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Tan SL, editor. Hepatitis C Viruses: Genomes and Molecular Biology. Norfolk (UK): Horizon Bioscience; 2006.

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Hepatitis C Viruses: Genomes and Molecular Biology.

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Chapter 4HCV Glycoproteins: Assembly of a Functional E1–E2 Heterodimer

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Abstract

The two HCV envelope glycoproteins E1 and E2 are released from HCV polyprotein by signal peptidase cleavages. These glycoproteins are type I transmembrane proteins with a highly glycosylated N-terminal ectodomain and a C-terminal hydrophobic anchor. After their synthesis, HCV glycoproteins E1 and E2 associate as a noncovalent heterodimer. The transmembrane domains of HCV envelope glycoproteins play a major role in E1–E2 heterodimer assembly and subcellular localization. The envelope glycoprotein complex E1–E2 has been proposed to be essential for HCV entry. However, for a long time, HCV entry studies have been limited by the lack of a robust cell culture system for HCV replication and viral particle production. Recently, a model mimicking the entry process of HCV lifecycle has been developed by pseudotyping retroviral particles with native HCV envelope glycoproteins, allowing the characterization of functional E1–E2 envelope glycoproteins. Here, we review our understanding to date on the assembly of the functional HCV glycoprotein heterodimer.

Introduction

As obligate intracellular parasites, all viruses have evolved ways of entering target cells to initiate replication and infection. The first step in virus entry is the recognition of host cells through cell surface receptor(s). This initial engagement can mediate attachment as well as act as a primer for subsequent conformational alteration, leading to virus entry into host cell. In many cases, interaction with a receptor is important for defining the tropism of a virus for a particular organism, tissue or cell type. Enveloped viruses possess a lipid bilayer that surrounds their nucleocapsid. The glycoproteins present in their envelope are involved in the receptor-binding step. After attachment, the entry of these viruses into cells requires the fusion of the viral and a cellular membrane by a process that is also driven by the viral envelope glycoproteins. To fulfill these functions, viral envelope glycoproteins have to adopt dramatically different conformations during the virus lifecycle. In addition, these conformational changes have to occur at a precise time of the virus lifecycle, and thus, have to be tightly modulated.

HCV encodes two envelope glycoproteins, named E1 and E2. For a long time, the lack of a cell culture system supporting efficient HCV replication and particle assembly has hampered the characterization of the envelope proteins present on the virion. Cell culture transient expression systems have allowed investigators to characterize the first steps in the biogenesis of HCV envelope glycoproteins (reviewed in: Op De Beeck et al., 2001). In addition, surrogate models have also been developed to study the entry steps of HCV lifecycle (reviewed in: Op De Beeck and Dubuisson, 2003). However, it is only recently that a model mimicking the entry process of HCV lifecycle has been developed. This has been achieved by pseudotyping retroviral particles with native HCV envelope glycoproteins (Bartosch et al., 2003b; Drummer et al., 2003; Hsu et al., 2003). This new tool allows, for the first time, the characterization of the assembly of functional HCV envelope glycoproteins.

Biogenesis of HCV Envelope Glycoproteins

Cleavage of HCV Glycoproteins from the Viral Polyprotein

As for the other members of the Flaviviridae family, the genome of HCV encodes a single polyprotein. This ~3010 amino acid polyprotein is processed by cellular (signal peptidase and signal peptide peptidase) and viral proteases (NS2-3 and NS3-4A) to generate at least 10 polypeptides (reviewed in: Penin et al., 2004). The nonstructural proteins are released from the polyprotein after cleavage by HCV proteases NS2-3 and NS3-4A, whereas the structural proteins are released by host endoplasmic reticulum (ER) signal peptidase(s) (Fig. 1)(reviewed in Reed and Rice, 2000). Further processing mediated by a signal peptide peptidase also occurs at the C-terminus of the capsid protein (McLauchlan et al., 2002). Most cleavages in the polyprotein precursor proceed to completion during or immediately after translation (Grakoui et al., 1993; Dubuisson et al., 1994; Lin et al., 1994; Mizushima et al., 1994; Dubuisson and Rice, 1996). Partial cleavages occur at the E2/p7 and p7/NS2 sites, leading to the production of an uncleaved E2p7NS2 molecule. While most of NS2 is progressively cleaved from the E2p7NS2 precursor, the cleavage between E2 and p7 does not change over time, at least for most HCV strains analyzed (Dubuisson, 2000). Thus, this results in cleavage products consisting of E2, E2p7, p7, and NS2.

Fig. 1. Processing of the N-terminal one-third of HCV polyprotein.

Fig. 1

Processing of the N-terminal one-third of HCV polyprotein. The arrows show host signal peptidase cleavages. Partial cleavages at E2/p7 and p7/NS2 sites are indicated by dotted arrows. Cleavage by the host cell signal peptide peptidase (SPP) is indicated (more...)

The sequences located immediately N-terminally of E2/p7 and p7/NS2 cleavage sites can efficiently function as signal peptides. Indeed, when fused to a reporter protein, the signal peptides of p7 and NS2 are efficiently cleaved (Carrère-Kremer et al., 2004). These data indicate that inefficiency of cleavage at E2/p7 and p7/NS2 sites is not due to the presence of suboptimal signal peptides. The p7 polypeptide is a polytopic membrane protein containing two transmembrane domains with both its N- and C-termini oriented toward the ER lumen (Fig. 1)(Carrère-Kremer et al., 2002). Interestingly, the presence of the first transmembrane domain of p7 reduces the efficiency of p7/NS2 cleavage (Carrère-Kremer et al., 2004). Sequence analyses and mutagenesis studies have also identified structural determinants responsible for the partial cleavage at both E2/p7 and p7/NS2 sites (Carrère-Kremer et al., 2004). In addition, the short distance between the cleavage site of E2/p7 or p7/NS2 and the predicted transmembrane α-helix located downstream of the cleavage sites might impose additional structural constraints to these cleavage sites (Fig. 1). Such constraints in the processing of a polyprotein precursor are likely essential for HCV to post-translationally regulate the kinetics and/or the level of expression of p7 as well as NS2 and E2 mature proteins.

The processing at the E2p7 site has been further explored. It has been reported to be more efficient in genotype 1b (strain BK) than in the genotype 1a (strain H77c) (Dubuisson et al., 1994; Lin et al., 1994). A sequence comparison of p7 signal peptides of these two viral strains has identified a difference of 3 amino acids and mutational analysis has shown that the V720L change in the H77c sequence substantially increases the efficiency of processing at the E2/p7 site (Isherwood and Patel, 2005). Although, when expressed alone, p7 protein has been shown to adopt a double membrane spanning topology with both extremities orientated luminally in the ER (Carrère-Kremer et al., 2002), the C-terminal part of E2p7 proteins has been found to be located in the cytosol (Isherwood and Patel, 2005). These data suggest that p7 can potentially adopt a dual transmembrane topology. It remains, however, to be shown whether an E2p7 with a cytosolic orientation of the C-terminus of p7 exists when this protein is expressed in the context of the polyprotein.

Since p7 and NS2 are not essential for HCV genomic replication (Lohmann et al., 1999; Blight et al., 2000), they will likely play their role in virion assembly, a process that is supposed to be tightly regulated. It has recently been shown that p7 reconstituted into artificial lipid membranes homo-oligomerizes and behaves as an ion channel protein (Griffin et al., 2003; Pavlovic et al., 2003; Premkumar et al., 2004). It is likely that, when bound to E2, p7 cannot oligomerize and function as an ion channel, and the existence of E2p7 would therefore reduce the amount of functional p7 molecules available. Production of precursors like E2p7NS2 and E2p7 might be a means to maintain p7 inactive during the phase of the accumulation of E2 molecules required for HCV envelope formation. Alternatively, such precursors might also control the temporal release of E2 and NS2.

Glycosylation of HCV Envelope Glycoproteins

N-linked glycosylation is one of the most common types of protein modification, and it occurs by the transfer of an oligosaccharide from a lipid intermediate to an Asn residue in the consensus sequence Asn-X-Thr/Ser of a nascent protein, where X is any amino acid except Pro (Kornfeld and Kornfeld, 1985; Gavel and von Heijne, 1990). The addition of this glycan is catalyzed by the oligosaccharyltransferase, which is closely associated with the translocon through which the nascent peptidic chains emerge in the ER lumen (Silberstein and Gilmore, 1996). However, not every tripeptide sequence in a protein sequence is used for carbohydrate addition (Gavel and von Heijne, 1990). In the early secretory pathway, the glycans play a role in protein folding, quality control and certain sorting events. Viral envelope proteins usually contain N-linked glycans that can play a major role in their folding, in their entry functions or in modulating the immune response (Hebert et al., 1997; Ohuchi et al., 1997a; Ohuchi et al., 1997b; van Kooyk and Geijtenbeek, 2003; von Messling and Cattaneo, 2003; Wei et al., 2003).

