François Helle

The Neutralizing Activity of Anti-Hepatitis C Virus Antibodies Is Modulated by Specific Glycans on the E2 Envelope Protein

ABSTRACT Hepatitis C virus (HCV) envelope glycoproteins are highly glycosylated, with up to 5 and 11 N-linked glycans on E1 and E2, respectively. Most of the glycosylation sites on HCV envelope glycoproteins are conserved, and some of the glycans associated with these proteins have been shown to play an essential role in protein folding and HCV entry. Such a high level of glycosylation suggests that these glycans can limit the immunogenicity of HCV envelope proteins and restrict the binding of some antibodies to their epitopes. Here, we investigated whether these glycans can modulate the neutralizing activity of anti-HCV antibodies. HCV pseudoparticles (HCVpp) bearing wild-type glycoproteins or mutants at individual glycosylation sites were evaluated for their sensitivity to neutralization by antibodies from the sera of infected patients and anti-E2 monoclonal antibodies. While we did not find any evidence that N-linked glycans of E1 contribute to the masking of neutralizing epitopes, our data demonstrate that at least three glycans on E2 (denoted E2N1, E2N6, and E2N11) reduce the sensitivity of HCVpp to antibody neutralization. Importantly, these three glycans also reduced the access of CD81 to its E2 binding site, as shown by using a soluble form of the extracellular loop of CD81 in inhibition of entry. These data suggest that glycans E2N1, E2N6, and E2N11 are close to the binding site of CD81 and modulate both CD81 and neutralizing antibody binding to E2. In conclusion, this work indicates that HCV glycans contribute to the evasion of HCV from the humoral immune response.

Subcellular Localization of Hepatitis C Virus Structural Proteins in a Cell Culture System That Efficiently Replicates the Virus

ABSTRACT 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.

Role of N-Linked Glycans in the Functions of Hepatitis C Virus Envelope Proteins Incorporated into Infectious Virions

ABSTRACT Hepatitis C virus (HCV) envelope glycoproteins are highly glycosylated, with generally 4 and 11 N-linked glycans on E1 and E2, respectively. Studies using mutated recombinant HCV envelope glycoproteins incorporated into retroviral pseudoparticles (HCVpp) suggest that some glycans play a role in protein folding, virus entry, and protection against neutralization. The development of a cell culture system producing infectious particles (HCVcc) in hepatoma cells provides an opportunity to characterize the role of these glycans in the context of authentic infectious virions. Here, we used HCVcc in which point mutations were engineered at N-linked glycosylation sites to determine the role of these glycans in the functions of HCV envelope proteins. The mutants were characterized for their effects on virus replication and envelope protein expression as well as on viral particle secretion, infectivity, and sensitivity to neutralizing antibodies. Our results indicate that several glycans play an important role in HCVcc assembly and/or infectivity. Furthermore, our data demonstrate that at least five glycans on E2 (denoted E2N1, E2N2, E2N4, E2N6, and E2N11) strongly reduce the sensitivity of HCVcc to antibody neutralization, with four of them surrounding the CD81 binding site. Altogether, these data indicate that the glycans associated with HCV envelope glycoproteins play roles at different steps of the viral life cycle. They also highlight differences in the effects of glycosylation mutations between the HCVpp and HCVcc systems. Furthermore, these carbohydrates form a “glycan shield” at the surface of the virion, which contributes to the evasion of HCV from the humoral immune response.

Cyanovirin-N inhibits hepatitis C virus entry by binding to envelope protein glycans.

Inhibition of viruses at the stage of viral entry provides a route for therapeutic intervention. Because of difficulties in propagating hepatitis C virus (HCV) in cell culture, entry inhibitors have not yet been reported for this virus. However, with the development of retroviral particles pseudotyped with HCV envelope glycoproteins (HCVpp) and the recent progress in amplification of HCV in cell culture (HCVcc), studying HCV entry is now possible. In addition, these systems are essential for the identification and the characterization of molecules that block HCV entry. The lectin cyanovirin-N (CV-N) has initially been discovered based on its potent activity against human immunodeficiency virus. Because HCV envelope glycoproteins are highly glycosylated, we sought to determine whether CV-N has an antiviral activity against this virus. CV-N inhibited the infectivity of HCVcc and HCVpp at low nanomolar concentrations. This inhibition is attributed to the interaction of CV-N with HCV envelope glycoproteins. In addition, we showed that the carbohydrate binding property of CV-N is involved in the anti-HCV activity. Finally, CV-N bound to HCV envelope glycoproteins and blocked the interaction between the envelope protein E2 and CD81, a cell surface molecule involved in HCV entry. These data demonstrate that targeting the glycans of HCV envelope proteins is a promising approach in the development of antiviral therapies to combat a virus that is a major cause of chronic liver diseases. Furthermore, CV-N is a new invaluable tool to further dissect the early steps of HCV entry into host cells.

Early steps of the hepatitis C virus life cycle

To replicate its genome, a virus needs to cross the plasma membrane of a host cell and get access to cytosolic and/or nuclear components. For an enveloped virus, this involves binding to the plasma membrane, followed by migration of the virion to a microdomain or an endosomal vesicle where fusion between the virion envelope and a host cell membrane occurs. Increasing evidences indicate that virus entry is a tightly regulated process. Although we are still far from understanding the details of hepatitis C virus (HCV) entry, recent data show that this virus enters into target cells in a slow and complex multistep process involving the presence of several entry factors. Initial attachment of the virion may involve glycosaminoglycans and the low‐density lipoprotein receptor, and it is followed by the sequential interaction with the scavenger receptor class B type I, the tetraspanin CD81 and tight junction protein Claudin‐1, ‐6 or ‐9. Furthermore, the identification of EWI‐2wint as a new partner of CD81 which blocks E2–CD81 interaction provides additional evidence of the complexity of the HCV entry process. The current knowledge accumulated on HCV entry is summarized in this review.

