With an estimated 170 million infected individuals, hepatitis C virus (HCV) has a major impact on public health.1 A vaccine protecting against HCV infection is not available, and current antiviral therapies are characterized by limited efficacy, high cost, and substantial side effects.2 HCV is a small enveloped positive-strand RNA virus that has been classified in a separate genus (Hepacivirus) of the Flaviviridae family. In vivo, HCV infects only humans and chimpanzees.3 Tree shrews and transgenic mice repopulated with human hepatocytes are susceptible to experimental infection.3, 4 The liver is the primary target organ of HCV infection. Apart from infection of hepatocytes, infection of B cells and dendritic cells has been described.5–7 Attachment 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.8 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 a new antiviral therapy.
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.)
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Model Systems for the Study of Viral Attachment and Entry
To study viral attachment, entry, and infection, several in vitro models have been developed. Inoculation of primary hepatocytes with serum-derived HCV is an elegant way to study HCV infection in vitro. Primary hepatocytes from humans, chimpanzees, or tree shrews can be successfully infected with serum-derived HCV.9, 10 However, limitations of these systems are the low level of HCV replication requiring reverse transcription polymerase chain reaction for detection of viral infection, the variable quality of the human hepatocytes obtained from surgical specimens, and the lack of a well-defined viral inoculum. Furthermore, the lack of suitable methods to measure cell surface–bound HCV have made it difficult to study important mechanisms of viral life cycle such as the attachment of the virion to the host cell, which is usually determined by an interaction between viral surface glycoproteins and specific cell surface molecule(s). To overcome these hurdles, alternative model systems have been developed to study the mechanisms and host functions involved in viral attachment, entry, replication, assembly, and releases, such as recombinant HCV envelope glycoproteins,11, 12 HCV-like particles,13 HCV pseudotyped particles,14–17 and, more recently, cell-culture–derived infectious HCV.18–20
Recombinant HCV envelope glycoproteins have been successfully used as a surrogate model to study virus–host cell interaction leading to the identification of putative HCV receptor candidates including CD81,11 scavenger receptor class B type I,12 and heparan sulfate.21 HCV-like particles (HCV-LPs) generated by self-assembly of the HCV structural proteins in insect cells have been shown to exhibit morphological, biophysical, and antigenic properties similar to putative virions isolated from HCV-infected patients.13 In contrast to individually expressed envelope glycoproteins E1 and E2, E1-E2 heterodimers of HCV-LPs are presumably presented in a native, virion-like conformation. HCV-LPs have been shown to bind and enter human hepatoma cells as well as primary hepatocytes and dendritic cells in a receptor-mediated manner, therefore representing a useful model system for the study of HCV–host cell interaction.10, 21–26
HCV pseudotyped particles (HCVpp) represent another approach to study viral entry. Infectious HCVpp consist of unmodified HCV envelope glycoproteins E1 and E2 assembled onto retroviral or lentiviral core particles. HCVpp are produced by transfecting cells with expression vectors encoding the full-length E1/E2 polyprotein, retroviral or lentiviral core proteins, and a packaging-competent retro- or lentiviral genome carrying a marker gene. The presence of a green fluorescent protein or luciferase reporter gene packaged within these HCVpp allows reliable and fast determination of infectivity mediated by the envelope glycoproteins. HCVpp are infectious for certain cell lines of hepatocyte origin, principally Huh-7 cells, as well as for human primary hepatocytes.14, 15 Neutralization of HCVpp infectivity has been demonstrated for anti-E2 antibodies as well as sera from human and chimpanzees infected with HCV, but not sera from healthy controls.14, 15, 27–31
Vesicular stomatitis virus (VSV)/HCV pseudotypes expressing HCV E1 or E2 chimeric proteins containing transmembrane and cytoplasmic domains of the VSV G glycoprotein16, 17 have been developed as another HCV pseudotype model system to study HCV binding and entry. VSV/HCV pseudotypes infect human hepatoma cell lines, and sera from HCV-infected chimpanzees or humans neutralize the pseudotype virus infectivity.16, 32 In contrast to retroviral HCV pseudotypes exhibiting a marked tropism for liver-derived cell lines, the VSV/HCV pseudotype system is limited by background infectivity of VSV and infection of cell lines not derived from the liver. It is also not known whether the presence of VSV domains within the HCV envelope proteins may alter E1/E2 conformation and fusion properties.
