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. 2009 Nov 17;4(11):e7870. doi: 10.1371/journal.pone.0007870

Figure 4. Differential expression of wild-type and mutant SARS S glycoprotein and SARS-CoV pseudotype production from 293T cells.

Figure 4

A, Different putative spike glycoprotein sequences from SARS-CoV isolates were obtained from NCBI. Name of SARS-CoV isolates and GenBank accession numbers are listed. Potential airway protease cleavage residues are highlighted in green. B, Three mutant constructs were made from the wild-type SARS-CoV Spike glycoprotein cDNA cloned in the vector pcDNA3.1 as described in Materials and Methods. C, Cell lysates were collected from 293T cells 48 h post transfection. Pseudotypes were collected from culture medium and concentrated as described previously [33]. Samples (cell lysates or pseduotypes) were denatured, reduced and analyzed by 4–12% Bis-Tris SDS-PAGE gel and Western Blot using M2 monoclonal antibody against the FLAG peptide. Sizes of molecular weight markers are indicated on the right. Wild-type SARS spike (lane 1), R667A (lane 2), R797A (lane 3), R797AR667A (lane 4) and pseudotype without envelope (lane 5). D, Analysis of various types of SARS-CoVpp for viral entry. SARS-CoVpp (wild-type or mutant SARS S glycoprotein) were incubated with 16HBE cells and transduction was measured by determination of the luciferase activity (LCPS). Experiments were performed in duplicates and data are presented as means±SE from two independent experiments.