Heidi Barth

Cellular binding of hepatitis C virus envelope glycoprotein E2 requires cell surface heparan sulfate.

The conservation of positively charged residues in the N terminus of the hepatitis C virus (HCV) envelope glycoprotein E2 suggests an interaction of the viral envelope with cell surface glycosaminoglycans. Using recombinant envelope glycoprotein E2 and virus-like particles as ligands for cellular binding, we demonstrate that cell surface heparan sulfate proteoglycans (HSPG) play an important role in mediating HCV envelope-target cell interaction. Heparin and liver-derived highly sulfated heparan sulfate but not other soluble glycosaminoglycans inhibited cellular binding and entry of virus-like particles in a dose-dependent manner. Degradation of cell surface heparan sulfate by pretreatment with heparinases resulted in a marked reduction of viral envelope protein binding. Surface plasmon resonance analysis demonstrated a high affinity interaction (KD 5.2 × 10–9 m) of E2 with heparin, a structural homologue of highly sulfated heparan sulfate. Deletion of E2 hypervariable region-1 reduced E2-heparin interaction suggesting that positively charged residues in the N-terminal E2 region play an important role in mediating E2-HSPG binding. In conclusion, our results demonstrate for the first time that cellular binding of HCV envelope requires E2-HSPG interaction. Docking of E2 to cellular HSPG may be the initial step in the interaction between HCV and the cell surface resulting in receptor-mediated entry and initiation of infection.

Initiation of Hepatitis C Virus Infection Is Dependent on Cholesterol and Cooperativity between CD81 and Scavenger Receptor B Type I

ABSTRACT In the past several years, a number of cellular proteins have been identified as candidate entry receptors for hepatitis C virus (HCV) by using surrogate models of HCV infection. Among these, the tetraspanin CD81 and scavenger receptor B type I (SR-BI), both of which localize to specialized plasma membrane domains enriched in cholesterol, have been suggested to be key players in HCV entry. In the current study, we used a recently developed in vitro HCV infection system to demonstrate that both CD81 and SR-BI are required for authentic HCV infection in vitro, that they function cooperatively to initiate HCV infection, and that CD81-mediated HCV entry is, in part, dependent on membrane cholesterol.

Scavenger receptor class B type I is a key host factor for hepatitis C virus infection required for an entry step closely linked to CD81.

Hepatitis C virus (HCV) is a major cause of chronic hepatitis worldwide. Scavenger receptor class B type I (SR‐BI) has been shown to bind HCV envelope glycoprotein E2, participate in entry of HCV pseudotype particles, and modulate HCV infection. However, the functional role of SR‐BI for productive HCV infection remains unclear. In this study, we investigated the role of SR‐BI as an entry factor for infection of human hepatoma cells using cell culture–derived HCV (HCVcc). Anti–SR‐BI antibodies directed against epitopes of the human SR‐BI extracellular loop specifically inhibited HCVcc infection in a dose‐dependent manner. Down‐regulation of SR‐BI expression by SR‐BI–specific short interfering RNAs (siRNAs) markedly reduced the susceptibility of human hepatoma cells to HCVcc infection. Kinetic studies demonstrated that SR‐BI acts predominately after binding of HCV at an entry step occurring at a similar time point as CD81–HCV interaction. Although the addition of high‐density lipoprotein (HDL) enhanced the efficiency of HCVcc infection, anti–SR‐BI antibodies and SR‐BI–specific siRNA efficiently inhibited HCV infection independent of lipoprotein. Conclusion: Our data suggest that SR‐BI (i) represents a key host factor for HCV entry, (ii) is implicated in the same HCV entry pathway as CD81, and (iii) targets an entry step closely linked to HCV–CD81 interaction. (HEPATOLOGY 2007.)

Viral and cellular determinants of the hepatitis C virus envelope-heparan sulfate interaction.

ABSTRACT Cellular binding and entry of hepatitis C virus (HCV) are the first steps of viral infection and represent a major target for antiviral antibodies and novel therapeutic strategies. We have recently demonstrated that heparan sulfate (HS) plays a key role in the binding of HCV envelope glycoprotein E2 to target cells (Barth et al., J. Biol. Chem. 278:41003-41012, 2003). In this study, we characterized the HCV-HS interaction and analyzed its inhibition by antiviral host immune responses. Using recombinant envelope glycoproteins, virus-like particles, and HCV pseudoparticles as model systems for the early steps of viral infection, we mapped viral and cellular determinants of HCV-HS interaction. HCV-HS binding required a specific HS structure that included N-sulfo groups and a minimum of 10 to 14 saccharide subunits. HCV envelope binding to HS was mediated by four viral epitopes overlapping the E2 hypervariable region 1 and E2-CD81 binding domains. In functional studies using HCV pseudoparticles, we demonstrate that HCV binding and entry are specifically inhibited by highly sulfated HS. Finally, HCV-HS binding was markedly inhibited by antiviral antibodies derived from HCV-infected individuals. In conclusion, our results demonstrate that binding of the viral envelope to a specific HS configuration represents an important step for the initiation of viral infection and is a target of antiviral host immune responses in vivo. Mapping of viral and cellular determinants of HCV-HS interaction sets the stage for the development of novel HS-based antiviral strategies targeting viral attachment and entry.