The ectodomains of HCV envelope glycoproteins E1 and E2 are highly modified by N-linked glycans. E1 and E2 possess up to 6 and 11 potential glycosylation sites, respectively (Fig. 2). Sequence analyses of E1 indicate that 5 potential N-glycosylation sites are strongly conserved among HCV genotypes (Goffard and Dubuisson, 2003; Zhang et al., 2004b). However, in one case the presence of a proline residue immediately downstream the glycosylation site is unfavorable for glycosylation, and it has been confirmed experimentally that this site is not glycosylated (Meunier et al., 1999). Interestingly, the glycosylation site of E1 at position 250 is poorly conserved; this site is indeed only observed in genotypes 1b and 6 (Fig. 2)(Goffard and Dubuisson, 2003). Most E2 glycosylation sites are also well conserved. Indeed, global sequence analyses of potential glycosylation sites in E2 indicate that nine of the eleven sites are strongly conserved (Goffard and Dubuisson, 2003; Zhang et al., 2004b). The two remaining sites, N5 and N7, show conservation levels of 75% and 89%, respectively (Goffard and Dubuisson, 2003).

Fig. 2. Schematic representation of E1 and E2 features.

Fig. 2

Schematic representation of E1 and E2 features. Positions of N-linked glycans are indicated as an N followed by a number related to the relative position of the potential glycosylation site in each glycoprotein. The numbers correspond to the positions (more...)

Mutants of E1 and E2 have been produced to characterize the glycosylation of these proteins (Meunier et al., 1999; Nakano et al., 1999; Slater-Handshy et al., 2004; Goffard et al., 2005). In the context of the H strain, the 4 potential glycosylation sites of E1 were shown to be occupied by glycans (Meunier et al., 1999; Goffard et al., 2005). In the case of E2, a first study has shown that mutation of some glycosylation sites in the context of a truncated form of E2 alters the recognition by sera from HCV patients (Nakano et al., 1999); however, these mutants were not characterized in terms of glycosylation and no clear conclusion can be drawn from this study. More recently, glycosylation mutants have been produced in the context of a truncated form of E2 ending at position 660 (Slater-Handshy et al., 2004). The E2 sequence of HCV isolate used in this study contains 10 instead of 11 potential glycosylation sites, the site N5 at position 476 being missing (Fig. 2). Interestingly, the last two glycosylation sites, N10 and N11, were not occupied in E2660 (Slater-Handshy et al., 2004). However, at least one of these sites was occupied in the context of full-length E2. A more recent mutagenesis study, in the context of an E2 glycoprotein containing 11 potential glycosylation sites, has shown that all the sites are occupied by glycans (Goffard et al., 2005). In this case, E2 was expressed as a polyprotein containing full-length E1 and E2. Altogether, these data indicate that full-length and truncated forms of E2 can have different properties.

The addition of the glycan precursor is catalyzed by the oligosaccharyltransferase, and this enzyme is thought to have access only to nascent chains as they emerge from the ribosome at the luminal face of the rough ER (Silberstein and Gilmore, 1996). The glycosylation process of HCV envelope glycoprotein E1 has been analyzed in the context of a Man-P-Dol-deficient cell line (B3F7) and it has been shown to occur post-translationally (Duvet et al., 2002), indicating that the oligosaccharyltransferase has also access to the E1 glycoprotein for more than an hour after its translation. A characterization of HCV glycoprotein E1 has also shown that, in the absence of E2, different glycoforms of E1 are produced and the glycosylation of E1 is improved by co-expression of E2 in cis (Dubuisson et al., 2000).

Folding of HCV Envelope Glycoproteins

HCV envelope glycoproteins have been shown to assemble as a noncovalent E1E2 heterodimer (Deleersnyder et al., 1997). However, at least in heterologous expression systems, HCV envelope glycoproteins have a tendency to also form misfolded aggregates stabilized by disulfide bonds (reviewed in (Dubuisson, 2000)). Analyses of HCV envelope glycoproteins with conformation-sensitive antibodies are therefore necessary to discriminate noncovalent heterodimers from misfolded complexes (Deleersnyder et al., 1997; Cocquerel et al., 2003b). Alternatively, such discrimination can also be made by analyzing disulfide-bond formation by migrating HCV envelope glycoproteins on SDS-PAGE under non-reducing conditions (Dubuisson and Rice, 1996; Brazzoli et al., 2005). Analyses of the formation of conformation-dependent epitopes and disulfide-bond formation indicate that folding of HCV envelope glycoproteins is a slow process (Deleersnyder et al., 1997; Dubuisson and Rice, 1996; Duvet et al., 1998; Brazzoli et al., 2005). Interestingly, the folding of E1 has been shown to be dependent on the co-expression of E2 (Michalak et al., 1997; Patel et al., 2001). In addition, it has also been shown that the folding of E2 is also dependent on the co-expression of E1 (Cocquerel et al., 2003a; Brazzoli et al., 2005). Altogether, these observations indicate that HCV envelope glycoproteins cooperate for the formation of a functional complex. These observations also indicate that, although some degree of folding can be observed in E2 expressed alone (Michalak et al., 1997; Cocquerel et al., 2003a), both glycoproteins need to be co-expressed to analyze their functional properties.

During their folding, HCV envelope glycoproteins have been shown to interact with calnexin (Dubuisson and Rice, 1996; Choukhi et al., 1998; Merola et al., 2001; Brazzoli et al., 2005), a lectin-like ER chaperone, which shows an affinity for monoglucosylated N-linked oligosaccharides (Trombetta and Helenius, 1998). Both E1 and E2 have been found to associate rapidly with calnexin and dissociate slowly, suggesting a role of this chaperone in the folding of HCV envelope glycoproteins (Dubuisson and Rice, 1996; Choukhi et al., 1998; Merola et al., 2001). However, more recent data suggest that only E1 interacts with calnexin (Brazzoli et al., 2005). Differences in the cell lines used and/or in the levels of expression of the envelope glycoproteins might potentially explain these discrepancies. Further experiments in cell cultures infected with native HCV particles will be needed to confirm the involvement of calnexin in the folding E2.

The presence of glycans on HCV envelope glycoproteins can potentially affect their folding either directly or through interaction with calnexin. Site-directed mutagenesis studies have indeed shown that the absence of some glycans in E1 (N1 and N4) and E2 (N8 and N10) leads to misfolding of HCV envelope glycoproteins (Fig. 2)(Meunier et al., 1999; Goffard et al., 2005). This alteration in folding was not due to the lack of interaction of HCV envelope glycoproteins with calnexin, suggesting that the mutations would rather have a direct effect on protein folding. The presence of a large polar saccharide is indeed known to affect the folding at least locally by orienting polypeptide segments toward the surface of protein domains (Imperiali and O’Connor, 1999; Wormald and Dwek, 1999).

Involvement of the Transmembrane Domains in the Biogenesis of E1E2 Heterodimer

Membrane Anchor and Signal Sequence

Due to their resistance to alkaline or salt extraction, HCV envelope glycoproteins have been confirmed to be membrane associated proteins (Ralston et al., 1993; Cocquerel et al., 2001). In addition, deletion of the C-terminal hydrophobic regions of these proteins leads to their secretion, indicating that these regions are involved in membrane anchoring (Michalak et al., 1997). Sequence analysis of a large number of HCV isolates has shown that the C-termini of E1 and E2 contain hydrophobic sequences that are less than 30 amino acid residues long (Fig. 2)(Cocquerel et al., 2000). As in other viruses of the Flaviviridae family, these regions are composed of two stretches of hydrophobic residues separated by a short segment containing at least one fully conserved positively charged residue (Cocquerel et al., 2000). Interestingly, when fused to a reporter protein the second hydrophobic stretch functions as a signal sequence (Cocquerel et al., 2002), which is in agreement with the observation that HCV envelope glycoproteins are released from the polyprotein precursor after cleavage by host signal peptidase(s) (Dubuisson et al., 2002). It is worth noting that in the context of HCV polyprotein, only the sequence located at the C-terminus of the immature form of the capsid protein is a true signal peptide that will interact with the signal recognition particle (Santolini et al., 1994). The sequence present at the C-terminus of E1 and E2 do not interact with the signal recognition particle, and they should be called signals of reinitiation of translocation (Fig. 1). Deletion of these signals leads to the secretion of E1 and E2, indicating that these signals are involved in their membrane anchoring (Cocquerel et al., 2000).

