Secretion of HCV Glycoproteins Figure 2. Kinetic of ApoB and HCV glycoprotein secretion during Caco-2 cell differentiation. (A) Caco-2 cells were grown on porous filters under asymmetric medium conditions as depicted for two weeks after seeding. Media in upper and lower chambers were changed every two days. Under these differentiation conditions, basolateral lipoprotein secretion begins after one week of culture [32]. (B) ApoB secreted by control and E1E2 transduced cells into the basal medium was measured by ELISA. Values indicate apoB (ng) secreted by one thousand cells for two days per mL of culture medium. Vertical bars indicate standard deviations. The kinetic is representative of three separate experiments. (C) Expression and secretion of E2 protein by E1E2-Caco-2 cells during differentiation. Content of E2 protein in 40 ml of basal medium, upper pannel, and, in cell lysate, lower panel, (per 20 mg total protein), was analysed every two days by Western blotting with the E2-specific H52 antibody. As indicated in Figure 1, E2 was detectable in seeded cells and remained present during the two weeks of culture. Its expression increased during the early phase of the differentiation until day 9 when a plateau was reached. E2 secretion into the basal medium began at day 7, concomitantly with apoB secretion. Secreted E2 appeared to have a higher molecular weight than the control intracellular E2 protein likely because of a higher glycosylated state. doi:10.1371/journal.pone.0004233.g002 assembly of the hybrid glycoproteins-TRL particles, Caco-2 cells were grown under conditions of differentiation for 11 days before the MTP inhibitor Cp 346086 was added [35]. MTP activity and the secretion of apoB and E2 were measured one and two days later. As shown in Figure 8A, MTP activity was inhibited in a dose dependant manner by 35 and 50% at one or two days after addition of the inhibitor, respectively. ApoB secretion was reduced in parallel in similar proportions (Figure 8B) while the secretion of MIP-1 beta in the basal medium was not affected suggesting that the MTP inhibitor specifically reduced the apoB lipoprotein secretion but not the secretion of unrelated proteins (data not shown). Most importantly if the intracellular expression of the two glycoproteins was not modified by the MTP inhibition, the quantity of E1 and E2 secreted into the basal medium after 24 h or 48 h incubation with 1 and 10 nM inhibitor, was greatly reduced and correlated with the concentration of secreted apoB (Figure 8C). The effect of MTP inhibition on the two glycoproteins secretion by E1E2-HepG2 cells was very similar to that observed with E1E2-Caco-2 cells (Figure 9A) with around 40% reduction of E2 secretion and 30% reduction of E1 secretion after 48 h in presence of 1 nM inhibitor. The same effect was also observed with E1E2- Huh-7.5 cells (Figure 9B) but to a lower extent with only 16% and 8% reduction of respectively E2 and E1 secretion in the same inhibitor conditions. The effect of MTP inhibition on the two glycoproteins secretion seemed also to depend on the lipoprotein pathway since the secretion of albumin by these two liver derived cell lines was not modified (data not shown). Discussion In both Caco-2 and HepG2 cell culture systems, the HCV glycoproteins are secreted with the apoB positive lipoproteins. This secretion is strictly dependent on an efficient apoB positive lipoprotein secretion pathway because, first, apoB negative epithelial Hela cells do not secrete HCV envelope proteins, second, Caco-2 cells only secrete the viral glycoproteins after having acquired the apoB synthesis and secretion capacity and, finally, inhibition of a normal lipidation of apoB with an MTP inhibitor reduced the amount of the secreted viral glycoproteins. The E1 and E2 envelope proteins thus contain the signals mandatory to interfere with a functional apoB pathway in liver and intestinal cells. It is worth noting that both systems seem to be interdependent. Indeed, the amount of cellular viral glycoproteins increases accordingly to apoB and conversely the presence of E1 and E2 likely delays the secretion of apoB. A metabolic syndrome frequently associated with hepatitis C includes insulin-resistance, liver steatosis and hypo-beta- lipoproteinemia. Decreased serum apoB concentrations have been attributed to inhibition of MTP activity by HCV core protein [36,37] and by the facilitated degradation of apoB by the non structural protein NS5A [38] which both could reduce the production of TRL. Here we found that expression of E1 and E2 delays apoB secretion by Caco-2 cells providing another mechanism for reduced apoB serum concentration. Transduction of the gene coding for E1 and E2 without any additional viral genes is sufficient to induce the assembly and the secretion of low density hybrid particles resembling TRL and PLoS ONE | www.plosone.org 4 January 2009 | Volume 4 | Issue 1 | e4233