HCVcc particles and HCV E2 glycoprotein block production of IFN-α and IFN-λ induced by exposure of pDCs to HCV-infected Huh7.5 cells. (A) Time course of production of HCVcc particles in cell-free supernatant of HCV-infected Huh7.5 cells. Huh7.5 cell monolayer at ∼ 70% confluency and containing ∼ 75% of HCV-infected cells was rinsed 3 times with a fresh medium and cultured at 37°C. Culture supernatant aliquots were collected at the indicated time points, filtered through 0.45-μm membrane filter, and stored at −80°C. HCV RNA was quantified by RT-PCR. In parallel, infectious titer of HCV was determined in Huh7.5 cells. (B-H) Inhibition of type I IFN production in pDCs by increasing quantity of HCVcc particles (MOI, left panels B,D,G) or sE2 glycoprotein (μg/mL, right panels C,E-F,H). pDCs exposed to HCVcc particles or sE2 for 1 hour were stimulated with TLR7/9 agonists. (B-C,G-H) CpG-A, influenza virus A/H3N2/Johannesburg (Flu, MOI_{Flu RNA} = 100), HCV-infected Huh7.5 cells (Huh-HCV), or control Huh7.5 cells (Huh). (D-E) A total of 0.05, 0.1, or 5μM R848. Production of IFN-α and IFN-λ in cell-free supernatant was determined by ELISA 20 hours after stimulation. (F) Inhibition of IFN-α production is abrogated by treatment of E2 glycoprotein with 1 μg of anti-E2 mAb (AP33) at 37°C for 1 hour. MOI is expressed as a quantity of HCV RNA copies per pDC. Ctl indicates supernatant of CHO cells transfected with empty vector. Data are mean ± SEM of 3-6 independent experiments with pDCs from different donors. Some error bars are too small to be visible compared with the size of the symbol.