Secretion of HCV Glycoproteins Figure 3. Epithelial Hela cells do not secrete HCV glycoproteins. (A) Analysis of HCV glycoprotein expression by E1E2-Hela by immunofluorescence with E2-specific antibody H52. Cells were grown under standard conditions and stained as described in materials and methods. Left panel, control cells, right panel, E1E2-Hela. E2 positive cells are stained in red. (B) Caco-2 and E1E2-Caco-2 cells were cultured under differentiating conditions for 11 days followed by 2 days in FCS free medium before baso-lateral medium and cells were collected. Hela and E1E2-Hela cells were cultured in standard conditions until 80% confluence and then kept for two days in FCS free medium before cells and supernatants were collected. Cellular and medium samples for western blotting were adjusted for the total cellular protein contents. HCV glycoproteins were detected in both E1E2-Caco-2 and E1E2-Hela cell and not in control cells (left panel) but the two glycoproteins were only secreted by E1E2-Caco-2 and not by E1E2-Hela and control cells (right panel). doi:10.1371/journal.pone.0004233.g003 bearing the viral glycoproteins on their surface. The current VLDL or chylomicron assembly model is envisioned as a two steps process which requires at least apoB and MTP (for reviews see [39–41] and Figure 10). During the first step, apoB is synthesized and translocated into the rough ER lumen forming a lipid binding cavity that is completed with MTP. MTP thus allows the recruitment of phospholipids and triglycerides (step 2). The ongoing translation of apoB concomitant with MTP mediated lipid transfer from the ER lipid droplets forms a neutral lipid core and conversion to a spherical emulsion particle (step 3). After release from the ribosome, the apoB precursors (precursor 1) move to a distal compartment in the secretory pathway (step 4) where they meet with apoB-free luminal lipid droplets or second precursor, formed by MTP in the smooth ER (step 5–6). The first and second precursors fuse to form mature TRL, a process that may be facilitated by apoE, another major component of TRL [42,43]. In that model, it seems unlikely that a transfer of the viral glycoproteins from the ER membrane where they reside to the apoB precursor may occur. Indeed such a leap would need energy and/or complex tertiary or quaternary rearrangement of the glycoproteins induced by an hypothetical binding to the nascent precursor and allowing the transmembrane domain to detach from the ER membrane. The formation of the apoB free precursor is less well understood (step 5–6). If it also depends on MTP, the mechanism leading to a luminal lipid droplet limited by a monolayer of phospholipids is not known. It is tempting to hypothesize that E1 and E2 may slide from the ER membrane to the budding apoB free precursor without any energetically costly conformational changes. In Caco-2 and HepG2 cells, the envelope proteins associated with the second precursor would then fuse with the apoB precursors to form mature hybrid particles. On the contrary, in Huh-7, the fusion likely does not occur and envelope proteins are secreted associated with lipids, and likely with the second precursors, a process that seems to depend on MTP since the secretion of both glycoproteins could be reduced by an MTP inhibitor but less efficiently than in Caco-2 and HepG2 cells. E1 and E2 might therefore be envisioned as markers of the second precursor supporting the two precursor model of lipoprotein synthesis. These data support the recently published hypothesis that some viral particles could result from co-assembly of viral particles and lipoproteins [25] and at least partially explain why apoB and/or MTP inhibition reduces the production of infectious viral particles in Huh-7 cells [28,29]. Chang and colleagues in addition showed that ApoE is required for the production of infectious virus in cell culture [27]. As apoE is involved directly or indirectly in the second precursor formation [43], the reduction of intracellular and secreted virions by apoE knockdown could result from the suppression of the second VLDL precursor assembly. Altogether, these data indicate that both steps of VLDL formation and PLoS ONE | www.plosone.org 5 January 2009 | Volume 4 | Issue 1 | e4233