Secretion of HCV Glycoproteins Figure 7. Characterization of particles secreted by Huh-7.5 cells. (A) Analysis of HCV glycoprotein expression by immunofluorescence with E2-specific antibody. Huh-7.5 were grown under standard conditions and stained as described in materials and methods. Left panel, control cells, right panel, E1E2 transduced Huh-7. E2 positive cells are stained in red. (B) Density of apoB and E1E2 positive fractions in the E1E2-Huh-7.5 supernatant. Three day culture supernatant was run on a iodixanol gradient and fractions were analysed by apoB ELISA, upper panel, and western blotting with E1 and E2-specific antibodies, lower panel. Envelope control proteins were deposited in the last right lane (+). E1 and E2 proteins were only present in apoB positive fractions. (C) Immuno-precipitation of E2 positive particles was performed as in figure 5 with Huh-7.5 supernatant collected three days after seeding. Basal culture medium of control and E1E2-Huh-7.5 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 H52 anti-E2 (upper panel), A4 anti-E1 (middle panel) and anti-apoB (lower panel) antibodies. Molecular weight are indicated on the left, control proteins were deposited in the + lane. doi:10.1371/journal.pone.0004233.g007 Immunofluorescence Cells were fixed with formaldehyde 4% in PBS (formaldehyde 37%, Merck) for 30 minutes at 4uC and permeabilized by triton X100 0.5% in PBS for 30 minutes at 4uC. Intracellular expression of HCV E2 protein was assayed by indirect immunofluorescence with anti-E2 H52 antibody (10 mg/ml) and Alexa Fluor 546 F(ab’)2 fragment of goat anti-mouse immunoglobulin G (1 mg/ml, Invitrogen). Cells were countercolored with Hoechst 33 342 (0,2 mg/ml) and observed with a Leica DMIRB microscope. ApoB ELISA ApoB concentrations in the medium of the lower compartment were determined by ELISA. Ninety-six-well flat-bottomed ELISA plates (Maxisorb; Nunc) were coated overnight at 4uC with 100 ml monoclonal anti-human apoB antibody (5 mg/ml; clone 1609) in PBS and then saturated with 2% BSA for 1 h. Samples were distributed at 100 ml per well. After 2 h incubation at 37uC and washing with PBS/ 0,05% Tween 20, 100 ml of peroxidase- conjugated goat anti-human apoB antibody (1.6 mg/ml) 100 ml per well in PBS/ 0.2% BSA were added for 90 min at 37uC. The plates were washed and o-phenylenediamine substrate was added (100 ml per well). The reaction was revealed for 30 min and absorbance was read at 450 nm. Standard curves were established with apoB dilutions ranging from 10 to 200 ng/ml apoB (ApoB kit, SFRI Diagnostics). To normalized quantity of apoB produced by 1000 cells, the number of viable cells in 0.3 cm2 transwells was determined by CellTiter 96H AQueous Non-Radioactive Cell Proliferation Assay (Promega). The MTS conversion into formazan was realized into transwells according to manufacturer recommandations. The absorbance at 490 nm of formazan product in transwells supernatants was measured after transfer to a 96-well plate. Iodixanol density gradients Iodixanol gradients were prepared as described by Nielsen et al. [18]. Isopycnic linear density gradients were prepared from 6% (wt/vol) (1.7 ml of 60% [wt/vol] iodixanol, 0.34 ml of 0.5 M Tris- HCl, pH 8.0, 0.34 ml of 0.1 M EDTA, pH 8.0, and 14.6 ml PLoS ONE | www.plosone.org 9 January 2009 | Volume 4 | Issue 1 | e4233