ER Retention Function

HCV envelope glycoproteins are retained in the ER (Dubuisson et al., 1994; Deleersnyder et al., 1997; Duvet et al., 1998), and ER retention signals are present in the transmembrane domains of E1 and E2 (Cocquerel et al., 1998; Cocquerel et al., 1999). In addition, the charged residues of the transmembrane domains of E1 (Lys) and E2 (Asp and Arg) play a key role in the ER retention of these glycoproteins (Cocquerel et al., 2000). It has been proposed that an additional ER retention signal might also be present in the ectodomain of E1 (Mottola et al., 2000). Interestingly, in some conditions of overexpression a small fraction of HCV envelope glycoproteins has been shown to accumulate at the plasma membrane (Bartosch et al., 2003b; Drummer et al., 2003; Hsu et al., 2003; Op De Beeck et al., 2004). Cell surface expression of E1 and E2 is likely due to the accumulation of small amounts of glycoproteins escaping the ER-retention machinery, due to saturation of this mechanism.

Role in Heterodimerization

In addition to their anchoring, signal sequence and ER retention functions, the transmembrane domains of HCV envelope glycoproteins have also been shown to play a major role in the assembly of E1E2 heterodimer. Indeed, deletion of the transmembrane domain of E2 or its replacement by the anchor signal of another protein abolishes the formation of E1E2 heterodimer (Selby et al., 1994; Michalak et al., 1997; Cocquerel et al., 1998; Patel et al., 2001). Other studies by site-directed mutagenesis or alanine scanning insertion mutagenesis (Cocquerel et al., 2000; Op De Beeck et al., 2000) have confirmed that the transmembrane domains of E1 and E2 play a direct role in E1E2 assembly. In addition, alanine scanning insertion mutagenesis allowed to identify two distinct segments in the transmembrane domain of E1 and one in the transmembrane domain of E2 that were specifically involved in E1E2 assembly (Fig. 2). Interestingly, at least one region located outside of the transmembrane domains has also been shown to be involved in heterodimerization (Drummer and Poumbourios, 2004).

Topological Change in the Transmembrane Domain of HCV Glycoproteins

The topology of the transmembrane domain of HCV envelope glycoproteins has given rise to some controversy. Indeed the presence of a first hydrophobic stretch and a signal sequence function separated by charged residues in the transmembrane domains of E1 and E2 has suggested that they might be composed of two membrane spanning segments with the charged residues facing the cytosol (Charloteaux et al., 2002). This type of organization has been observed in the C-terminal region of the envelope glycoprotein E2 of the alphaviruses as well as for the envelope proteins of the flaviviruses (Strauss and Strauss, 1994; Op De Beeck et al., 2003; Zhang et al., 2003). However sequence analysis and data of alanine scanning insertion mutagenesis were in favor of a single spanning topology of E1 and E2 transmembrane domain (Cocquerel et al., 2000; Op De Beeck et al., 2000). A study of the topology of the transmembrane domains of HCV envelope proteins has been performed by determining the accessibility of their N- and C-termini in selectively permeabilized cells (Cocquerel et al., 2002). This work has shown that before signal sequence cleavage at their C-terminus, the transmembrane domains form a hairpin structure (Fig. 1). However, after cleavage between E1 and E2 or between E2 and p7, the second C-terminal hydrophobic stretch is reoriented towards the cytosol, leading to the formation of a single membrane-spanning domain. Here again, the charged residues located in the middle of the transmembrane domains were shown to play a crucial role in their structural dynamics (Cocquerel et al., 2002).

Role of HCV Envelope Glycoproteins in Virus Entry

For most viruses, entry into the cytosol is a multistep process, during which the host cell assists the incoming virus. Viruses first attach themselves to components of the plasma membrane, which they use as non-specific attachment factors or as specific cell surface receptors. Viral attachment is mediated by the binding of a protein present at the surface of the virion to a molecule on the cell surface acting as a virus receptor. The envelope glycoprotein complex E1E2 is the viral component thought to be present at the surface of HCV particles and it is therefore the obvious candidate ligand for cellular receptors. Receptor binding can activate cellular endocytic pathways through which viruses are internalized in endosomes. When they reach the appropriate intracellular location, viruses are activated for penetration by cellular signals and make their way through the membrane of the endosome, or through the plasma membrane for those that do not enter by endocytosis. Enveloped viruses fuse their lipid envelope with the plasma membrane or the membrane of an endosome, resulting in the release of the nucleocapsid into the cytosol.

Models to Study HCV Entry

In the absence of a robust cell culture system to amplify HCV, several models have been developed to study HCV entry. In a first approach, a soluble form of HCV glycoprotein E2 has been used to identify cell surface proteins potentially involved in HCV entry (Rosa et al., 1996; Pileri et al., 1998). Although this approach is potentially interesting in protein-protein interactions studies, it cannot be used to study the entire entry process. In addition, as discussed above, due to their cooperative role in folding, both glycoproteins need to be co-expressed to analyze their functional properties. To study the role of E1E2 envelope glycoproteins in HCV entry, several surrogate models of HCV particles have therefore been developed. As a first approach, virus-like particles have been produced in insect cells infected by a recombinant baculovirus containing the cDNA of HCV structural proteins (Baumert et al., 1998). However these particles are not infectious and they are retained in an intracellular compartment. It is therefore difficult to evaluate how close these virus-like particles are to native virion. In addition, due to the absence of infectivity, these particles cannot be used to study the fusion process. Another approach to study HCV entry has been to produce virosomes by incorporating E1E2 heterodimers into liposomes (Lambot et al., 2002). These virosomes can be used to study the interactions between E1E2 heterodimers and cell surface receptors. However, it has not been shown whether the envelope glycoproteins incorporated into these liposomes can induce fusion.

Other models have been based on pseudotyping of viral vectors. The first model that has been developed was based on vesicular stomatitis virus (VSV) pseudotyped with modified E1 and/or E2 glycoproteins (Lagging et al., 1998; Matsuura et al., 2001). In these particles, the transmembrane domains of HCV envelope glycoproteins have been replaced by the transmembrane domain and cytoplasmic tail of the VSV envelope glycoprotein G. This allows the export of HCV envelope glycoproteins to the cell surface (Takikawa et al., 2000). However, some doubts have been raised on the infectivity of such VSV pseudotyped particles (Buonocore et al., 2002). In addition, replacement of HCV envelope glycoproteins has been shown to alter their entry function (Hsu et al., 2003).

More recently, retroviruses have also been used to produce pseudotyped particles containing HCV envelope glycoproteins (Bartosch et al., 2003b; Drummer et al., 2003; Hsu et al., 2003). Murine leukemia virus (MLV) or human immunodeficiency virus (HIV) vectors were used. Retroviruses are indeed well known to be able to incorporate in their envelope a variety of cellular and viral glycoproteins (Ott, 1997; Sandrin et al., 2002). In addition, they can easily package and integrate genetic markers into host cell DNA (Negre et al., 2002). All these properties were exploited to produce viral pseudoparticles expressing E1E2 at their surface and packaging a reporter gene that allows to monitor viral infection of the target cell. HCV pseudoparticles (HCVpp) are produced by transfecting 293T cells with three expression vectors encoding the E1E2 polyprotein, the retroviral core proteins and a packaging-competent retrovirus-derived genome containing a marker gene (Fig. 3). Because MLV and HIV are supposed to assemble at the plasma membrane and HCV glycoproteins are retained in the ER, a first approach has been to modify the transmembrane domains of E1 and E2 to re-address them at the plasma membrane (Hsu et al., 2003; Pohlmann et al., 2003). However pseudoparticles bearing such modified HCV envelope glycoproteins were not infectious. Surprisingly, in the absence of any modification of HCV envelope glycoproteins, infectious pseudoparticles were produced (Bartosch et al., 2003b; Drummer et al., 2003; Hsu et al., 2003). Interestingly, due to saturation of the ER retention machinery, the cells used to produce HCVpp were shown to express a small fraction of HCV envelope glycoproteins at the plasma membrane (Bartosch et al., 2003b; Drummer et al., 2003; Hsu et al., 2003; Op De Beeck et al., 2004). This accumulation at the plasma membrane might therefore be sufficient to incorporate native HCV envelope glycoproteins into retroviral pseudotyped particles.

Fig. 3. Production of HCV pseudoparticules (HCVpp).

Fig. 3

Production of HCV pseudoparticules (HCVpp). For the production of HCVpp, human embryo kidney cells 293T are transfected with three expression vectors. The first vector encodes retroviral Gag and Pol proteins. Gag proteins are responsible for particle (more...)

The data that have been accumulated on these pseudoparticles strongly suggest that they mimic the early steps of HCV infection. Indeed, they exhibit a preferential tropism for hepatic cells and they are specifically neutralized by anti-E2 monoclonal antibodies as well as sera from HCV-infected patients (Bartosch et al., 2003b; Hsu et al., 2003; Op De Beeck et al., 2004). These HCVpp therefore represent the best tool available to study functional HCV envelope glycoproteins. An analysis of the glycoproteins associated with HCVpp has shown the heterogeneous nature of E1 and E2 incorporated into HCVpp (Flint et al., 2004). This highlights the difficulty in identifying forms of the HCV glycoproteins that initiate infection. However, characterization of HCVpp envelope glycoproteins with conformation-sensitive neutralizing monoclonal antibodies has shown that the functional unit is a noncovalent E1E2 heterodimer (Op De Beeck et al., 2004). In addition, coexpression of both envelope glycoproteins has been shown to be necessary to produce infectious pseudoparticles (Bartosch et al., 2003b), confirming that only the E1E2 heterodimer is functional.

