FIG. 2.

HCVcc acquisition by mDCs and pDCs. (A) Role of HCV entry factors in HCVcc acquisition by pDCs and mDCs. DC subsets were preincubated with mouse anti-CD81 (5 μg/ml), rat anti-SR-BI (1:50 dilution), rat anti-claudin 1 (1:50 dilution), anti-DC-SIGN (5 μg/ml), irrelevant mouse IgG (5 μg/ml), or rat IgG (1:50 dilution) for 45 min at 37°C. DC subsets were then incubated with HCVcc (multiplicity of infection [MOI], ∼0.01) for 2 h at 37°C. After extensive washing, the presence of HCVcc RNA was measured by real-time quantitative RT-PCR. As negative controls, DC subsets were incubated with phosphate-buffered saline (PBS) or control iodixanol preparations obtained from culture supernatants of noninfected Huh7.5.1 cells (NC [negative control]). The data represent the means ± standard deviations for triplicate samples from one experiment. The results shown are representative of three independent experiments, using three different donors. (B) Visualization of HCVcc uptake into pDCs and mDCs. For HCVcc internalization, DC subsets were incubated with iodixanol gradient-purified HCVcc (MOI, ∼10) for 3 h at 37°C. Following fixation and permeabilization, cells were stained for HCVcc E2 protein by use of mouse anti-E2 monoclonal antibody (AP33; green), and the nucleus was stained with DAPI (4′,6-diamidino-2-phenylindole; blue). Arrows indicate detectable HCVcc E2 protein. To confirm the specificity of HCV E2 protein labeling, DC subsets were incubated with control iodixanol preparations obtained from culture supernatants of noninfected Huh7.5.1 cells and subsequently labeled with anti-E2 (AP33) antibody (NC [negative control]). (C) For HCVcc double labeling, DC subsets were incubated with mouse anti-E2 (AP33; green) and rabbit anti-core antibody (red). The yellow areas in the overlays indicate colocalization of these two proteins in mDCs and pDCs. (D) HCVcc uptake by pDCs and mDCs was quantified by counting the number of cells that stained positive for E2 protein relative to the total number of cells (n = 300). The results for pDCs and mDCs isolated from five different donors are shown. Statistical analysis was performed using the Mann-Whitney U test. Tests of significance were two-sided, and a P value of ≤0.05 was considered significant. (E) Inhibition of HCVcc uptake in the presence of human anti-HCV serum (1:50 dilution) and antibodies targeting the HCV entry factors (rabbit anti-CD81, 6 μg/ml; rat anti-SR-BI and rat anti-CLDN1, 1:50 dilution). HCVcc uptake by DC subsets was analyzed as described for panel D. The results are shown as the percent inhibition of HCVcc uptake compared to HCVcc uptake in the presence of the respective isotype controls (irrelevant IgG or human control serum) for pDCs and mDCs from four different donors.