Hepatitis C virus entry: Molecular biology and clinical implications†

With an estimated 170 million infected individuals, hepatitis C virus (HCV) has a major impact on public health. A vaccine protecting against HCV infection is not available, and current antiviral therapies are characterized by limited efficacy, high costs, and substantial side effects. Binding of the virus to the cell surface followed by viral entry is the first step in a cascade of interactions between virus and the target cell that is required for the initiation of infection. Because this step represents a critical determinant of tissue tropism and pathogenesis, it is a major target for host cell responses such as antibody‐mediated virus‐neutralization—and a promising target for new antiviral therapy. The recent development of novel tissue culture model systems for the study of the first steps of HCV infection has allowed rapid progress in the understanding of the molecular mechanisms of HCV binding and entry. This review summarizes the impact of recently identified viral and host cell factors for HCV attachment and entry. Clinical implications of this important process for the pathogenesis of HCV infection and novel therapeutic interventions are discussed. (HEPATOLOGY 2006;44:527–535.)

CD81 Expression Is Important for the Permissiveness of Huh7 Cell Clones for Heterogeneous Hepatitis C Virus Infection

ABSTRACT Huh7 cells constitute a permissive cell line for cell culture of hepatitis C virus (HCV) particles. However, our Huh7 line shows limited permissiveness for HCV. Thus, in this study we set out to determine which host factors are important for conferring permissiveness. To analyze the limited permissiveness of our Huh7 cells, 70 clones were obtained after single-cell cloning of parental Huh7 cells. The cloned Huh7 cells exhibited various levels of HCV pseudoparticles and JFH-1 virus infection efficiency, and some clones were not permissive. A subgenomic replicon was then transfected into the cloned Huh7 cells. While the replication efficiencies differed among the cloned Huh7 cells, these efficiencies did not correlate with infectious permissibility. Flow cytometry showed that CD81, scavenger receptor class B type I, and low-density-lipoprotein receptor expression on the cell surfaces of the Huh7 clones differed among the clones. Interestingly, we found that all of the permissive cell clones expressed CD81 while the nonpermissive cell clones did not. To confirm the importance of CD81 expression for HCV permissiveness, CD81 was then transiently and stably expressed on a nonpermissive Huh7 cell clone, which was consequently restored to HCV infection permissiveness. Furthermore, permissiveness was down-regulated upon transfection of CD81 silencing RNA into a CD81-positive cell clone. In conclusion, CD81 expression is an important determinant of HCV permissiveness of Huh7 cell clones harboring different characteristics.

Binding of Hepatitis C Virus-Like Particles Derived from Infectious Clone H77C to Defined Human Cell Lines

ABSTRACT Hepatitis C virus (HCV) is a leading cause of chronic hepatitis in the world. The study of viral entry and infection has been hampered by the inability to efficiently propagate the virus in cultured cells and the lack of a small-animal model. Recent studies have shown that in insect cells, the HCV structural proteins assemble into HCV-like particles (HCV-LPs) with morphological, biophysical, and antigenic properties similar to those of putative virions isolated from HCV-infected humans. In this study, we used HCV-LPs derived from infectious clone H77C as a tool to examine virus-cell interactions. The binding of partially purified particles to human cell lines was analyzed by fluorescence-activated cell sorting with defined monoclonal antibodies to envelope glycoprotein E2. HCV-LPs demonstrated dose-dependent and saturable binding to defined human lymphoma and hepatoma cell lines but not to mouse cell lines. Binding could be inhibited by monoclonal anti-E2 antibodies, indicating that the HCV-LP-cell interaction was mediated by envelope glycoprotein E2. Binding appeared to be CD81 independent and did not correlate with low-density lipoprotein receptor expression. Heat denaturation of HCV-LPs drastically reduced binding, indicating that the interaction of HCV-LPs with target cells was dependent on the proper conformation of the particles. In conclusion, our data demonstrate that insect cell-derived HCV-LPs bind specifically to defined human cell lines. Since the envelope proteins of HCV-LPs are presumably presented in a virion-like conformation, the binding of HCV-LPs to target cells may allow the study of virus-host cell interactions, including the isolation of HCV receptor candidates and antibody-mediated neutralization of binding.

