In infected cells, hepatitis C virus (HCV) induces the formation of membrane alterations referred to as membranous webs, which are sites of RNA replication. In addition, HCV RNA replication also occurs in smaller membrane structures that are associated with the endoplasmic reticulum. However, cellular mechanisms involved in the formation of HCV replication complexes remain largely unknown. Here, we used brefeldin A (BFA) to investigate cellular mechanisms involved in HCV infection. BFA acts on cell membranes by interfering with the activation of several members of the family of ADP-ribosylation factors (ARF), which can lead to a wide range of inhibitory actions on membrane-associated mechanisms of the secretory and endocytic pathways. Our data show that HCV RNA replication is highly sensitive to BFA. Individual knockdown of the cellular targets of BFA using RNA interference and the use of a specific pharmacological inhibitor identified GBF1, a guanine nucleotide exchange factor for small GTPases of the ARF family, as a host factor critically involved in HCV replication. Furthermore, overexpression of a BFA-resistant GBF1 mutant rescued HCV replication in BFA-treated cells, indicating that GBF1 is the BFA-sensitive factor required for HCV replication. Finally, immunofluorescence and electron microscopy analyses indicated that BFA does not block the formation of membranous web-like structures induced by expression of HCV proteins in a nonreplicative context, suggesting that GBF1 is probably involved not in the formation of HCV replication complexes but, rather, in their activity. Altogether, our results highlight a functional connection between the early secretory pathway and HCV RNA replication.Hepatitis C virus (HCV) is an important human pathogen. It mainly infects human hepatocytes, and this often leads to chronic hepatitis, cirrhosis, or hepatocarcinoma. HCV studies have been hampered for many years by the difficulty in propagating this virus in vitro. Things have recently changed with the development of a cell culture model referred to as HCVcc (34,60,65), which allows the study of the HCV life cycle in cell culture and facilitates studies of the interactions between HCV and the host cell.HCV is an enveloped positive-strand RNA virus belonging to the family Flaviviridae (35). The viral genome contains a single open reading frame, which is flanked by two noncoding regions that are required for translation and replication. All viral proteins that are produced after proteolytic processing of the initially synthesized polyprotein are membrane associated (15,43). This reflects the fact that virtually all steps of the viral life cycle occur in close association with cellular membranes.Interactions of HCV with cell membranes begin during entry. Several receptors, coreceptors, and other entry factors have been discovered over the years, which link HCV entry to specialized domains of the plasma membrane, such as tetraspanin-enriched microdomains and tight junctions (8,16,59). The internalization of the viral particle...

The neutralizing activity of anti-hepatitis C virus (HCV) antibodies is attenuated by a factor present in human sera, which has been proposed to be high-density lipoproteins (HDLs). HDLs have also been shown to facilitate the entry of HCV pseudoparticles (HCVpp) into target cells. Here, the aim of the study was to determine whether HDL-mediated facilitation of HCVpp and infectious HCV (HCVcc) entry and attenuation of neutralization are two related phenomena. The data indicated that HDLs attenuate neutralization at a constant rate. In addition, as for HDL-mediated facilitation of HCVpp entry, attenuation of neutralization depended on the expression of the scavenger receptor BI (SR-BI) and its selective lipid-uptake function. Finally, kinetic experiments showed that HDL-mediated facilitation of HCVpp entry is more rapid than virus neutralization. Altogether, these observations indicate that HCV is exploiting the physiological activity of SR-BI for promoting its entry into target cells, which consequently also protects the virus against neutralizing antibodies.Detection of the presence of neutralizing antibodies in the sera of patients infected by Hepatitis C virus (HCV) has been hampered by the difficulties in establishing a robust and reliable cell-culture system for HCV propagation. Interestingly, the recent development of HCV pseudoparticles (HCVpp) (Bartosch et al., 2003b;Drummer et al., 2003;Hsu et al., 2003) has allowed confirmation of the presence of neutralizing antibodies in sera of chronically infected chimpanzees and humans (Bartosch et al., 2003a, b;Hsu et al., 2003;Lavillette et al., 2005b;Logvinoff et al., 2004;Meunier et al., 2005;Yu et al., 2004). However, it has been shown that the neutralization activity of antibodies from HCV-infected patients is attenuated by a factor present in human serum, which has been proposed to be high-density lipoproteins (HDLs) (Bartosch et al., 2005;Lavillette et al., 2005a;Meunier et al., 2005). At present, neither the intensity nor the mechanism of attenuation of neutralization by HDLs has been clearly determined. Interestingly, we and others have shown that HDLs are also able to facilitate HCVpp entry through a mechanism depending on the expression of the scavenger receptor BI (SR-BI) and its selective lipid-uptake function (Bartosch et al., 2005;Meunier et al., 2005; Voisset et al., 2005). Here, we sought to determine whether HDLmediated facilitation of HCVpp entry and attenuation of neutralization are two related phenomena.HCVpp containing subtype 1a envelope glycoproteins (strain H) and infectious JFH1 virus (HCVcc) were generated as described previously (Bartosch et al., 2003b;Rouillé et al., 2006;Wakita et al., 2005). Infection experiments were performed on Huh-7 cells (Nakabayashi et al., 1982). HCVpp and HCVcc were produced in lipoprotein-free medium (2 % fetal calf lipoprotein-depleted serum, LPDS) and neutralization experiments were also performed in medium supplemented with 2 % LPDS. Human HDL (density, 1?13-1?18 g ml 21 ) fractions from fresh human plas...