high density particles, in vivo for chimpanzees and in vitro in the Huh-7 cell culture system [11,20,21]. A transmission case of hepatitis C suggests that low density viral particles are also infectious in humans [22]. It is not clear however, whether every circulating HCV particles are associated with apoB, the triglyc- eride content of the particle being the parameter changing the density, or whether only the low density particles are apoB positive and triglyceride rich viral complexes. Because of their association with TRL, the low density particles have been assigned the name of lipo-viro-particles (LVP) [10]. The proportion of LVP amongst the circulating viral particles varies from patient to patient, but on average almost half of HCV RNA is detected in the circulating plasma fractions with density lower than 1.06 g/mL. LVPs are recognized by host antibodies and these immunoglobulin positive particles can be purified by protein A precipitation. Electron microscopy studies identified purified LVPs as globular particles that are heterogeneous in size with an average diameter of 100 nm. These contain higher amounts of triglycerides than lipoproteins isolated from the same density fractions and they contain apolipoproteins (B, CII, CIII, and E, but not the HDL-associated apoA) as well as the viral RNA, core protein and envelope glycoproteins E1 and E2 [10,18]. Treatment of LVP with detergent does not destroy the association of HCV RNA with apoB [18]. Surprisingly, the two apoB isoforms, apoB 100 and apoB 48, are present in LVP with comparatively more apoB 48 in LVP than in the plasma [16]. While apoB 100 is produced by the liver, apoB 48 is only synthesized by enterocytes and is essential for the formation of chylomicrons [23]. This is in keeping with the existence of an intestinal site of HCV assembly and maturation that is supported by the detection of viral non structural proteins in enterocytes of chronically infected patients and the change in neutral lipid composition of LVP content early after a fat rich meal [16,24]. The nature of LVP however remains poorly defined and the process leading to the coassembly of lipoproteins and virus hybrid complexes is not understood. It has been suggested that LVP formation occurs at the ER membrane where TRL synthesis takes place [25] since HCV RNA can already be immunoprecipitated by anti-apoB antibodies in chronically infected liver macerates [26]. In support to this hypothesis, it was recently shown that in vitro production of HCV in the Huh-7 cell line depends on the assembly of VLDL and on the expression of apoE [27–29]. It has also been documented that HCV envelope glycoproteins E1 and E2 are retained in the endoplasmic reticulum (ER) by retention signals in their transmembrane domains (for review see [30,31]). Concerning LVP, E1 and E2 appear to be exposed on the surface of purified LVP since they can be recognized by anti-envelope antibodies under non denaturating conditions [16]. The envelope glycoproteins may thus play a pivotal role in the formation of LVP. To better understand the interaction between envelope glycoproteins and TRL and the assembly of LVP, E1 and E2 proteins were stably expressed in cell lines secreting apoB positive lipoproteins. It is shown here that the viral glycoproteins are secreted from these cells only when the TRL synthesis and secretion pathway is functional. Moreover, the resulting hybrid TRL particles bear E1 and E2 glycoproteins at their surface. These findings reveal that HCV envelope glycoproteins have the intrinsic capacity to assemble into TRL. Results Establishment and characterization of Caco-2 cells expressing HCV envelope glycoproteins The human intestinal cell line Caco-2 does not synthesize apoB and MTP when cultured under standard conditions and therefore Secretion of HCV Glycoproteins does not secrete apoB-containing lipoproteins. Caco-2 cells maintained under these conditions were infected with the E1E2- HIV-SIN lentiviral vector. After transduction, E1 and E2 proteins with the expected molecular weight could be detected in cell lysates by Western blotting (Figure 1A). Immunofluorescence analysis indicated that almost all cells expressed the glycoproteins (Figure 1B) and that the expression was stable for at least 40 passages. However, when cultured on porous filter allowing asymmetrical culture conditions with serum free medium in the upper chamber and complete medium in the lower chamber of the culture device, Caco-2 cells partially differentiate into polarized cells and regain the capacity to synthesize and secrete TRL at their basal membrane into the lower chamber a few days after cells reach confluence (Figure 2A) [32,33]. As shown in the time course experiment in Figure 2B and in agreement with previous reports [32,33], apoB could only be detected in the basal compartment of the culture 7 days after cell seeding. Concentrations of apoB then increased until day 13. Interestingly, in E1E2 expressing cells, the apoB secretion was significantly delayed but still reached a similar high level at day 13. Although E2 was always detected in cell lysates (Figure 2C), a fraction of E2 was detected in the basal medium concomitant with the apoB secretion, suggesting that HCV envelope proteins are secreted with apoB. Cell differentiation was also correlated with an increased amount of intracellular E2 (Figure 2C). HCV glycoproteins are not secreted by the apoB negative epithelial Hela cell line The secretion of HCV glycoproteins was only detected concomitantly with the synthesis and secretion of apoB positive lipoproteins by E1E2-Caco-2 cells suggesting that a functional apoB pathway is necessary for E1E2 secretion. We then tested if the two glycoproteins could nevertheless be also secreted by epithelial cell line not from hepatic or intestine origin and not producing lipoproteins. Hela cells were transduced by the E1E2 lentiviral vector and were found, as Caco-2 cells, to stably express the glycoproteins as shown by immunostaining (Figure 3A). By contrast, the glycoproteins were not detected in the supernatant of E1E2-Hela cells as they were in E1E2-Caco-2 basal medium (Figure 3B). Characterization of the apoB and HCV envelope glycoprotein complexes To further investigate the association of glycoproteins and TRL, E1E2 expressing and control Caco-2 cells were cultured under differentiation conditions and the basal medium collected at day 13 was analyzed in an iodixanol density gradient. Figure 4 shows that the glycoprotein E2 and isoforms of apoB and apoE were detected in fractions with the lowest density (#1.05 g/mL) with the maximum concentration in the 1.035 g/mL fraction. Con- cerning apoB, the two isoforms, apoB100 and apoB48, were secreted in comparable molar concentrations. This further confirms the differentiation state of the cells that can edit, at least partially, the apoB mRNA to synthetize apoB48 [32]. The distribution of the two glycoproteins E1 and E2 in a highly resolved density gradient is shown in Figure 5. Both glycoproteins co-sedimented in apoB containing fractions from E1E2-Caco-2 cells but not in corresponding fractions from control Caco-2 cells (Figure 5 A and B). Interestingly, although apoB was still present in fractions with the lowest density of about 1.02 g/mL, the two glycoproteins and particularly E1 were confined to the apoB positive fractions with a density from 1.07 to 1.05 g/mL suggesting that different lipoproteins were secreted into the basal PLoS ONE | www.plosone.org 2 January 2009 | Volume 4 | Issue 1 | e4233