Most recently, several laboratories succeeded in establishing the efficient production of infectious HCV particles using a unique clone derived from a viral isolate of a Japanese patient with fulminant hepatitis C (JFH-1).18–20 Successful infection of naïve Huh-7 and Huh-7–derived hepatoma cells with cell-culture–derived HCV (HCVcc) was demonstrated by detection of viral proteins and a highly reproducible time-dependent increase of viral RNA in infected cells.18–20 Virus production in Huh-7 cells was dependent on an active viral polymerase and expression of a functional viral envelope containing the HCV envelope glycoproteins E1 and E2.18–20 Inoculation of naïve chimpanzees with JFH-1 or chimeric J6/JFH-1–derived HCV particles synthesized in vitro resulted in viral infection in vivo,18, 33 demonstrating the biological significance of this model system. The ability to generate infectious HCVcc of different genotypes—such as the development of chimeric 1a/2a HCVcc or HCVcc derived from HCV genotype prototype 1a strain H7734—certainly improves the scope of the cell culture system for HCV infection.
Viral Determinants: Impact of Envelope Glycoproteins E1 and E2
The HCV genome encodes a single polyprotein of just above 3,000 amino acids that is processed into functional proteins by host and viral proteases. The structural proteins constitute the core protein, which is the major component of the viral nucleocapsid, and two envelope glycoproteins, E1 and E2. In analogy to other members of the Flaviviridae family such as dengue or tick-borne encephalitis virus, HCV is thought to adopt a classical icosahedral scaffold in which the two envelope glycoproteins are anchored to the host cell–derived double-layer lipid envelope.35 E1 and E2 are type I transmembrane glycoproteins, with N-terminal ectodomains and a short C-terminal transmembrane domain and assemble as noncovalent heterodimers. Underneath the membrane is the nucleocapsid composed of multiple copies of the core proteins in complex with the genomic RNA.35
The study of early steps of viral infection using the above-mentioned model systems has shown that both envelope glycoproteins E1 and E2 are essential for host cell entry. HCVpp assembled with either E1 or E2 glycoproteins demonstrated significantly less HCVpp infectivity of target cells than HCVpp containing both envelope glycoproteins.15 Furthermore, an in-frame deletion of the HCV envelope protein coding sequence in infectious HCV clones resulted in the abolishment of particle infectivity.18
Monoclonal or polyclonal antibodies targeting both linear and conformational epitopes of envelope glycoprotein E2 have been shown to inhibit cellular binding of HCV-LP binding, entry of HCVpp, and infection of HCVcc,14, 15, 18–20, 22–25, 31, 36–39 suggesting that envelope glycoprotein E2 plays a key role in host cell surface interaction. Within the E2 envelope glycoprotein sequence, hypervariable regions have been identified. These regions differ more than 80% among HCV genotypes as well as subtypes of the same genotype. The N-terminal 27 residues of E2 (aa 384-410) shows a very high degree of variation, and this portion of the sequence has been termed hypervariable region 1 (HVR-1).40 Deletion of HVR-1 has been shown to reduce HCVpp infectivity in cell culture,41, 42 supporting an important role of this domain in host cell entry. Moreover, antibodies that target regions within HVR1 have been shown to exhibit inhibition of cellular binding of recombinant E212, 43 and HCV-LP23, 24 as well as entry of HCVpp,14, 30 supporting the importance of HVR-1 in host cell recognition and attachment. Whether E1 directly interacts with cell surface molecules or its main function is related to proper folding and processing of E2 is unknown. Interestingly, antibodies targeting the N-terminal of E1 region have been shown to inhibit HCV-LP binding23, 25 as well as HCV infection of a B-cell–derived cell line,44 suggesting that E1–cell surface interaction may contribute to viral binding and entry.