HCV Receptors

As a first approach to identify potential HCV receptor(s), a soluble form of HCV glycoprotein E2 has been used. This allowed to identify the CD81 tetraspanin (Levy and Shoham, 2005) as a putative receptor for HCV (Pileri et al., 1998). A very similar approach identified the scavenger receptor class B type I (SR-BI) (Scarselli et al., 2002), a high-density lipoprotein (HDL)-binding molecule (Connelly and Williams, 2004), and the mannose binding lectins DC-SIGN and L-SIGN (van Kooyk and Geijtenbeek, 2003) as additional candidate receptors for HCV (Gardner et al., 2003; Lozach et al., 2003; Pohlmann et al., 2003; Ludwig et al., 2004). Heparan sulfate has also been shown to interact with HCV glycoprotein E2, suggesting that this type of molecule can play a role in HCV entry (Barth et al., 2003). An approach using virus-like particles produced in insect cells has led to the identification of the asialoglycoprotein receptor as another candidate receptor for HCV (Saunier et al., 2003). Finally, because of the physical association of HCV with low- or very-low-density lipoproteins (LDL or VLDL) in serum, the LDL receptor has also been proposed as another candidate receptor for HCV. (Agnello et al., 1999; Monazahian et al., 1999).

A number of cell-surface molecules bind viral envelope glycoproteins without mediating entry, and validation of a viral receptor or co-receptor requires proof that the putative receptor is necessary for infection. This is not easy for HCV due to the absence of a robust cell culture system to amplify this virus. The recent development of HCVpp has allowed to further investigate the role of candidate receptors in virus entry. Among all the candidate receptors, only CD81 and SR-BI have been shown to play a direct role in HCVpp entry. Indeed, antibodies directed against CD81 or SR-BI as well as siRNA targeting these receptors reduce HCVpp infectivity (Bartosch et al., 2003b; Bartosch et al., 2003c; Hsu et al., 2003; Cormier et al., 2004b; Zhang et al., 2004a; Lavillette et al., 2005b). A soluble domain of CD81 is also able to compete with HCVpp infectivity (Bartosch et al., 2003b; Hsu et al., 2003). In addition, HDL, the natural ligands of SR-BI, are able to markedly enhance HCVpp entry (Meunier et al., 2005; Voisset et al., 2005). This HDL-mediated enhancement of HCVpp entry involves a complex interplay between SR-BI, HDL and HCV envelope glycoproteins (Voisset et al., 2005). Interestingly, the involvement of CD81 and SR-BI in HCVpp entry seems to be conserved among all the HCV genotypes (McKeating et al., 2004; Lavillette et al., 2005b).

Interactions between viral envelope glycoproteins and potential receptors can have other consequences than virus entry. It has been shown that intracellular interaction between HCV envelope glycoproteins and CD81 can lead to secretion of exosomes containing E1 and E2 glycoproteins (Masciopinto et al., 2004). Interestingly, a soluble form of E2 is also able to bind CD81 at the surface of natural killer cells, and this interaction inhibits cytotoxicity and cytokine production by these cells (Crotta et al., 2002; Tseng and Klimpel, 2002). Binding of a soluble form of E2 can also provide a co-stimulatory signal for T cells (Wack et al., 2001; Soldaini et al., 2003;) and up-regulate matrix metalloproteinase-2 in human hepatic stellate cells (Mazzocca et al., 2005). It remains however to be determined whether HCV glycoprotein expressed in the context of native particles will have the same effects on cell functions.

HCVpp have also been used to investigate the role of other candidate receptors in HCV entry. HCVpp as well as native HCV particles have been shown to bind to cells expressing L-SIGN and DC-SIGN (Gardner et al., 2003; Pohlmann et al., 2003; Lozach et al., 2004). Although these molecules are not expressed on hepatocytes, HCV interactions with L-SIGN and DC-SIGN may contribute to establishment or persistence of infection both by the capture and delivery of virus to the liver and by modulating dendritic cell functions as recently suggested (Cormier et al., 2004a; Lozach et al., 2004). Finally, there is no clear evidence that the LDL receptor is a major receptor for HCVpp (Bartosch et al., 2003b).

Interestingly, all the cells permissive to HCVpp co-express CD81 and SR-BI and are of liver origin (Bartosch et al., 2003c; Hsu et al., 2003; Zhang et al., 2004a). However, there are some other cell lines coexpressing CD81 and SR-BI that are non-permissive to infection and which are of non-hepatic origin. These results suggest that additional molecule(s), expressed in hepatic cells only, are necessary for HCV entry. Further investigations with HCVpp should allow to identify such molecule(s).

Functional Regions of HCV Envelope Glycoproteins

HCVpp have been used to investigate the functional role of some regions of HCV envelope glycoproteins in virus entry. Mutagenesis studies of the transmembrane domains of HCV envelope glycoproteins have shown that some mutations can affect the entry function of HCVpp without alteration in the biogenesis of E1E2 heterodimer and their incorporation into HCVpp (Ciczora Y, Callens N, Montpellier C, Bartosch B, Cosset FL, Op De Beeck A, Dubuisson J, unpublished data). This suggests that in addition to their role in E1E2 heterodimerization, the transmembrane domains of HCV glycoproteins might play a role in coordinating protein reorganization for the fusion process to occur.

Studies of E2-CD81 interactions and identification of epitopes recognized by antibodies that inhibit these interactions suggest that the CD81-binding region consists of discrete segments of E2 that are rearranged within the same domain during E2 folding (Flint et al., 1999a; Forns et al., 2000a; Yagnik et al., 2000; Owsianka et al., 2001; Clayton et al., 2002; Hsu et al., 2003). Besides this putative binding region, the hypervariable region 1 (HVR1)(Weiner et al., 1991), a 27-amino acid long segment found at the N-terminus of E2 (Fig. 2), has also been suggested to play a role in cell attachment (Penin et al., 2001; Scarselli et al., 2002). This region evolves rapidly in infected individuals, suggesting that it is under strong immune pressure (reviewed in Mondelli et al., 2003). Although an HCV clone lacking HVR1 was shown to be infectious in chimpanzee, this mutant virus was attenuated, suggesting that HVR1 plays a facilitating role in HCV infectivity (Forns et al., 2000b). In addition, deletion of HVR1 reduces HCVpp infectivity (Bartosch et al., 2003c) and abolishes HDL-mediated enhancement of HCVpp infectivity (Voisset et al., 2005). Despite strong amino acid sequence variability related to strong pressure towards change, the chemicophysical properties and conformation of HVR1 are highly conserved, and HVR1 is a globally basic stretch, with basic residues located at specific sequence positions. Functional studies of HCVpp containing mutations in HVR1 indicate that infectivity increases with the number of basic residues in HVR1 (Callens N, Ciczora Y, Bartosch B, Vu-Dac N, Cosset FL, Pawlotsky JM, Penin F, Dubuisson J, unpublished data). In addition, a shift in position of some charged residues modulates infectivity. These data suggest that HVR1 is a region involved in interaction with a host molecule involved in HCV entry. However, it remains to be determined whether SR-BI or another putative receptor is involved in this interaction.

HCV envelope glycoproteins are highly glycosylated and some maturation of these glycans has been observed on HCV envelope glycoproteins associated with HCVpp (Flint et al., 2004; Lozach et al., 2004; Op De Beeck et al., 2004). Mutation of some glycosylation sites in HCV envelope glycoproteins can reduce or abolish HCVpp infectivity without apparently affecting folding and incorporation of the glycoproteins into the particles (Goffard et al., 2005). N-linked glycans at position N2 and N4 of E2 have indeed been shown to be essential for the entry functions of HCV envelope glycoproteins (Fig. 2). In addition, some other glycans (N2 of E1 and N5, N6 and N11 of E2) can also modulate HCVpp entry. Further studies will be necessary to determine whether these mutations affect receptor binding or the fusion properties of HCV envelope glycoproteins.

