Figure 5

Susceptibility to ADE is dependent on an intact cytosolic domain. (A) Schematic representation of wild-type and mutated constructs that were utilized to produce stably transduced ST486 cell lines (see Materials and methods); from top to bottom: human FcγRIIA-His131 (hFcγRIIA-H), FcγRIIA-Arg131 (hFcγRIIA-R), FcγRIIB1 (hFcγRIIB1) and FcγRIIB2 (hFcγRIIB2). B) Surface expression in stably transduced ST486 cell lines was evaluated by flow cytometry using a mouse monoclonal anti-FcγRII antibodies (dark grey) or isotype control (light grey), and expressed as mean fluorescence intensity in Arbitrary Units (AU) as described under Materials and methods. Constructs are identified by Arabic numbers and grouped from top to bottom as indicated in A. C) Immune complex-binding ability of wild-type, truncated and chimeric FcγR receptors. SARS-CoVpp were incubated with purified mouse anti-Spike IgG (dark grey) or control IgG (light grey) to form immune complexes, which were then added to the ST486 transfectants (see Materials and methods). After washing and fixation, bound immune complexes were detected by flow cytometry with an FITC-conjugated goat anti-mouse F(ab’)2, and results shown as in B. Constructs are identified by Arabic numbers and grouped from top to bottom as indicated in A. D) Susceptibility of ST486 transfectants to ADE of infection by SARS-CoVpp. SARS-CoVpp were incubated with purified mouse anti-Spike IgG (dark grey) or control IgG (light grey) and then used to infect cells (see Materials and methods). Cells were washed and three days post infection incubation was stopped by adding luciferase substrate to measure enzymatic activity, which was expressed as Relative Luminescence Units (RLU). Constructs are identified by Arabic numbers and grouped from top to bottom as indicated in A. Results are shown as the means ± SEM of three (B and C) or 4–5 independent experiments (D). *P < 0.05; **P < 0.01 by the unpaired Student’s t test.