Cellular Determinants: Receptors, Fusion and Signal Transduction
Several cell surface molecules—the tetraspanin CD81,11 scavenger receptor class B type I (SR-BI),12 heparan sulfate (HS),21 DC-SIGN/L-SIGN,45–47 and the low-density lipoprotein (LDL) receptor48 have been proposed to play a role in mediating HCV attachment and entry (Table 1).
|Cell surface molecule||Putative function during HCV infection||Tissue expression||References|
|DC-SIGN/L-SIGN||Capture/Binding||LSEC, Kupfer cell||68, 69|
|Highly sulfated HS||Capture/Binding||Hepatocyte||21, 65, 66|
|SR-BI||Binding/Entry||Hepatocyte, steroidogenic tissue, dendritic cell||10, 57, 62|
|CD81||Entry||Ubiquitous (except erythrocytes and platelets)||18, 50, 51|
Expression cloning using a cDNA library derived from a subclone of the human T cell lymphoma cell line Molt-4, which exhibits a high E2-binding capacity,43 allowed the identification of CD81 as an E2 binding molecule.11 The tetraspanin CD81 is a widely expressed 25-kd cell surface protein that is involved in pleiotropic activities such as cell adhesion, motility, metastasis, cell activation, and signal transduction.49 Anti-CD81 antibodies have been shown to inhibit HCVpp and HCVcc entry into Huh-7 hepatoma cells and human hepatocytes.14, 15, 18, 19, 50–52 Furthermore, silencing of CD81 expression in hepatoma cells by small interfering RNAs inhibited HCVpp entry and expression of CD81 in hepatoma cell lines that are resistant to HCVpp and HCVcc infection conferred susceptibility to HCV infection.41, 50, 51 Interestingly, recent studies using the HCVpp and HCVcc model system provided evidence that inhibition of viral entry by anti-CD81 antibodies appears to occur at a step post HCV attachment.50, 53 Taken together these findings indicate that CD81 plays an important role as an HCV cell entry receptor and probably functions at a post-binding step during the initiation of the infection.
However, the almost ubiquitous expression of CD81 and the ability of CD81 to bind E2 in species known to be refractory to HCV infection54 suggests that CD81 expression on target cells is not the sole determinant of HCV tissue and species specificity. Human hepatoma HepG2 cells that do not express endogenously CD81 but efficiently recognize E2 have been used to identify alternative cell surface molecules implicated in HCV binding and entry. Cross-linking studies using recombinant C-terminally truncated E2 as an HCV surrogate ligand isolated an 82-kd E2-binding protein on HepG2 cells identified as SR-BI.12 SR-BI is involved in bidirectional cholesterol transport at the cell membrane and can bind both native high-demsity lipoprotein (HDL) and LDL as well as modified lipoproteins such as oxidized LDL. SR-BI is highly expressed in liver and steroidogenic tissues55 as well as in human monocyte-derived dendritic cells but not on any other peripheral blood mononuclear cells.56 Antibodies directed against cell surface–expressed SR-BI have been shown to inhibit HCV-LP binding to primary hepatocytes10 as well as HCVpp entry.41 Furthermore, silencing of cellular SR-BI expression by small interfering RNAs markedly reduced HCVpp entry,57 although this reduction appeared to be HDL-dependent as shown by other investigators.58, 59 Finally, pre-incubation of HCVcc with oxidized LDL—a well-characterized SR-BI ligand—inhibited HCVcc infection in a dose-dependent manner.60 These data clearly indicate that SR-BI plays an important role in HCV attachment and entry.