Mechanisms of HCV Entry

Virus attachment to receptors initiates a series of events that lead to virus entry. For enveloped viruses, the entry process is controlled by viral surface glycoproteins that undergo triggered conformational changes from a metastable state to a lower energy state. This structural change leads to the exposure of a buried functional element, named the fusion peptide and is believed to provide the energy required for the merging of the lipid bilayers (reviewed in Colman and Lawrence, 2003). So far, viral fusion proteins have been shown to fall into two different structural classes designated as class I and II (reviewed in Earp et al., 2005). Class I fusion proteins possess N-terminal or N-proximal fusion peptides, and they are synthesized as a precursor that is cleaved into two subunits by host cell proteases. In some cases (e.g., influenza HA), the two subunits remain associated through a disulfide bond, whereas in others (e.g. HIV Env) the two subunits remain associated through noncovalent interactions. The proteolytic processing event creates the metastable state of the fusion protein (Colman and Lawrence, 2003). In their native metastable conformation, class I fusion proteins form trimeric spikes at the surface of the virions with the fusion subunit being highly helical. Upon a fusion trigger event (receptor binding at the cell surface or low pH in endosomes), the trimeric proteins transiently form an extended conformation allowing the hydrophobic fusion peptide to insert into the target membrane. Protein refolding leads then to the formation of very stable trimeric structures in which both the N-proximal fusion peptide and the C-proximal membrane anchor are juxtaposed at the same end to allow virus and cell membrane connection and hemifusion (reviewed in Colman and Lawrence, 2003).

Class II viral fusion proteins have a completely different structure. They are predominantly non-helical, instead having a beta-sheet type structure; they are not cleaved during biosynthesis; and they possess an internal fusion peptide with a loop conformation (reviewed in Earp et al., 2005). The proteins are oriented parallel to the membrane, and they have a three-domain architecture with domain I beginning at the N-terminus, domain II containing the internal fusion loop, and domain III being at the C-terminus. In addition, class II fusion proteins are synthesized as a complex with a second membrane glycoprotein (prM for flaviviruses; pE2 for alphaviruses). Newly synthesized E and prM proteins of the tick borne encephalitis virus associate to form noncovalent heterodimers (Fig. 4) that are incorporated into immature virions by budding into the ER lumen (Allison et al., 1995; Mackenzie and Westaway, 2001). These particles are then transported through the secretory pathway and shortly before release from the cell, the activation of the fusogenic potential occurs by the cleavage of the accessory protein prM by a cellular furin protease in the trans-Golgi network (Stadler et al., 1997). After prM cleavage, the E protein exists as a metastable homodimers at the virion surface. The ectodomains of the dimers are orientated antiparallel to one another (Rey et al., 1995; Lescar et al., 2001; Modis et al., 2003). The architecture of the alphavirus Semliki Forest virus spike is similar to that of tick borne encephalitis virus E, but in this case, the metastable oligomer is a heterodimer of the fusion protein E1 and the companion protein E2 with an associated small protein E3 (Lescar et al., 2001). In addition, contrary to flaviviruses, alphaviruses have been shown to bud from the plasma membrane.

Fig. 4. Comparison of flavivirus and hepacivirus envelope proteins.

Fig. 4

Comparison of flavivirus and hepacivirus envelope proteins. In the Flaviviridae family, class II fusion proteins (depicted in light grey) have been described in the flaviviruses (E protein of tick born encephalitis and dengue viruses). They are synthesized (more...)

Both alphaviruses and flaviviruses enter target cells by receptor-mediated endocytosis. The receptor recognition function is carried by the fusion protein itself for the flaviviruses (E) and by the companion protein (E2) for the alphaviruses. Exposure to the acidic pH of the endosomes triggers a major conformational change of the envelope involving dissociation of the native homodimer (for flaviviruses) or heterodimer (for alphaviruses) and the irreversible formation of homotrimers of the fusion proteins (Earp et al., 2005; Mukhopadhyay et al., 2005).

Based on its classification in the Flaviviridae family, HCV envelope has been proposed to contain a class II fusion protein (Yagnik et al., 2000). As found in the case of alphaviruses and flaviruses, HCVpp entry is pH dependent (Bartosch et al., 2003c; Hsu et al., 2003). These observations indicate that HCV may enter the cells through endocytosis. The cell surface receptor(s) recognized by HCV should therefore traffic cell-bound virions to endosomal compartments. However, characterization of the route of HCV entry needs further investigations. Contrary to what is observed for other class II envelope proteins, there is no evidence that HCV envelope glycoproteins are matured by a cellular endoprotease during their transport through the secretory pathway (Op De Beeck et al., 2004). In addition, HCV envelope glycoproteins are highly glycosylated, whereas other described class II envelope proteins contain a very low number of glycans (Fig. 4). Interestingly, some of the glycans present on HCV envelope glycoproteins seem to be involved in controlling HCV entry (Goffard et al., 2005).

There remains some controversy on the identity of HCV fusion protein. It has been proposed that E1 might be a good candidate because sequence analyses suggest that it might contain a putative fusion peptide in its ectodomain (Flint et al., 1999b; Garry and Dash, 2003). On the other hand, potential structural homology with other class II fusion proteins suggests that E2 could be the fusion protein (Yagnik et al., 2000). Mutagenesis studies in the putative fusion peptides of the envelope glycoproteins associated with HCVpp as described for the flavivirus envelope protein E (Allison et al., 2001), should be helpful for further characterization of HCV fusion protein. In addition, a high-resolution structure of HCV envelope glycoproteins would also help understanding the fusion mechanism of the virus.

Inhibition of HCV Envelope Glycoprotein Functions by Neutralizing Antibodies

Because they are exposed at the surface of the virion, the envelope proteins are targets of neutralizing antibodies. These antibodies block a viral infection by inhibiting virion binding or membrane fusion. Understanding the mechanisms of neutralization needs therefore a good knowledge of the mechanism of entry. The role of neutralizing antibodies in HCV infection and disease progression remained unclear for a long time, largely because of the lack of assays to measure and quantify their activity. Previous experiments showed that serum from a chronically infected patient could neutralize HCV infectivity in a chimpanzee model, giving evidence for antibody-mediated neutralization of HCV (Farci et al., 1994). Neutralizing antibodies could also be identified by their ability to prevent HCV replication in a lymphoid cell line (Shimizu et al., 1994; Shimizu et al., 1996).

The recent development of HCVpp offers the possibility to study HCV neutralization with defined sequences of HCV envelope glycoproteins, and the use of HCVpp in neutralization studies has been validated (Bartosch et al., 2003a). As determined with HCVpp, it seems that the majority of chronically infected patients have cross-reactive neutralizing antibodies (Logvinoff et al., 2004; Meunier et al., 2005). In contrast, neutralizing antibodies have not been detected in several cases of acute resolving infection (Logvinoff et al., 2004; Meunier et al., 2005), and the detection of neutralizing antibodies in acutely infected individuals did not seem to be associated with viral clearance (Logvinoff et al., 2004). However, another study has shown in some patients a progressive emergence of a relatively strong neutralizing response in correlation with a decrease in viremia (Lavillette et al., 2005a). Further investigations on a large number of acutely infected patients will be necessary to determine the role of neutralizing antibodies in controlling HCV infections. Interestingly, it has been observed that HCVpp infectivity is enhanced by human sera, and this enhancement of infectivity can partly mask the presence of neutralizing antibodies (Lavillette et al., 2005a; Meunier et al., 2005). In addition, HDL have been identified as the component responsible for serum-mediated enhancement of infectivity (Meunier et al., 2005; Voisset et al., 2005).

For a long time, the HVR1 sequence of E2 has been proposed to be a major target for neutralizing antibodies (Kato et al., 1993; Farci et al., 1996). However, data obtained with the HCVpp model indicate that neutralizing epitopes located outside of HVR1 also exist (Bartosch et al., 2003a). Interestingly, characterization of HCVpp with monoclonal antibodies has allowed to identify conformation-dependent and -independent neutralizing epitopes outside of HVR1 (Fig. 2)(Bartosch et al., 2003b; Hsu et al., 2003; Keck et al., 2004; Op De Beeck et al., 2004). Conformation-dependent human monoclonal antibodies have also allowed to identify three immunogenic domains in E2 with neutralizing antibodies being restricted to two of these domains (Keck et al., 2004). Whether E2 domains identified with these monoclonal antibodies are similar to the antigenic structural and functional domains of the envelope protein E of the flaviviruses (Rey et al., 1995) remains to be determined.

Conclusion

Studies of the biogenesis of HCV envelope glycoproteins have shown the pivotal role of the transmembrane domains in the assembly of a noncovalent E1E2 heterodimer in the ER. More recently, the development of the HCVpp model has allowed to investigate the role of E1E2 heterodimer in virus entry. Functional regions in HCV envelope glycoproteins can now be identified and potential receptors can also be validated. Entry is an essential step in the life cycle of a virus, which can potentially be blocked by neutralizing antibodies or antiviral drugs that target the envelope proteins of the virus. Understanding the viral and cellular components involved in HCV invasion into the host cell, combined with a comprehension of the mechanisms that govern this process, should therefore open the possibility of developing new therapeutic approaches.

