Due to the recent development of a cell culture model, hepatitis C virus (HCV) can be efficiently propagated in cell culture. This allowed us to reinvestigate the subcellular localization of HCV structural proteins in the context of an infectious cycle. In agreement with previous reports, confocal immunofluorescence analysis of the subcellular localization of HCV structural proteins indicated that, in infected cells, the glycoprotein heterodimer is retained in the endoplasmic reticulum. However, in contrast to other studies, the glycoprotein heterodimer did not accumulate in other intracellular compartments or at the plasma membrane. As previously reported, an association between the capsid protein and lipid droplets was also observed. In addition, a fraction of labeling was consistent with the capsid protein being localized in a membranous compartment that is associated with the lipid droplets. However, in contrast to previous reports, the capsid protein was not found in the nucleus or in association with mitochondria or other well-defined intracellular compartments. Surprisingly, no colocalization was observed between the glycoprotein heterodimer and the capsid protein in infected cells. Electron microscopy analyses allowed us to identify a membrane alteration similar to the previously reported "membranous web." However, no virus-like particles were found in this type of structure. In addition, dense elements compatible with the size and shape of a viral particle were seldom observed in infected cells. In conclusion, the cell culture system for HCV allowed us for the first time to characterize the subcellular localization of HCV structural proteins in the context an infectious cycle.
Recently, the characterization of a cell culture system allowing the amplification of an authentic virus, named hepatitis C virus cell culture (HCVcc), has been reported by several groups. To obtain higher HCV particle productions, we investigated the potential effect of some amino acid changes on the infectivity of the JFH-1 isolate. As a first approach, successive infections of naïve Huh-7 cells were performed until high viral titres were obtained, and mutations that appeared during this selection were identified by sequencing. Only one major modification, N534K, located in the E2 glycoprotein sequence was found. Interestingly, this mutation prevented core glycosylation of E2 site 6. In addition, JFH-1 generated with this modification facilitated the infection of Huh-7 cells. In a second approach to identify mutations favouring HCVcc infectivity, we exploited the observation that a chimeric virus containing the genotype 1a core protein in the context of JFH-1 background was more infectious than wild-type JFH-1 isolate. Sequence alignment between JFH-1 and our chimera, led us to identify two major positions, 172 and 173, which were not occupied by similar amino acids in these two viruses. Importantly, higher viral titres were obtained by introducing these residues in the context of wild-type JFH-1. Altogether, our data indicate that a more robust production of HCVcc particles can be obtained by introducing a few specific mutations in JFH-1 structural proteins. INTRODUCTIONThe Hepatitis C virus (HCV) is the only member of the genus Hepacivirus of the family Flaviviridae. HCV is a major cause of chronic hepatitis, liver cirrhosis, hepatocellular carcinoma (Major et al., 2001) as well as several extrahepatic diseases (Houghton, 1996). An estimation of about 170 million people infected with HCV worldwide has been reported (Poynard et al., 2003;Thomas, 2000).HCV is an enveloped single-strand, positive-sense RNA virus and its genome encodes a unique open reading frame that is flanked by two structured non-translated regions in 59 and 39 ends of HCV genome (59NTR and 39NTR). Mediated by an internal ribosome entry site (TsukiyamaKohara et al., 1992), the translation of HCV RNA genome results in polyprotein synthesis that is processed by cellular and viral proteases into at least 10 structural and nonstructural (NS) proteins (Grakoui et al., 1993;Hijikata et al., 1991). In the viral particle, HCV genomic RNA is complexed with the highly basic capsid protein. On its surface, the viral particle bears two envelope glycoproteins E1 and E2 that are anchored in the lipid bilayer. Both these proteins have been shown to accumulate in the endoplasmic reticulum (ER), where the particles are probably assembled (Op De Beeck et al., 2001). A small integral membrane protein, p7, has been reported to function as an ion channel (Griffin et al., 2003; Pavlovic et al., 2003). Among the NS proteins NS2, NS3, NS4A, NS4B, NS5A and NS5B, which coordinate the intracellular processes of the virus life cycle, only proteins NS3 through to 5B are...
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