Secretion of HCV Glycoproteins Figure 5. Characterization of the apoB and envelope containing particles secreted into Caco-2 basal medium. Cells were cultured as in Figure 4. Basal medium was collected at day 13 after two day incubation in serum free medium. Density of apoB and E1E2 glycoprotein positive fractions in the E1E2-Caco-2 basal medium. Basal medium was run on a iodixanol gradient and fractions were analysed by apoB Elisa (A) and western blotting (B, upper panel) stained with envelope specific H52 and A4 antibodies. E1 and E2 proteins were present in apoB positive fractions with the higher density leaving the upper apoB fractions free of envelope glycoproteins. E1 was only detected in the fractions with the highest density. Intracellular E1 and E2 control proteins were deposited in the last right lane (+). To further assess the specificity of the antibody, fractions obtained from E1E2-Caco-2 and control Caco-2 grown in the same conditions were normalized for apoB concentrations and ran in parallel in one western blot (B, lower panel). Envelope glycoproteins were only detected in fractions obtained from E1E2-Caco-2. (C) Immuno-precipitation of E2 positive particles. Basal culture medium of control Caco-2 and E1E2-Caco-2 cells were collected at day 13 of differentiation after two days of culture in serum free medium. E2 positive particles were captured by E2-specific H48 monoclonal antibody and immuno-precipitated particles were analysed by western blotting and stained with anti-E2 H52 (upper panel), anti-E1 A4 (middle panel) and anti-apoB (lower panel) antibodies as described in materials and methods section. Molecular weights are indicated on the left, control proteins were deposited in the + lane. Identical volume of precipitated material either from E1E2-Caco-2 or control Caco-2 basal medium were deposited in lanes ‘‘E1E2’’ and ‘‘control’’ respectively for every western blot. doi:10.1371/journal.pone.0004233.g005 Materials and Methods Materials Unless indicated, all chemicals were from Sigma. Monoclonal antibodies anti-E1 (clone A4) or E2 (clones H52 and H48) mAbs were obtained from Dr J. Dubuisson (Institut de Biologie de Lille- Institut Pasteur de Lille, France). Anti-apoB (clone 1D1) mAb was from the Heart Institute (University of Ottawa, Ontario, Canada), anti-apoB (clone 1609) monoclonal antibodies and peroxidase- conjugated goat anti-apoB antibodies were from Biodesign (Saco, ME, USA). Anti-apoE (clone 3D12) was obtained from Millipore (Molsheim, France). The MTP inhibitor CP346086 [35] was kindly provided by G. Luo (Department of Microbiology,Univer- sity of Kentucky College of Medicine, Lexington, Kentucky). Cell culture reagents were obtained from Invitrogen (Cergy-Pontoise, France) and FCS (foetal calf serum) from PAN-Biotech GmbH (Aidenbach, Germany). Microporous polyethylene terephthalate membrane inserts were from Becton Dickinson (Le Pont de Claix, France) (4.2 or 0.3 cm2, 1 mm pore size). Lentiviral particles production and cell transduction A lentiviral vector E1E2-HIV-SIN for the transduction of the HCV glycoproteins E1 and E2 was constructed. The gene coding the E1 and E2 from HCV genotype 1a H77 strain (AJ 318514, nucleotides 733 to 2579) were introduced at the Bsrg I and BamHI restriction sites of the HIV-1 self-inactivating vector backbone pRRL-SIN18-cPPT-hPGK-EGFP-WPRE (pHIV-hPGK-GFPW+, [48]) under the control of the internal human phosphoglycerate kinase (hPGK) promoter. Retrovirus carrying the E1E2-HIV-SIN sequence was produced by co-transfection of the Gag pol packaging construct pCMVdeltaR8.91 [49] and glycoprotein expression construct pCMV-VSV-G (AJ 318514) in 293T cells (ATCC CRL-1573). 2.46106 293T cells per 55 cm2 culture dish (Corning) were seeded 24 h prior to transfection in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 2 mM glutamine, 100 i.u./ ml penicillin, 100 mg/ml streptomycin and 10% heat-inactivated FCS in a 95% humidified incubator containing 5% CO2 in air at 37uC. For the production of E1E2-HIV particles, 13.2 mg of PLoS ONE | www.plosone.org 7 January 2009 | Volume 4 | Issue 1 | e4233