FIG. 5.

Decreased C5aR1 surface expression results in reduced CCR5-mediated HIV infection of THP-1 cells. (a) THP-1 cells were treated with C5aR1 siRNA or control siRNA for 24 h, and then differentiated with PMA for an additional 24 h. Efficiency of C5aR1 silencing was measured by flow cytometry. (b) Transfected cells were loaded with the CCF2-AM dye and infected with YK-JRCSF-BlaM-Vpr. Fusion was measured 6 h later by flow cytometry. Dot plots show the levels of cleaved-CCF2-AM in control siRNA-treated cells (left panel) versus C5aR1 siRNA-transfected cells (right panel). Percentages of positive/negative cells are indicated; the positivity gate was set up based on the fluorescence level in uninfected cells loaded with CCF2-AM. The MFI of cleaved CCF2-AM was measured in both experimental conditions, and was 338 and 247 for control siRNA and C5aR1 siRNA-transfected cells, respectively. The data shown are representative of two independent experiments. (c) Cells gated based on cleaved CCF2-AM fluorescence were assessed for CCR5 and C5aR1 expression. The dot plots represent the frequency of CCR5⁺C5aR1⁺ HIV-infected cells in control siRNA-transfected (left panel) versus C5aR1 siRNA-transfected cells (right panel). The data shown are representative of two independent experiments.