Future Trends

The development of the HCVpp model has allowed to initiate the characterization of the entry function of HCV envelope glycoproteins. The use of HCVpp will continue to provide additional information on the role of HCV envelope glycoproteins in viral entry. However, the recent development of a full-length clone that is infectious in cell culture (see chapter 16) provides new opportunities to study the functions of HCV envelope glycoproteins. A comparison of the properties of HCV envelope glycoproteins produced in HCVpp and in this infectious clone will be very useful to validate the data that have been generated during the past three years. In addition, this infectious clone will allow for the first time to decipher the role of HCV envelope glycoproteins in virion assembly. Finally, obtaining a high-resolution structure of HCV envelope glycoproteins will also be necessary to understand the fusion mechanism of this virus.

Acknowledgments

We thank Sophana Ung for preparing the illustrations. Our research was supported by EU grant QLRT-2000-01120 and QLRT-2001-01329 and grants from the "Agence Nationale de Recherche sur le Sida et les hépatites virales" (ANRS), INSERM "ATC-Hépatite C" and the "Association pour la Recherche sur le Cancer" (ARC).

References

  1. Agnello V, Abel G, Elfahal M, Knight GB, Zhang Q-X. Hepatitis C virus and other flaviviridae viruses enter cells via low density lipoprotein receptor. Proc Natl Acad Sci USA. 1999;96:12766–12771. [PMC free article: PMC23090] [PubMed: 10535997]
  2. Allison SL, Schalich J, Stiasny K, Mandl CW, Heinz FX. Mutational evidence for an internal fusion peptide in flavivirus envelope protein E. J Virol. 2001;75:4268–4275. [PMC free article: PMC114172] [PubMed: 11287576]
  3. Allison SL, Schalich J, Stiasny K, Mandl CW, Kunz C, Heinz FX. Oligomeric rearrangement of tick-borne encephalitis virus envelope proteins induced by an acidic pH. J Virol. 1995;69:695–700. [PMC free article: PMC188630] [PubMed: 7529335]
  4. Barth H, Schafer C, Adah MI, Zhang F, Linhardt RJ, Toyoda H, Kinoshita-Toyoda A, Toida T, Van Kuppevelt TH, Depla E, et al. Cellular binding of hepatitis C virus envelope glycoprotein E2 requires cell surface heparan sulfate. J Biol Chem. 2003;278:41003–41012. [PubMed: 12867431]
  5. Bartosch B, Bukh J, Meunier JC, Granier C, Engle RE, Blackwelder WC, Emerson SU, Cosset FL, Purcell RH. In vitro assay for neutralizing antibody to hepatitis C virus: evidence for broadly conserved neutralization epitopes. Proc Natl Acad Sci USA. 2003a;100:14199–14204. [PMC free article: PMC283569] [PubMed: 14617769]
  6. Bartosch B, Dubuisson J, Cosset FL. Infectious hepatitis C pseudo-particles containing functional E1E2 envelope protein complexes. J Exp Med. 2003b;197:633–642. [PMC free article: PMC2193821] [PubMed: 12615904]
  7. Bartosch B, Vitelli A, Granier C, Goujon C, Dubuisson J, Pascale S, Scarselli E, Cortese R, Nicosia A, Cosset FL. Cell entry of hepatitis C virus requires a set of co-receptors that include the CD81 tetraspanin and the SR-B1 scavenger receptor. J Biol Chem. 2003c;278:41624–41630. [PubMed: 12913001]
  8. Baumert TF, Ito S, Wong DT, Liang TJ. Hepatitis C virus structural proteins assemble into virus-like particles in insect cells. J Virol. 1998;72:3827–3836. [PMC free article: PMC109606] [PubMed: 9557666]
  9. Blight KJ, Kolykhalov AA, Rice CM. Efficient initiation of HCV RNA replication in cell culture. Science. 2000;290:1972–1974. [PubMed: 11110665]
  10. Brazzoli M, Helenius A, Foung SK, Houghton M, Abrignani S, Merola M. Folding and dimerization of hepatitis C virus E1 and E2 glycoproteins in stably transfected CHO cells. Virology. 2005;332:438–453. [PubMed: 15661174]
  11. Buonocore L, Blight KJ, Rice CM, Rose JK. Characterization of vesicular stomatitis virus recombinants that express and incorporate high levels of hepatitis C virus glycoproteins. J Virol. 2002;76:6865–6872. [PMC free article: PMC136334] [PubMed: 12072487]
  12. Carrère-Kremer S, Montpellier C, Lorenzo L, Brulin B, Cocquerel L, Belouzard S, Penin F, Dubuisson J. Regulation of hepatitis C virus polyprotein processing by signal peptidase involves structural determinants at the p7 sequence junctions. J Biol Chem. 2004;279:41384–41392. [PubMed: 15247249]
  13. Carrère-Kremer S, Montpellier-Pala C, Cocquerel L, Wychowski C, Penin F, Dubuisson J. Subcellular localization and topology of the p7 polypeptide of hepatitis C virus. J Virol. 2002;76:3720–3730. [PMC free article: PMC136108] [PubMed: 11907211]
  14. Charloteaux B, Lins L, Moereels H, Brasseur R. Analysis of the C-terminal membrane anchor domains of hepatitis C virus glycoproteins E1 and E2: toward a topological model. J Virol. 2002;76:1944–1958. [PMC free article: PMC135876] [PubMed: 11799189]
  15. Choukhi A, Ung S, Wychowski C, Dubuisson J. Involvement of endoplasmic reticulum chaperones in folding of hepatitis C virus glycoproteins. J Virol. 1998;72:3851–3858. [PMC free article: PMC109609] [PubMed: 9557669]
  16. Clayton RF, Owsianka A, Aitken J, Graham S, Bhella D, Patel AH. Analysis of antigenicity and topology of E2 glycoprotein present on recombinant hepatitis C virus-like particles. J Virol. 2002;76:7672–7682. [PMC free article: PMC136371] [PubMed: 12097581]
  17. Cocquerel L, Duvet S, Meunier J-C, Pillez A, Cacan R, Wychowski C, Dubuisson J. The transmembrane domain of hepatitis C virus glycoprotein E1 is a signal for static retention in the endoplasmic reticulum. J Virol. 1999;73:2641–2649. [PMC free article: PMC104019] [PubMed: 10074109]
  18. Cocquerel L, Kuo C-C, Dubuisson J, Levy S. CD81-dependent binding of Hepatitis C virus E1E2 heterodimers. J Virol. 2003a;77:10677–10683. [PMC free article: PMC228455] [PubMed: 12970454]
  19. Cocquerel L, Meunier J-C, Pillez A, Wychowski C, Dubuisson J. A retention signal necessary and sufficient for endoplasmic reticulum localization maps to the transmembrane domain of hepatitis C virus glycoprotein E2. J Virol. 1998;72:2183–2191. [PMC free article: PMC109514] [PubMed: 9499075]
  20. Cocquerel L, Meunier JC, Op De Beeck A, Bonte D, Wychowski C, Dubuisson J. Coexpression of hepatitis C virus envelope proteins E1 and E2 in cis improves the stability of membrane insertion of E2. J Gen Virol. 2001;82:1629–1635. [PubMed: 11413374]
  21. Cocquerel L, Op de Beeck A, Lambot M, Roussel J, Delgrange D, Pillez A, Wychowski C, Penin F, Dubuisson J. Topologic changes in the transmembrane domains of hepatitis C virus envelope glycoproteins. EMBO J. 2002;21:2893–2902. [PMC free article: PMC125386] [PubMed: 12065403]
  22. Cocquerel L, Quinn ER, Flint M, Hadlock KG, Foung SK, Levy S. Recognition of native hepatitis C virus E1E2 heterodimers by a human monoclonal antibody. J Virol. 2003b;77:1604–1609. [PMC free article: PMC140849] [PubMed: 12502876]
  23. Cocquerel L, Wychowski C, Minner F, Penin F, Dubuisson J. Charged residues in the transmembrane domains of Hepatitis C virus glycoproteins play a key role in the processing, subcellular localization and assembly of these envelope proteins. J Virol. 2000;74:3623–3633. [PMC free article: PMC111872] [PubMed: 10729138]
  24. Colman PM, Lawrence MC. The structural biology of type I viral membrane fusion. Nat Rev Mol Cell Biol. 2003;4:309–319. [PubMed: 12671653]
  25. Connelly MA, Williams DL. Scavenger receptor BI: a scavenger receptor with a mission to transport high density lipoprotein lipids. Curr Opin Lipidol. 2004;15:287–295. [PubMed: 15166784]
