Secretion of HCV Glycoproteins Figure 4. Density and nature of apolipoproteins and HCV glycoproteins secreted into Caco-2 basal medium. E1E2-Caco-2 cells were grown on porous filters under asymmetric medium conditions for 11 days. Upper and lower culture medium were then changed and replaced with foetal calf serum free medium. Cells were incubated for two additional days. Basal medium was collected and run on an iodixanol gradient. (A) Density fractions were collected and apoB concentrations per mL of basal medium were determined by ELISA and expressed in ng/ml. ApoB was only present in fractions with density #1.05 g/mL. (B) Density fractions were analysed by western blotting using specific antibodies against apoB, apoE and E2 glycoprotein. ApoB positive fractions contained the two apoB isoforms, apoB100 and apoB48. ApoE and E2 were only detected in the apoB positive fractions. Control proteins were deposited in lane +. doi:10.1371/journal.pone.0004233.g004 secretion are needed for efficient virion production and suggest that secreted virions are associated with apoB while other particles are degraded [28]. Interestingly however the latter data were obtained in Huh-7 that is shown here to be defective for proper assembly of VLDL with a defect either in the second precursor formation or in the two precursor fusion step since apoB and glycoproteins are present on separate low density molecular complexes. Virions produced in Huh-7 might benefit from the second precursor to assemble and to be associated with lipid in an MTP dependent manner, but could only marginally fused with the apoB precursor leading to particles with some density heteroge- neity that cannot be immunoprecipitated by anti-apoB antibody [29]. The pathways for biosynthesis of TRL triglyceride both in liver and intestine involve several key enzymes [44,45] which may not be fully functional in Huh-7. In our experimental system, the formation of the hybrid lipoproteins only required the presence of the envelope glycopro- teins. Conversely, the formation of infectious virions may benefit from the budding of the hybrid precursor. Recently, it was proposed that HCV core localizes on the monolayer membrane of cytosolic lipid droplets and then recruits replication complexes associated with the ER membrane forming a membranous local environment critical for producing infectious viruses [46,47]. It is thus tempting to speculate that, in this local environment, assembling capsids might profit of the budding hybrid second precursor driven by the envelope glycoproteins and MTP to translocate within the precursor lipid core to form an uncompleted viral particle. That particle could secondly fuse in competent cells with the first precursor in the presence of apoE forming the lipo- viro-particles. Translocation of the nucleocapsid limited by the core hydrophobic domains into the lipid core may be energetically favored by the phospholipid monolayer surrounding the hybrid precursor.It is likely that many defective particles circulate in the blood of chronically infected patients since particles containing only E1 and E2 can be produced in large amount as observed in this system. Previous LVP studies reported that purified LVP contained more apoB than HCV RNA molecules suggesting that some hybrid particles recognized by endogenous antibody are defective viral particles [10,16]. This study shows that viral envelope proteins interfere with the apolipoprotein B pathway to be secreted and provides further evidences on the nature and on the assembly of LVP. Hepatitis C virus also appears as a valuable tool for studying the successive steps of VLDL and chylomicron synthesis and secretion. PLoS ONE | www.plosone.org 6 January 2009 | Volume 4 | Issue 1 | e4233