Several studies have demonstrated that HCV particles isolated from patients are associated with plasma lipoproteins such as LDL, VLDL, and HDL.48, 61 The association of HCV with lipoproteins in the blood suggests that HCV might “hitch a ride” into hepatocytes by binding to lipoproteins. This hypothesis is supported by the observation that HDL and apolipoprotein C1 alone or associated with HDL were able to enhance entry of HCVpp.29, 58, 62 HDL-mediated enhancement of HCVpp entry was dependent on cellular SR-BI expression, suggesting a complex interplay between SR-BI, HDL, and HCV envelope glycoproteins for HCV entry.58, 62 Interestingly, there is no evidence for a direct interaction between HDL and HCVpp.58, 62 Because HCVpp infectivity was enhanced when HDL58, 62 or inhibited when oxidized LDL60 was added to HCVpp prebound to target cells, it is likely that interaction of lipoproteins with HCV may modify viral entry during steps after viral attachment.60 Further studies are needed to dissect the mechanisms of HCV-lipoprotein-scavenger receptor interaction.
Interestingly, many human cell lines co-expressing CD81 and SR-BI are non-permissive for HCVpp infection.15, 41, 51 This observation suggests that additional liver-specific cell surface molecules are required for HCV entry. Another cell surface molecule proposed to mediate tissue-specific attachment of the virion to hepatocytes is highly sulfated heparan sulfate. Glycosaminoglycan chains on cell surface proteoglycans have been shown to represent primary docking sites for the binding of various viruses to eukaryotic cells and vary with respect to their composition and quantity among different species, cell types, tissues, and cellular development stages.63 Liver-derived heparan sulfate is characterized by a unique composition of highly sulfated disaccharides.64 Envelope glycoprotein E2 has been shown to bind specifically to highly sulfated heparan sulfate expressed on the cell surface of human hepatoma cell lines.21 Incubation of serum-derived HCV or VSV/HCV pseudotypes with heparin—a structural homolog of highly sulfated heparan sulfate—markedly inhibited virus binding to target cells.65, 66 These observations are corroborated by recent studies showing that HCVcc binding or infection is dose-dependently inhibited by heparin or pre-treatment of cells with heparinases.53 Thus, a tissue- and species-specific heparan sulfate pattern—as shown for liver heparan sulfate—may contribute to the initiation of HCV infection and HCV cell tropism.21
Finally, the mannose binding C-type lectins DC-SIGN and L-SIGN have been shown to bind envelope glycoprotein E2 with high affinity.45–47 L-SIGN is highly expressed in liver sinusoidal endothelial cells (LSEC) but not in hepatocytes. DC-SIGN, is expressed in Kupffer cells, which are immobile liver macrophages localized close to LSEC and hepatocytes.67 L-SIGN and DC-SIGN have been shown to capture and transmit HCVpp.68, 69 Capture of circulating HCV particles by these cells may facilitate viral infection of hepatocytes and therefore may contribute to the pathogenesis of viral infection.
In summary, HCV envelope glycoproteins participate in a complex cascade of interactions with specific cell surface molecules resulting in viral entry. CD81, SR-BI, heparan sulfate, and DC-SIGN/L-SIGN have been shown to be important players in this complex dialog between virus and the host cell. Binding of HCV to these cell surface molecules might be the first step in a cascade of interactions that is required for efficient viral entry and initiation of infection (Fig. 1).
