Viral envelope glycoproteins and HCV-LP binding to DCs. (A) Inhibition of HCV-LP binding to imDCs by anti-HCV antibodies. HCV-LPs (1 μg HCV-LP E2/mL) were preincubated with anti-E2 (AP33, 2F10; 50 μg/mL), anti-E1 antibody (11B7; 50μg/mL), or an anti-HCV antiserum (dilution 1:50) containing high-titer antibodies against the HCV envelope glycoprotein E2 for 1 hour at 37°C. Incubation of HCV-LPs with control IgG (50 μg/mL) or anti-HCV–negative serum (dilution 1:50) served as negative controls. HCV-LP binding was analyzed by flow cytometry using chimpanzee anti-E2 mAb (49F3) and PE–conjugated anti–human IgG. Denaturation of HCV-LPs was performed by heat treatment as described.^27,35  Mean ± SD of 3 experiments is shown (HCV-LP binding in the absence of antibody = 100%). (B) Analysis of envelope glycoprotein glycosylation. HCV-LPs were digested with EndoH or PNGaseF for 2 hours at 37°C. Glycosidase (+) and mock-digested HCV-LP envelope proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, followed by immunoblotting with anti-E1 and anti-E2 mAbs as described previously.^27,35  Sizes of molecular weight (MW) markers in kilodaltons are indicated on the left (E1+ and E2+ indicate deglycosylated forms of E1 and E2). (C) Impact of envelope glycoprotein glycosylation for HCV-LP binding. ImDCs were incubated with increasing concentrations of glycosidase-digested (+) and mock-digested HCV-LPs, and particle binding was analyzed by flow cytometry as described in Figure 1.