximately 30 kDa of the apparent protein mass. The detection of S-protein by immunoassays was difficult using human convalescent sera, suggesting that the protein may not elicit strong humoral immune response in virus-infected patients. We were able to pseudotype murine leukemia virus particles with S-protein and produce SARS pseudoviruses. Pseudoviruses infected Vero E6 cells in a pH-independent manner and the infection could be specifically inhibited by convalescent sera. Consistent with low levels of antibodies against S-protein, neutralizing activity was weak with 50% neutralization titers ranging between 1:15 to 1:25. To facilitate quantifying pseudovirus-infected cells, which are stained blue with X-Gal, we devised an automated procedure using an ELISPOT analyzer. The high-throughput capacity of this procedure and the safety of using SARS pseudoviruses should make possible large-scale analyses of neutralizing antibody responses against SARS-CoV.
Han
Dong P
DP
Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA.
Kim
Hyung G
HG
Kim
Young B
YB
Poon
Leo L M
LL
Cho
Michael W
MW
D000818:Animals; D000914:Antibodies, Viral / Q000032:analysis; D002522:Chlorocebus aethiops; D003289:Convalescence; D005822:Genetic Vectors; D006031:Glycosylation; D006367:HeLa Cells; D006801:Humans; D009052:Leukemia Virus, Murine / Q000235:genetics; D008358:Mannose / Q000737:chemistry; D008562:Membrane Glycoproteins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics*; D008970:Molecular Weight; D009500:Neutralization Tests / Q000379:methods*; D009844:Oligosaccharides / Q000737:chemistry; D016865:Reassortant Viruses / Q000302:isolation & purification / Q000472:pathogenicity; D045473:Severe acute respiratory syndrome-related coronavirus / Q000235:genetics / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D045169:Severe Acute Respiratory Syndrome / Q000097:blood / Q000175:diagnosis*; D013045:Species Specificity; D064370:Spike Glycoprotein, Coronavirus; D014709:Vero Cells; D014759:Viral Envelope Proteins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics*
D000914:Antibodies, Viral; D008562:Membrane Glycoproteins; D009844:Oligosaccharides; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins; C578557:spike glycoprotein, SARS-CoV; C578553:spike protein, mouse hepatitis virus; D008358:Mannose