Knowledge about the steps following the first encounter of the HCV envelope with target cell surface membrane is still limited. Cell attachment of other members of the Flaviviridae family such as flaviviruses leads to endocytosis of bound virions.70 The mildly acidic pH in the late endosome provides an essential cue that triggers penetration and uncoating. Penetration of enveloped virus occurs by membrane fusion catalyzed by fusion peptides embedded in the viral envelope glycoproteins. The low pH in the late endosomes induces conformational changes in the envelope glycoproteins, which exposes the fusion peptide, leading to fusion between viral and endosomal membranes.8 Two classes of viral fusion proteins (types I and II) mediating entry of enveloped viruses have been defined. Type II fusion proteins occur in flaviviruses and alpha viruses. In the mature virion, the type II fusion protein exists as a metastable dimer that dissociates at the acidic pH in the endosome and is converted into a more stable trimeric conformation that destabilizes the target cell membrane and, finally, leads to the formation of a fusion pore.8, 71 Structural homology with fusion proteins from flaviviruses suggests that HCV envelope glycoproteins may belong to type II fusion proteins.35, 72 Although—in contrast to flaviviruses—HCV glycoproteins are not matured by a cellular endoprotease during their transport through the secretory pathway,35, 72–74 similar membrane fusion mechanisms may operate in HCV. This hypothesis is corroborated by the observation that HCVpp entry into Huh-7 cells is pH-dependent.14, 15, 75 Finally, HCVcc infection was markedly inhibited by agents preventing the acidification of endosomal compartments, suggesting that a pH-dependent membrane fusion process may be required for delivery of the HCV capsid into the host cell cytosol.76
In recent years, it has become clear that interaction of viral envelope glycoproteins with cell surface molecules leads to an intensive dialog between the incoming viruses and the host cell. Virus-activated cell signaling during the entry process facilitates viral uptake, appropriate intracellular targeting, and evasion strategies and neutralizes host defenses.77 Using gene expression profiling, a recent study has demonstrated evidence that binding of envelope glycoproteins to human hepatoma cells results in a cascade of intracellular signals modulating cellular gene expression, which may condition the cell for support of viral propagation.78
Because CD81, as well as other tetraspanin molecules, act as molecular facilitators for downstream intracellular signaling,79 interaction of HCV envelope glycoprotein E2 with CD81 on target cells may play a crucial role in cell signaling. Indeed, CD81 engagement by E2 inhibits natural killer (NK) cell activation,80, 81 activates T82 and B cells,83 and leads to hypermutation in the V(H) genes of B cells.84 These findings indicate that interaction of HCV with CD81 on the cell surface of T and B cells mediates not only viral attachment but also generates signals that may modulate host's innate or adaptive immune responses, which allow the virus to establish persistent infection.
Viral Entry: Target of Antibody-Mediated Virus Neutralization
Viral attachment and entry—representing the first encounter of the virus with the host cell—is a major target of adaptive host cell defences. Virus-specific neutralizing antibodies are defined mechanistically for their ability to block viral entry into cells and are an important defense mechanism for the control of viral spread. This observation is reflected by the fact that many successful antiviral vaccines are based on the induction of neutralizing antibodies. Neutralizing antibodies may target different stages of virus–host cell interactions, such as virus attachment/binding, virus–host membrane fusion, as well as post-entry processes including penetration and uncoating. Isolation and characterization of antibodies targeting distinct steps of viral entry is an important strategy for protection and provides a rational basis for HCV vaccine development.
Several observations suggest that antibody-mediated neutralization occurs during HCV infection in vivo. Using the chimpanzee model, antibodies with neutralizing properties have been described.85, 86 These antibodies were directed against epitopes in the E2 HVR-1 and appeared to be isolate-specific. Antibody-mediated neutralization has also been suggested by study of patients undergoing liver transplantation for HCV- and hepatitis B virus–related liver cirrhosis. Infusion of anti-HBs hyperimmune globulin containing anti-HCV appeared to reduce HCV infection in the transplanted liver.87 Finally, HCV-infected patients with primary antibody deficiencies have been reported to have accelerated rates of disease progression.88, 89 However, HCV infection is established despite the induction of an humoral immune response that is targeted against various epitopes of the HCV envelope glycoproteins.24, 27, 29, 30, 90
Initial functional studies analyzing the neutralizing antibody response during acute and chronic HCV infection using the mentioned surrogate model systems demonstrated a lack of neutralizing antibodies in most patients with acute HCV infection.24, 27, 30, 91 However, these studies were limited by the fact that the viral surrogate ligand was derived from a different isolate than the virus present in the infected patient, thus precluding the detection of isolate-specific antibodies. Functional studies using well-defined nosocomial or a single-source HCV outbreaks with a defined inoculum resulted in different findings: Two studies using the HCVpp model system demonstrated that neutralizing antibodies are induced in the early phase of infection and may play a role in control of viral infection28 or viral clearance (Pestka JM, Zeisel MB, Schürmann P, Cosset FL, Meisel H, Rispeter K, et al. Presentation O-03. 12th International Symposium on Hepatitis C Virus and Related Viruses, Montreal, Canada, October 2-6, 2005).
