Secretion of HCV Glycoproteins Figure 6. Characterization of particles secreted by HepG2. (A) Analysis of HCV glycoprotein expression by immunofluorescence with E2- specific antibody H52. HepG2 cells were grown under standard conditions and stained as described in materials and methods. Left panel, control cells, right panel, E1E2 transduced HepG2 cells. E2 positive cells are stained in red. (B) Density of apoB and E1E2 positive fractions in the E1E2-HepG2 supernatant. Three day culture supernatant was run on an iodixanol gradient and fractions were analysed by apoB ELISA, upper panel, and western blotting with E2-specific antibody, lower panel. Envelope control proteins were deposited in the last right lane (+). E2 proteins were only present in apoB positive fractions. (C) Immuno-precipitation of E2 positive particles was performed as in figure 5 with HepG2 supernatant collected three days after seeding. Basal culture medium of control and E1E2-HepG2 were collected 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 E2- specific H52 antibody (upper panel), E1-specific A4 antibody (middle panel) and apoB-specific antibody (lower panel). Molecular weight are indicated on the left, control proteins were deposited in the + lane. doi:10.1371/journal.pone.0004233.g006 pCMV-VSV-G envelope plasmid, 10.2 mg of pCMVdeltaR8.91 packaging plasmid and 13.2 mg of E1E2-HIV-SIN derived vector plasmid were co-transfected by calcium phosphate precipitation. Medium was replaced with 0.1 ml/cm2 of fresh culture medium without serum 14–16 h post-transfection.Viral supernatants were harvested 24 h later, cleared by low-speed centrifugation, and filtered through a 0.45 mm low binding protein filter (Millex filter unit, Millipore) prior to concentration. High-titer viral stocks were prepared by concentrating viral supernatants about 100 fold through ultracentrifugation for 2 h in a Beckman LE 70 ultracentrifuge at 26 000 rpm at 4uC in an SW28 rotor. Viral stocks were aliquoted in PBS 1% glycerol and stored at 280uC. 7 ng of p24 antigen (HIV P24 II Vidas, Vidas apparatus, BioMe´rieux) with 8 mg/ml polybrene in culture medium was used to infect overnight target cells, plated at 100 000 in a 2 cm2 well the day before. Cell culture Caco-2 cells (ATCC HTB-37) were plated on inserts at a density of 250 000 cells /4.2 cm2 transwell and grown until confluency (,6 days post seeding) in DMEM (Dulbecco’s modified Eagle’s medium) containing 25 mM glucose and 2 mM glutamine, supplemented with penicillin (100 i.u./ml) and streptomycin (100 mg/ml), 1% non-essential amino acids and 20%(v/v) heat- inactivated FCS. Cells were then cultured for an additional week under asymmetrical conditions with media containing FCS in the lower compartment and media without FCS in the upper compartment. Media were changed every 48 hours. For incuba- tions with MTP inhibitor CP346086, fresh medium without FCS was added to the lower and upper compartment at the final concentrations of inhibitor for 24 or 48 hours. HepG2 (ATCC HB-8065), Huh-7.5 (from Charles Rice, Rockefeller University, New York, New York), Huh-7 cells (from John Mac Lauchlan, MRC, Glasgow, UK), and Hela (ATCC CCL-2) were plated in 9.5 cm2 wells at a density of 400 000 cells/well and grown for 24 h or 48 hours (until 80% confluence) in DMEM containing 25 mM glucose and 2 mM glutamine, supplemented with penicillin (100 i.u./ml), streptomycin (100 mg/ml), 1% non- essential amino acids and 10% (v/v) heat-inactivated FCS before the following experiments. For experiments with the MTP inhibitor CP346086, fresh medium without FCS was added to the 9.5 cm2 wells containing 0, 1 or 10 nM inhibitor for 48 hours. PLoS ONE | www.plosone.org 8 January 2009 | Volume 4 | Issue 1 | e4233