  26. Cormier EG, Durso RJ, Tsamis F, Boussemart L, Manix C, Olson WC, Gardner JP, Dragic T. L-SIGN (CD209L) and DC-SIGN (CD209) mediate transinfection of liver cells by hepatitis C virus. Proc Natl Acad Sci USA. 2004a;101:14067–14072. [PMC free article: PMC521122] [PubMed: 15371595]
  27. Cormier EG, Tsamis F, Kajumo F, Durso RJ, Gardner JP, Dragic T. CD81 is an entry coreceptor for hepatitis C virus. Proc Natl Acad Sci USA. 2004b;101:7270–7274. [PMC free article: PMC409908] [PubMed: 15123813]
  28. Crotta S, Stilla A, Wack A, D’Andrea A, Nuti S, D’Oro U, Mosca M, Filliponi F, Brunetto RM, Bonino F, et al. Inhibition of natural killer cells through engagement of CD81 by the major hepatitis C virus envelope protein. J Exp Med. 2002;195:35–41. [PMC free article: PMC2196014] [PubMed: 11781363]
  29. Deleersnyder V, Pillez A, Wychowski C, Blight K, Xu J, Hahn YS, Rice CM, Dubuisson J. Formation of native hepatitis C virus glycoprotein complexes. J Virol. 1997;71:697–704. [PMC free article: PMC191102] [PubMed: 8985401]
  30. Drummer HE, Maerz A, Poumbourios P. Cell surface expression of functional hepatitis C virus E1 and E2 glycoproteins. FEBS Lett. 2003;546:385–390. [PubMed: 12832074]
  31. Drummer HE, Poumbourios P. Hepatitis C virus glycoprotein E2 contains a membrane-proximal heptad repeat sequence that is essential for E1E2 glycoprotein heterodimerization and viral entry. J Biol Chem. 2004;279:30066–30072. [PubMed: 15136562]
  32. Dubuisson J. Folding, assembly and subcellular localization of HCV glycoproteins. Curr Top Microbiol Immunol. 2000;242:135–148. [PubMed: 10592659]
  33. Dubuisson J, Duvet S, Meunier JC, Op De Beeck A, Cacan R, Wychowski C, Cocquerel L. Glycosylation of the hepatitis C virus envelope protein E1 is dependent on the presence of a downstream sequence on the viral polyprotein. J Biol Chem. 2000;275:30605–30609. [PubMed: 10882734]
  34. Dubuisson J, Hsu HH, Cheung RC, Greenberg HB, Russell DG, Rice CM. Formation and intracellular localization of hepatitis C virus envelope glycoprotein complexes expressed by recombinant vaccinia and Sindbis viruses. J Virol. 1994;68:6147–6160. [PMC free article: PMC237034] [PubMed: 8083956]
  35. Dubuisson J, Penin F, Moradpour D. Interaction of hepatitis C virus proteins with host cell membranes and lipids. Trends Cell Biol. 2002;12:517–523. [PubMed: 12446113]
  36. Dubuisson J, Rice CM. Hepatitis C virus glycoprotein folding: disulfide bond formation and association with calnexin. J Virol. 1996;70:778–786. [PMC free article: PMC189879] [PubMed: 8551615]
  37. Duvet S, Cocquerel L, Pillez A, Cacan R, Verbert A, Moradpour D, Wychowski C, Dubuisson J. Hepatitis C virus glycoprotein complex localization in the endoplasmic reticulum involves a determinant for retention and not retrieval. J Biol Chem. 1998;273:32088–32095. [PubMed: 9822684]
  38. Duvet S, Op De Beeck A, Cocquerel L, Wychowski C, Cacan R, Dubuisson J. Glycosylation of the hepatitis C virus envelope protein E1 occurs posttranslationally in a mannosylphosphoryldolichol-deficient CHO mutant cell line. Glycobiology. 2002;12:95–101. [PubMed: 11886842]
  39. Earp LJ, Delos SE, Park HE, White JM. The many mechanisms of viral membrane fusion proteins. Curr Top Microbiol Immunol. 2005;285:25–66. [PubMed: 15609500]
  40. Farci P, Alter HJ, Wong DC, Miller RH, Govindarajan S, Engle R, Shapiro M, Purcell RH. Prevention of hepatitis C virus infection in chimpanzees after antibody-mediated in vitro neutralization. Proc Natl Acad Sci USA. 1994;91:7792–7796. [PMC free article: PMC44488] [PubMed: 7519785]
  41. Farci P, Shimoda A, Wong D, Cabezon T, De Gioannis D, Strazzera A, Shimizu Y, Shapiro M, Alter HJ, Purcell RH. Prevention of hepatitis C virus infection in chimpanzees by hyperimmune serum against the hypervariable region 1 of the envelope 2 protein. Proc Natl Acad Sci USA. 1996;93:15394–15399. [PMC free article: PMC26415] [PubMed: 8986822]
  42. Flint M, Logvinoff C, Rice CM, McKeating JA. Characterization of infectious retroviral pseudotype particles bearing hepatitis C virus glycoproteins. J Virol. 2004;78:6875–6882. [PMC free article: PMC421632] [PubMed: 15194763]
  43. Flint M, Maidens C, Loomis-Price LD, Shotton C, Dubuisson J, Monk P, Higginbottom A, Levy S, McKeating JA. Characterization of hepatitis C virus E2 glycoprotein interaction with a putative cellular receptor, CD81. J Virol. 1999a;73:6235–6244. [PMC free article: PMC112700] [PubMed: 10400713]
  44. Flint M, Thomas JM, Maidens CM, Shotton C, Levy S, Barclay WS, McKeating JA. Functional analysis of cell surface-expressed hepatitis C virus E2 glycoprotein. J Virol. 1999b;73:6782–6790. [PMC free article: PMC112763] [PubMed: 10400776]
  45. Forns X, Allander T, Rohwer-Nutter P, Bukh J. Characterization of modified hepatitis C virus E2 proteins expressed on the cell surface. Virology. 2000a;274:75–85. [PubMed: 10936090]
  46. Forns X, Thimme R, Govindarajan S, Emerson SU, Purcell RH, Chisari FV, Bukh J. Hepatitis C virus lacking the hypervariable region 1 of the second envelope protein is infectious and causes acute resolving or persistent infection in chimpanzees. Proc Natl Acad Sci USA. 2000b;97:13318–13323. [PMC free article: PMC27222] [PubMed: 11078521]
  47. Gardner JP, Durso RJ, Arrigale RR, Donovan GP, Maddon PJ, Dragic T, Olson WC. L-SIGN (CD 209L) is a liver-specific capture receptor for hepatitis C virus. Proc Natl Acad Sci USA. 2003;100:4498–4503. [PMC free article: PMC153584] [PubMed: 12676990]
  48. Garry RF, Dash S. Proteomics computational analyses suggest that hepatitis C virus E1 and pestivirus E2 envelope glycoproteins are truncated class II fusion proteins. Virology. 2003;307:255–265. [PubMed: 12667795]
  49. Gavel Y, von Heijne G. Sequence differences between glycosylated and non-glycosylated Asn-X-Thr/Ser acceptor sites: implications for protein engineering. Protein Eng. 1990;3:433–442. [PubMed: 2349213]
  50. Goffard A, Callens N, Bartosch B, Wychowski C, Cosset FL, Montpellier-Pala C, Dubuisson J. Role of N-linked glycans in the functions of hepatitis C virus envelope glycoproteins. J Virol. 2005;79:8400–8409. [PMC free article: PMC1143753] [PubMed: 15956584]
  51. Goffard A, Dubuisson J. Glycosylation of hepatitis C virus envelope proteins. Biochimie. 2003;85:295–301. [PubMed: 12770768]
  52. Grakoui A, Wychowski C, Lin C, Feinstone SM, Rice CM. Expression and identification of hepatitis C virus polyprotein cleavage products. J Virol. 1993;67:1385–1395. [PMC free article: PMC237508] [PubMed: 7679746]
  53. Griffin SD, Beales LP, Clarke DS, Worsfold O, Evans SD, Jaeger J, Harris MP, Rowlands DJ. The p7 protein of hepatitis C virus forms an ion channel that is blocked by the antiviral drug, Amantadine. FEBS Lett. 2003;535:34–38. [PubMed: 12560074]
  54. Hebert DN, Zhang JX, Chen W, Foellmer B, Helenius A. The number and location of glycans on influenza hemagglutinin determine folding and association with calnexin and calreticulin. J Cell Biol. 1997;139:613–623. [PMC free article: PMC2141715] [PubMed: 9348279]
  55. Hsu M, Zhang J, Flint M, Logvinoff C, Cheng-Mayer C, Rice CM, McKeating JA. Hepatitis C virus glycoproteins mediate pH-dependent cell entry of pseudotyped retroviral particles. Proc Natl Acad Sci USA. 2003;100:7271–7276. [PMC free article: PMC165865] [PubMed: 12761383]
  56. Imperiali B, O'Connor SE. Effect of N-linked glycosylation on glycopeptide and glycoprotein structure. Curr Opin Chem Biol. 1999;3:643–649. [PubMed: 10600722]