Scavenger Receptor Class B Type I and Hepatitis C Virus Infection of Primary Tupaia Hepatocytes

ABSTRACT Hepatitis C virus (HCV) is a major cause of chronic hepatitis worldwide. The study of early steps during HCV infection has been hampered by the lack of suitable in vitro or in vivo models. Primary Tupaia hepatocytes (PTH) have been shown to be susceptible to HCV infection in vitro and in vivo. Human scavenger receptor class B type I (SR-BI) represents an HCV receptor candidate mediating the cellular binding of E2 glycoprotein to HepG2 hepatoma cells. However, the function of SR-BI for viral infection of hepatocytes is unknown. In this study, we used PTH to assess the functional role of SR-BI as a putative HCV receptor. Sequence analysis of cloned tupaia SR-BI revealed a high homology between tupaia and human SR-BI. Transfection of CHO cells with human or tupaia SR-BI but not mouse SR-BI cDNA resulted in cellular E2 binding, suggesting that E2-binding domains between human and tupaia SR-BI are highly conserved. Preincubation of PTH with anti-SR-BI antibodies resulted in marked inhibition of E2 or HCV-like particle binding. However, anti-SR-BI antibodies were not able to block HCV infection of PTH. In conclusion, our results demonstrate that SR-BI represents an important cell surface molecule for the binding of the HCV envelope to hepatocytes and suggest that other or additional cell surface molecules are required for the initiation of HCV infection. Furthermore, the structural and functional similarities between human and tupaia SR-BI indicate that PTH represent a useful model system to characterize the molecular interaction of the HCV envelope and SR-BI on primary hepatocytes.

Scavenger Receptor Class B Is Required for Hepatitis C Virus Uptake and Cross-Presentation by Human Dendritic Cells

ABSTRACT Class B scavenger receptors (SR-Bs) bind lipoproteins and play an important role in lipid metabolism. Most recently, SR-B type I (SR-BI) and its splicing variant SR-BII have been found to mediate bacterial adhesion and cytosolic bacterial invasion in mammalian cells. In this study, we demonstrate that SR-BI is a key host factor required for hepatitis C virus (HCV) uptake and cross-presentation by human dendritic cells (DCs). Whereas monocytes and T and B cells were characterized by very low or undetectable SR-BI expression levels, human DCs demonstrated a high level of cell surface expression of SR-BI similar to that of primary human hepatocytes. Antibodies targeting the extracellular loop of SR-BI efficiently inhibited HCV-like particle binding, uptake, and cross-presentation by human DCs. Moreover, human high-density lipoprotein specifically modulated HCV-like particle binding to DCs, indicating an interplay of HCV with the lipid transfer function of SR-BI in DCs. Finally, we demonstrate that anti-SR-BI antibodies inhibit the uptake of cell culture-derived HCV (HCVcc) in DCs. In conclusion, these findings identify a novel function of SR-BI for viral antigen uptake and recognition and may have an important impact on the design of HCV vaccines and immunotherapeutic approaches aiming at the induction of efficient antiviral immune responses.

Inhibition of hepatitis C virus-like particle binding to target cells by antiviral antibodies in acute and chronic hepatitis C.

ABSTRACT Hepatitis C virus (HCV) is a leading cause of chronic viral hepatitis worldwide. The study of antibody-mediated virus neutralization has been hampered by the lack of an efficient and high-throughput cell culture system for the study of virus neutralization. The HCV structural proteins have been shown to assemble into noninfectious HCV-like particles (HCV-LPs). Similar to serum-derived virions, HCV-LPs bind and enter human hepatocytes and hepatoma cell lines. In this study, we developed an HCV-LP-based model system for a systematic functional analysis of antiviral antibodies from patients with acute or chronic hepatitis C. We demonstrate that cellular HCV-LP binding was specifically inhibited by antiviral antibodies from patients with acute or chronic hepatitis C in a dose-dependent manner. Using a library of homologous overlapping envelope peptides covering the entire HCV envelope, we identified an epitope in the N-terminal E2 region (SQKIQLVNTNGSWHI; amino acid positions 408 to 422) as one target of human antiviral antibodies inhibiting cellular particle binding. Using a large panel of serum samples from patients with acute and chronic hepatitis C, we demonstrated that the presence of antibodies with inhibition of binding activity was not associated with viral clearance. In conclusion, antibody-mediated inhibition of cellular HCV-LP binding represents a convenient system for the functional characterization of human anti-HCV antibodies, allowing the mapping of envelope neutralization epitopes targeted by naturally occurring antiviral antibodies.