During the chronic phase of HCV infection, most HCV-infected patients develop high-titer and even cross-neutralizing antibodies.18, 24, 27, 30 Viral escape from antibody-mediated neutralization in these individuals may occur on several levels. First, the presence of HCV as a highly variable pool of rapidly mutating viral quasispecies in an individual patient may contribute to viral escape from antibody-mediated virus neutralization as shown for epitopes of HVR-1.24, 86 Second, the virus may have developed highly specific escape strategies to evade antibody-mediated neutralization. These strategies may include conformational masking of receptor binding sites after envelope–antibody interaction92 or alteration in envelope N-glycosylation motifs as described recently for human immunodeficiency virus.93 Another very interesting recent observation suggests that escape from neutralizing antibodies may also occur through HCV/HDL/SR-BI ternary interactions.94 Further studies using the novel tissue culture model systems and patient cohorts with well-defined viral isolates will allow one to address these questions in the future.
During the last few years, several viral epitopes targeted by neutralizing antibodies have been identified. These include epitopes of the E2 HVR-1 region (aa 384-410),14, 43, 62 an epitope adjacent to the N-terminal region HVR-1 region (aa 408-422),24, 36 the E2 CD81 binding region (aa 474-494 and aa 522-551),14, 36, 51 and conformational epitopes within glycoprotein E2.31, 38 It is conceivable that these epitopes may represent candidate targets for antibodies in passive immunoprophylaxis. In deed, two studies have demonstrated that monoclonal antibodies directed against conformational epitopes31 or epitope aa 412-423 exhibited broad cross-neutralizing activity among all major genotypes of HCVpp entry36 as well as HCVcc infectivity.95 Further studies in the chimpanzee model are needed to assess the relevance of these findings for antibody-mediated prevention of HCV infection in vivo.
Viral Entry: Implication for Prevention and Treatment of HCV Infection
In the past, the search for antiviral drugs was focused mainly on inhibition of HCV replication.2 Recent advances in the early steps of HCV–host cell interactions have provide the necessary opportunities to consider new approaches for antiviral therapy and vaccine development. Blocking of HCV binding, entry and post-entry processes serves as an attractive and novel antiviral target in vivo.
HCV-specific antibodies are usually detectable approximately 7 to 8 weeks after HCV infection.96 Passive immunization with anti-HCV immune globulin preparation might be useful in preventing or controlling HCV infection in particular in the setting of liver transplantation, which is invariably associated with re-infection of the graft. Immune globulin prepared from patients with chronic HCV infection can prevent hepatitis C in recipients when administrated before exposure to the virus, and such a protection has been linked to presence of antibodies that can neutralize HCVpp infectivity.97–99 However, it has to be mentioned that reinfection by homologous virus still occurs in HCV-infected chimpanzees with high levels of circulating anti-HCV antibodies.100 Nevertheless, the identification of monoclonal antibodies that neutralize HCV binding and entry in vitro by targeting conserved epitopes in HCV envelope glycoproteins provides a rational basis for the development of an antibody-based vaccine. Passive immunization with human neutralizing antibodies with well-defined epitope specificity alone or in combination with other neutralizing antibodies may be effective when the antibodies are present before infection and before virus diversification occurs. It is important to know that passive immunization with human monoclonal antibodies targeted conserved human immunodeficiency virus (HIV) envelope epitopes completely prevented HIV infection in primates (for review, see Ferrantelli and Ruprecht101).
Finally, the knowledge of HCV binding and entry processes offers another potential target for antiviral strategies such as (i) the use of inhibitors that block envelope glycoprotein/cellular receptor and co-receptor interactions, and (ii) inhibitors that inhibit viral envelope fusion processes. The systematic generation and screening of heparan sulfate–like molecules and semisynthetic derivatives is currently explored as an antiviral approach against dengue virus infection.102 Furthermore, peptides that mimic conserved regions of E2 interacting with cell entry receptors may provide an interesting approach to prevent HCV infection. Peptide-based entry inhibitors have been established for the treatment of HIV infection. Enfuvirtide, which blocks HIV fusion to host cells, is the first compound of this family approved for clinical use.103