Hepatitis C virus (HCV) encodes two envelope glycoproteins, E1 and E2, that assemble as a noncovalent heterodimer which is mainly retained in the endoplasmic reticulum. Because assembly into particles and secretion from the cell lead to structural changes in viral envelope proteins, characterization of the proteins associated with the virion is necessary in order to better understand how they mature to be functional in virus entry. There is currently no efficient and reliable cell culture system to amplify HCV, and the envelope glycoproteins associated with the virion have therefore not been characterized yet. Recently, infectious pseudotype particles that are assembled by displaying unmodified HCV envelope glycoproteins on retroviral core particles have been successfully generated. Because HCV pseudotype particles contain fully functional envelope glycoproteins, these envelope proteins, or at least a fraction of them, should be in a mature conformation similar to that on the native HCV particles. In this study, we used conformation-dependent monoclonal antibodies to characterize the envelope glycoproteins associated with HCV pseudotype particles. We showed that the functional unit is a noncovalent E1E2 heterodimer containing complex or hybrid type glycans. We did not observe any evidence of maturation by a cellular endoprotease during the transport of these envelope glycoproteins through the secretory pathway. These envelope glycoproteins were recognized by a panel of conformation-dependent monoclonal antibodies as well as by CD81, a molecule involved in HCV entry. The functional envelope glycoproteins associated with HCV pseudotype particles were also shown to be sensitive to low-pH treatment. Such conformational changes are likely necessary to initiate fusion.

Although biological and biochemical data have been accumulated on most hepatitis C virus proteins, the structure and function of the 63-amino-acid p7 polypeptide of this virus have never been investigated. In this work, sequence analyses predicted that p7 contains two transmembrane passages connected by a short hydrophilic segment. The C-terminal transmembrane domain of p7 was predicted to function as a signal sequence, which was confirmed experimentally by analyzing the translocation of a reporter glycoprotein fused at its C terminus. The p7 polypeptide was tagged either with the ectodomain of CD4 or with a Myc epitope to study its membrane integration, its subcellular localization, and its topology. Alkaline extraction studies confirmed that p7 is an integral membrane polypeptide. The CD4-p7 chimera was detected by immunofluorescence on the surface of nonpermeabilized cells, indicating that it is exported to the plasma membrane. However, pulse-chase analyses showed that only approximately 20% of endoglycosidase H-resistant CD4-p7 was detected after long chase times, suggesting that a large proportion of p7 stays in an early compartment of the secretory pathway. Finally, by inserting a Myc epitope in several positions of p7 and analyzing the accessibility of this epitope on the plasma membrane of HepG2 cells, we showed that p7 has a double membrane-spanning topology, with both its N and C termini oriented toward the extracellular environment. Altogether, these data indicate that p7 is a polytopic membrane protein that could have a functional role in several compartments of the secretory pathway.

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.

For most membrane proteins, the transmembrane domain (TMD) is more than just an anchor to the membrane. The TMDs of hepatitis C virus (HCV) envelope proteins E1 and E2 are extreme examples of the multifunctionality of such membrane-spanning sequences. Indeed, they possess a signal sequence function in their C-terminal half, play a major role in endoplasmic reticulum localization of E1 and E2, and are potentially involved in the assembly of these envelope proteins. These multiple functions are supposed to be essential for the formation of the viral envelope. As for the other viruses of the family Flaviviridae , these anchor domains are composed of two stretches of hydrophobic residues separated by a short segment containing at least one fully conserved charged residue. Replacement of these charged residues by an alanine in HCV envelope proteins led to an alteration of all of the functions performed by their TMDs, indicating that these functions are tightly linked together. These data suggest that the charged residues of the TMDs of HCV glycoproteins play a key role in the formation of the viral envelope.

Several cellular molecules have been identified as putative receptors forHepatitis C virus(HCV): CD81 tetraspanin, scavenger receptor class B type I (SR-BI), mannose-binding lectins DC-SIGN and L-SIGN, low-density lipoprotein receptor, heparan sulphate proteoglycans and the asialoglycoprotein receptor. Due to difficulties in propagating HCV in cell culture, most of these molecules have been identified by analysing their interaction with a soluble, truncated form of HCV glycoprotein E2. A recent major step in investigating HCV entry was the development of pseudoparticles (HCVpp), consisting of unmodified HCV envelope glycoproteins assembled onto retroviral core particles. This system has allowed the investigation of the role of candidate receptors in the early steps of the HCV life cycle and the data obtained can now be confirmed with the help of a newly developed cell-culture system that allows efficient amplification of HCV (HCVcc). Interestingly, CD81 and SR-BI have been shown to play direct roles in HCVpp and/or HCVcc entry. However, co-expression of CD81 and SR-BI in non-hepatic cell lines does not lead to HCVpp entry, indicating that other molecule(s), expressed only in hepatic cells, are necessary for HCV entry. In this review, the molecules that have been proposed as potential HCV receptors are described and the experimental data indicating that CD81 and SR-BI are potentially involved in HCV entry are presented.