Hepatitis C virus entry into host cells

Abstract.The recent development of functional models to analyze the early steps of the hepatitis C virus (HCV) life cycle has highlighted that HCV entry is a slow and complex multistep process involving the presence of several entry factors. Initial host cell attachment may involve glycosaminoglycans and the low-density lipoprotein receptor, after which the particle appears to interact sequentially with three entry factors: the scavenger receptor class B type I, the tetraspanin CD81 and the tight-junction protein claudin-1. Several serum components may also modulate HCV entry, while the recently discovered CD81 partner EWI-2wint can block the interaction of the viral particle with CD81, potentially preventing infection in the cell types in which it is expressed. After binding to the host cell, the HCV particle is internalized by clathrin-mediated endocytosis, with fusion likely occuring in early endosomes. This review summarizes our current knowledge on HCV entry.

The CD81 Partner EWI-2wint Inhibits Hepatitis C Virus Entry

Two to three percent of the worlds population is chronically infected with hepatitis C virus (HCV) and thus at risk of developing liver cancer. Although precise mechanisms regulating HCV entry into hepatic cells are still unknown, several cell surface proteins have been identified as entry factors for this virus. Among these molecules, the tetraspanin CD81 is essential for HCV entry. Here, we have identified a partner of CD81, EWI-2wint, which is expressed in several cell lines but not in hepatocytes. Ectopic expression of EWI-2wint in a hepatoma cell line susceptible to HCV infection blocked 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. This is the first example of a pathogen gaining entry into host cells that lack a specific inhibitory factor.

Immunogenicity of CIGB-230, a therapeutic DNA vaccine preparation, in HCV-chronically infected individuals in a Phase I clinical trial

Summary.  Hepatitis C virus (HCV) is a worldwide health problem. No vaccine is available against this pathogen and therapeutic treatments currently in use are of limited efficacy. In the present study, the immunogenicity of the therapeutic vaccine candidate CIGB‐230, based on the mixture of pIDKE2, a plasmid expressing HCV structural antigens, with a recombinant HCV core protein, Co.120, was evaluated. CIGB‐230 was administered by intramuscular injection on weeks 0, 4, 8, 12, 16 and 20 to 15 HCV‐chronically infected individuals, non‐responders to previous treatment with interferon (IFN) plus ribavirin. Interestingly, following the final immunization, neutralizing antibody responses against heterologous viral pseudoparticles were modified in eight individuals, including six de novo responders. In addition, 73% of vaccinees exhibited specific T cell proliferative response and T cell IFN‐gamma secretory response 24 weeks after primary immunization with CIGB‐230. Furthermore, 33.3% of individuals developed de novo cellular immune response against HCV core and the number of patients (46.7% at the end of treatment) with cellular immune response against more than one HCV structural antigen increased during vaccination (P = 0.046). In addition, despite persistent detection of HCV RNA, more than 40% percent of vaccinated individuals improved or stabilized liver histology, particularly reducing fibrosis, which correlated with cellular immune response against more than one HCV antigen (P = 0.0053). In conclusion, CIGB‐230 is a promising candidate for effective therapeutic interventions based on its ability for enhancing the immune response in HCV chronically infected individuals.

Ceramide enrichment of the plasma membrane induces CD81 internalization and inhibits hepatitis C virus entry

Virus entry is a major step in which host‐cell lipids can play an essential role. In this report, we investigated the importance of sphingolipids in hepatitis C virus (HCV) entry. For this purpose, sphingomyelin present in the plasma membrane of target cells was hydrolysed into ceramide by sphingomyelinase treatment. Interestingly, ceramide enrichment of the plasma membrane strongly inhibited HCV entry. To understand how ceramide affected HCV entry, we analysed the effect of ceramide enrichment of the plasma membrane on three cell‐surface molecules identified as entry factors for HCV: CD81 tetraspanin, scavenger receptor BI and Claudin‐1. These proteins, which we found to be mainly associated with detergent‐soluble membranes in Huh‐7 cells, were not relocated in detergent‐resistant microdomains after sphingomyelin hydrolysis into ceramide. Importantly, ceramide enrichment of the plasma membrane led to a 50% decrease in cell‐surface CD81, which was due to its ATP‐independent internalization. Our results strongly suggest that the ceramide‐induced internalization of CD81 is responsible for the inhibitory effect of ceramide on HCV entry. Together, these data indicate that some specific lipids of the plasma membrane are essential for HCV entry and highlight plasma membrane lipids as potential targets to block HCV entry.

The hepatitis C virus glycan shield and evasion of the humoral immune response.

Despite the induction of effective immune responses, 80% of hepatitis C virus (HCV)-infected individuals progress from acute to chronic hepatitis. In contrast to the cellular immune response, the role of the humoral immune response in HCV clearance is still subject to debate. Indeed, HCV escapes neutralizing antibodies in chronically infected patients and reinfection has been described in human and chimpanzee. Studies of antibody-mediated HCV neutralization have long been hampered by the lack of cell-culture-derived virus and the absence of a small animal model. However, the development of surrogate models and recent progress in HCV propagation in vitro now enable robust neutralization assays to be performed. These advances are beginning to shed some light on the mechanisms of HCV neutralization. This review summarizes the current state of knowledge of the viral targets of anti-HCV-neutralizing antibodies and the mechanisms that enable HCV to evade the humoral immune response. The recent description of the HCV glycan shield that reduces the immunogenicity of envelope proteins and masks conserved neutralizing epitopes at their surface constitutes the major focus of this review.