More than 120 million people worldwide are chronically infected by with the hepatitis C virus (HCV), a major cause of chronic hepatitis, cirrhosis, and hepatocellular carcinoma. In addition, chronic HCV infection has become the most common indication for a liver transplant. HCV is a small enveloped virus that belongs to the Hepacivirus genus of the Flaviviridae family. Its genome encodes a single polyprotein precursor of about 3,000 amino acid residues, which is cleaved by cellular and viral proteases to yield at least 10 mature products, among which are a capsid protein and two envelope glycoproteins, E1 and E2. The envelope glycoproteins are present at the surface of HCV particles, and they play an essential role in virus entry.

Owing to the lack of a robust cell culture system to amplify HCV, the entry process for this virus was difficult to study until recently. A major advance in investigating HCV entry was achieved in 2003 by the development of pseudoparticles (HCVpp), which consist of unmodified HCV envelope glycoproteins assembled onto retroviral core particles. Extensive characterization of HCVpp has shown that they mimic the early steps of the HCV life cycle. In 2005, a cell culture system that allows a relatively efficient amplification of HCV (HCVcc) was also finally developed. This cell culture system permits, for the first time, the study of the whole life cycle of HCV and is an essential tool for the study of HCV entry.

Our earlier work largely contributed to understanding the biogenesis of HCV envelope glycoproteins. These proteins contain a large N-terminal ectodomain and a C-terminal hydrophobic transmembrane region. After translocation of their ectodomain into the lumen of the endoplasmic reticulum (ER), HCV envelope glycoproteins are cleaved from the polyprotein by a host signal peptidase. Interestingly, HCV glycoproteins E1 and E2 assemble as a noncovalent heterodimer, which is retained in the ER. The transmembrane domains of HCV envelope glycoproteins play a major role in the assembly of E1E2 heterodimer and in its retention in the ER, as do charged residues located in the middle of the transmembrane domains of HCV envelope glycoproteins.

Recently, we identified some residues of the transmembrane domains of E1 and E2 that are directly involved in E1-E2 interactions. We have observed that a GXXXG oligomerization motif present in the transmembrane domain of E1 is essential for the formation of a functional E1-E2 heterodimer. Furthermore, by using a recently developed in vitro fusion assay involving fluorescently labeled liposomes, we showed that mutations reducing HCVpp infectivity without altering E1E2 heterodimerization affect the fusion properties of HCV envelope glycoproteins. In conclusion, we have identified residues involved in E1E2 heterodimerization and demonstrated that the transmembrane domains of HCV envelope glycoproteins play a major role in the fusion properties of these proteins.

Given that HCV envelope glycoproteins contain a large number of conserved N-linked glycans, we investigated the role of these glycans in the entry process. We showed that a few specific glycans play an essential role in protein folding or HCV entry and that N-linked glycans can be a potential target for the development of new antiviral molecules against HCV. Indeed, we demonstrated that the lectin cyanovirin-N (CV-N) inhibits HCV entry at low nanomolar concentrations. By interacting with the HCV glycoprotein E2, CV-N blocks the interaction between the envelope glycoprotein E2 and CD81 tetraspanin, a cell surface molecule involved in HCV entry. The high level of glycosylation of HCV envelope glycoproteins also suggests that these glycans can limit the immunogenicity of HCV envelope glycoproteins and restrict the binding of some antibodies to their epitopes. We therefore investigated whether these glycans can modulate the neutralizing activity of anti-HCV antibodies and showed that glycans on E2 reduce the sensitivity of HCV to antibody neutralization. Furthermore, these glycans also reduce the access of CD81 to its E2 binding site. The data indicate that the glycans are close to the binding site of CD81 and modulate both CD81 and neutralizing antibody binding to E2. We conclude that, in addition to their role in protein folding and virus entry, HCV glycans contribute to the evasion of HCV from the humoral immune response.

Several cellular molecules have been identified as putative receptors for HCV. Among these molecules, CD81 tetraspanin, scavenger receptor BI (SR-BI), and more recently claudin 1 have been shown to play a direct role in HCV entry. However, it is not known what role these molecules play in HCV entry. To better understand the role of SR-BI, we analyzed the effects of some of its physiological ligands on HCV entry. We showed that, when HCVpp are incubated in the presence of high-density lipoproteins (HDL), which are physiological ligands of SR-BI, infectivity is augmented rather than reduced. In addition, HDL-mediated facilitation of HCVpp entry depends on the lipid-transfer property of SR-BI. These observations indicate that HCV exploits the physiological activity of SR-BI for promoting its entry into target cells and, furthermore, thus protecting the virus against neutralizing antibodies.

We have also analyzed the role of serum amyloid A (SAA), another ligand of SR-BI, on HCV entry. SAA is an acute-phase protein mainly produced by the liver immediately after infection, tissue damage, or inflammation. The high concentration of SAA during the acute-phase response suggests that this protein has a beneficial role in host defense. In contrast to HDL, SAA inhibits HCV entry in a dose-dependent manner. SAA blocks virus entry by interacting with the viral particle. In addition, the antiviral activity of SAA is strongly reduced when HDL are incubated with SAA. However, HDL have only a slight effect on the antiviral activity of SAA when HCVpp are preincubated with SAA. Taken together, our data demonstrate an antiviral activity for SAA and suggest a tight relationship between SAA and HDL in modulating HCV infectivity.

More recently, we investigated the role of CD81 in HCV entry. Like other members of the tetraspanin family, CD81 interacts with other tetraspanin molecules and with other transmembrane proteins, thus building membrane multi-molecular complexes, collectively referred to as the tetraspanin web. Within this network of interactions, tetraspanins form primary complexes with a limited number of proteins called tetraspanin partners. We therefore investigated the potential role of CD81 partners in HCV entry and recently identified a partner of CD81, EWI-2wint, which is expressed in several cell lines but not in hepatocytes. Furthermore, the ectopic expression of EWI-2wint blocks the interaction between HCV envelope glycoproteins and CD81. To further characterize the role of EWI-2wint in inhibiting the interactions between the envelope glycoproteins and CD81, we generated Huh-7 cells (a human hepatoma cell line in which HCV replicates) expressing EWI-2wint and demonstrated that expression of this molecule in Huh-7 cells inhibits viral entry by inhibiting the interaction between the HCV envelope glycoproteins and CD81. This finding suggests that, in addition to the presence of specific entry factors in the hepatocytes, the lack of a specific inhibitor can contribute to the hepatotropism of HCV.

Last updated November 2008