Secretion of HCV Glycoproteins Figure 1. Expression of the HCV glycoproteins E1 and E2 in Caco-2 cells. Caco-2 cells maintained under standard culture conditions after transduction with the E1E2-HIV-SIN lentiviral vector express E1 and E2 proteins. (A) Western blot analysis showing E1 and E2 revealed as single bands by anti-E1 antibody, (lane 2), and anti-E2 antibody (lane 4), at their expected molecular weight in transduced cells, but not in control cells (lanes 1 and 3, respectively). (B) Expression of envelope glycoprotein remains stable. Staining of transduced cells with anti-E2 antibody (in red) shows that almost all cells still expressed the envelope protein after at least 40 passages. doi:10.1371/journal.pone.0004233.g001 medium; one group of very light lipoproteins that are not associated with the glycoproteins and the group of hybrid lipoproteins which had a higher density presumably because of the additional viral proteins. E1 was mainly detected as monomer (apparent MW of ,30 kD) in the fractions with the highest density (fraction 16 and 17) and not in the other fractions where E2 was also detected. Some additional bands with molecular weight of 70 kD that may correspond to E1 trimers [34] were also detected in the other E2 positive fractions (data not shown). However, these high molecular weight bands co-migrated with a non specific cross reacting protein present in apoB positive fractions leaving open the question of the existence of E1 trimers associated with apoB particles of lower density. Alternatively, the weakest reactivity of anti-E1 antibody on western blot than the anti-E2 antibody reactivity might limit the detection of E1. Finally both glycopro- teins might not be secreted with the same efficiency. To confirm that both glycoproteins are associated with lipoproteins, complexes from unfractionnated basal medium were immuno-precipitated using an E2-specific conformational mono- clonal antibody. Only immuno-precipitated complexes from E1E2-Caco-2 cells but not from control cell basal medium (Figure 5C) contained E1 and the two isoforms of apoB in addition to E2. Taken together these data indicate that E1 and E2 glycoproteins can be integrated into at least some TRL that are secreted at the basolateral side of differentiated epithelial cells and that they are present on the surface of the lipoprotein complexes. HepG2 but not Huh-7 cell lines secretes HCV glycoproteins as hybrid TRL To extend the results found with intestinal cells to liver cells, and as HCV predominantly replicates in the liver, the viral glycoproteins were expressed by transduction with the lentiviral particles in the human hepatic cell line HepG2, often used as a model for TRL secretion. As observed with the Caco-2 model, E1 and E2 secreted in the supernatant were only found in low density fractions with apoB (Figure 6B). Similarly to the Caco-2 model, the E1E2-HepG2 cells secreted the glycoproteins as hybrid apoB positive particles, which could be immuno-precipitated with the E2-specific monoclonal antibody (Figure 6C). However, as expected, apoB was only found as the apoB100 isoform since human liver cells cannot edit the apoB mRNA. When testing E1E2-Huh-7.5 cells that support HCVcc replication, we also found that E1 and E2 were secreted into low density fractions of the supernatant (Figure 7B) indicating that these proteins were associated with lipids. But in strong contrast with the Caco-2 and HepG2 models and despite many attempts, if it was still possible to co-immunoprecipitate E1 with E2-specific antibody, it was not possible to co-immunoprecipitate apoB and, in the best cases as shown on Figure 8C, only traces of apoB, and not much more than from control cell supernatant, could be detected on these E1 and E2 positive low density complexes. We obtained the same lack of association of envelope glycoproteins and apoB with a parental Huh-7 cell line (data not shown). These data suggest that in Huh-7 cell lines, apoB positive lipoproteins and lipid associated envelope glycoproteins could likely not fuse in single hybrid particles. Inhibition of MTP decreases apoB and viral glycoproteins secretion MTP (microsomal triglyceride transfer protein) is an essential protein in the synthesis and assembly of TRL in both the liver and intestine. To evaluate the role of MTP in the synthesis and PLoS ONE | www.plosone.org 3 January 2009 | Volume 4 | Issue 1 | e4233