  57. Isherwood BJ, Patel AH. Analysis of the processing and transmembrane topology of the E2p7 protein of hepatitis C virus. J Gen Virol. 2005;86:667–676. [PubMed: 15722527]
  58. Kato N, Sekiya H, Ootsuyama Y, Nakazawa T, Hijikata M, Ohkoshi S, Shimotohno K. Humoral immune response to hypervariable region 1 of the putative envelope glycoprotein (gp70) of hepatitis C virus. J Virol. 1993;67:3923–3930. [PMC free article: PMC237759] [PubMed: 7685404]
  59. Keck ZY, Op De Beeck A, Hadlock KG, Xia J, Li TK, Dubuisson J, Foung SK. Hepatitis C virus E2 has three immunogenic domains containing conformational epitopes with distinct properties and biological functions. J Virol. 2004;78:9224–9232. [PMC free article: PMC506923] [PubMed: 15308717]
  60. Kornfeld R, Kornfeld S. Assembly of asparagine-linked oligosaccharides. Annu Rev Biochem. 1985;54:631–664. [PubMed: 3896128]
  61. Lagging LM, Meyer K, Owens RJ, Ray R. Functional role of hepatitis C virus chimeric glycoproteins in the infectivity of pseudotyped virus. J Virol. 1998;72:3539–3546. [PMC free article: PMC109573] [PubMed: 9557633]
  62. Lambot M, Fretier S, Op De Beeck A, Quatannens B, Lestavel S, Clavey V, Dubuisson J. Reconstitution of hepatitis C virus envelope glycoproteins into liposomes as a surrogate model to study virus attachment. J Biol Chem. 2002;277:20625–20630. [PubMed: 11937498]
  63. Lavillette D, Morice Y, Germanidis G, Donot P, Soulier A, Pagkalos E, Sakellariou G, Intrator L, Bartosch B, Pawlotsky JM, Cosset FL. Human serum facilitates hepatitis C virus infection, and neutralizing responses inversely correlate with viral replication kinetics at the acute phase of hepatitis C virus infection. J Virol. 2005a;79:6023–6034. [PMC free article: PMC1091689] [PubMed: 15857988]
  64. Lavillette D, Tarr AW, Voisset C, Donot P, Bartosch B, Bain C, Patel AH, Dubuisson J, Ball JK, Cosset FL. Characterization of host-range and cell entry properties of hepatitis C virus of major genotypes and subtypes. Hepatology. 2005b;41:265–274. [PubMed: 15660396]
  65. Lescar J, Roussel A, Wien MW, Navaza J, Fuller SD, Wengler G, Rey FA. The Fusion glycoprotein shell of Semliki Forest virus: an icosahedral assembly primed for fusogenic activation at endosomal pH. Cell. 2001;105:137–148. [PubMed: 11301009]
  66. Levy S, Shoham T. The tetraspanin web modulates immune-signalling complexes. Nat Rev Immunol. 2005;5:136–148. [PubMed: 15688041]
  67. Lin C, Lindenbach BD, Pragai BM, McCourt DW, Rice CM. Processing in the hepatitis C virus E2-NS2 region: identification of p7 and two distinct E2-specific products with different C termini. J Virol. 1994;68:5063–5073. [PMC free article: PMC236449] [PubMed: 7518529]
  68. Logvinoff C, Major ME, Oldach D, Heyward S, Talal A, Balfe P, Feinstone SM, Alter H, Rice CM, McKeating JA. Neutralizing antibody response during acute and chronic hepatitis C virus infection. Proc Natl Acad Sci USA. 2004;101:10149–10154. [PMC free article: PMC454180] [PubMed: 15220475]
  69. Lohmann V, Körner F, Koch J-O, Herian U, Theilmann L, Bartenschlager R. Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science. 1999;285:110–113. [PubMed: 10390360]
  70. Lozach PY, Amara A, Bartosch B, Virelizier JL, Arenzana-Seisdedos F, Cosset FL, Altmeyer R. C-type Lectins L-SIGN and DC-SIGN capture and transmit infectious hepatitis C virus pseudotype particles. J Biol Chem. 2004;279:32035–32045. [PubMed: 15166245]
  71. Lozach PY, Lortat-Jacob H, De Lacroix De Lavalette A, Staropoli I, Foung S, Amara A, Houles C, Fieschi F, Schwartz Virelizier, J, et al. DC-SIGN and L-SIGN are high-affinity binding receptors for hepatitis C Virus glycoprotein E2. J Biol Chem. 2003;278:20358–20366. [PubMed: 12609975]
  72. Ludwig IS, Lekkerkerker AN, Depla E, Bosman F, Musters RJ, Depraetere S, van Kooyk Y, Geijtenbeek TB. Hepatitis C virus targets DC-SIGN and L-SIGN to escape lysosomal degradation. J Virol. 2004;78:8322–8332. [PMC free article: PMC446128] [PubMed: 15254204]
  73. Mackenzie JM, Westaway EG. Assembly and maturation of the flavivirus Kunjin virus appear to occur in the rough endoplasmic reticulum and along the secretory pathway, respectively. J Virol. 2001;75:10787–10799. [PMC free article: PMC114660] [PubMed: 11602720]
  74. Masciopinto F, Giovani C, Campagnoli S, Galli-Stampino L, Colombatto P, Brunetto M, Yen TS, Houghton M, Pileri P, Abrignani S. Association of hepatitis C virus envelope proteins with exosomes. Eur J Immunol. 2004;34:2834–2842. [PubMed: 15368299]
  75. Matsuura Y, Tani H, Suzuki K, Kimura-Someya T, Suzuki R, Aizaki H, Ishii K, Moriishi K, Robison CS, Whitt MA, Miyamura T. Characterization of pseudotype VSV possessing HCV envelope proteins. Virology. 2001;286:263–275. [PubMed: 11485395]
  76. Mazzocca A, Sciammetta SC, Carloni V, Cosmi L, Annunziato F, Harada T, Abrignani S, Pinzani M. Binding of hepatitis C virus envelope protein E2 to CD81 up-regulates matrix metalloproteinase-2 in human hepatic stellate cells. J Biol Chem. 2005;280:11329–11339. [PubMed: 15611113]
  77. McKeating JA, Zhang LQ, Logvinoff C, Flint M, Zhang J, Yu J, Butera D, Ho DD, Dustin LB, Rice CM, Balfe P. Diverse hepatitis C virus glycoproteins mediate viral infection in a CD81-dependent manner. J Virol. 2004;78:8496–8505. [PMC free article: PMC479078] [PubMed: 15280458]
  78. McLauchlan J, Lemberg MK, Hope RG, Martoglio B. Intramembrane proteolysis promotes trafficking of hepatitis C virus core protein to lipid droplets. EMBO J. 2002;21:3980–3988. [PMC free article: PMC126158] [PubMed: 12145199]
  79. Merola M, Brazzoli M, Cocchiarella F, Heile JM, Helenius A, Weiner AJ, Houghton M, Abrignani S. Folding of hepatitis C virus E1 glycoprotein in a cell-free system. J Virol. 2001;75:11205–11217. [PMC free article: PMC114700] [PubMed: 11602760]
  80. Meunier J-C, Fournillier A, Choukhi A, Cahour A, Cocquerel L, Dubuisson J, Wychowski C. Analysis of the glycosylation sites of hepatitis C virus (HCV) glycoprotein E1 and the influence of E1 glycans on the formation of the HCV glycoprotein complex. J Gen Virol. 1999;80:887–896. [PubMed: 10211957]
  81. Meunier JC, Engle RE, Faulk K, Zhao M, Bartosch B, Alter H, Emerson SU, Cosset FL, Purcell RH, Bukh J. Evidence for cross-genotype neutralization of hepatitis C virus pseudo-particles and enhancement of infectivity by apolipoprotein C1. Proc Natl Acad Sci USA. 2005;102:4560–4565. [PMC free article: PMC555507] [PubMed: 15767578]
  82. Michalak J-P, Wychowski C, Choukhi A, Meunier J-C, Ung S, Rice CM, Dubuisson J. Characterization of truncated forms of hepatitis C virus glycoproteins. J Gen Virol. 1997;78:2299–2306. [PubMed: 9292018]
  83. Mizushima H, Hijikata M, Asabe S-I, Hirota M, Kimura K, Shimotohno K. Two hepatitis C virus glycoprotein E2 products with different C termini. J Virol. 1994;68:6215–6222. [PMC free article: PMC237041] [PubMed: 8083961]
  84. Modis Y, Ogata S, Clements D, Harrison SC. A ligand-binding pocket in the dengue virus envelope glycoprotein. Proc Natl Acad Sci USA. 2003;100:6986–6991. [PMC free article: PMC165817] [PubMed: 12759475]
  85. Monazahian M, Bohme I, Bonk S, Koch A, Scholz C, Grethe S, Thomssen R. Low density lipoprotein receptor as a candidate receptor for hepatitis C virus. J Med Virol. 1999;57:223–229. [PubMed: 10022791]
  86. Mondelli MU, Cerino A, Meola A, Nicosia A. Variability or conservation of hepatitis C virus hypervariable region 1? Implications for immune responses. J Biosci. 2003;28:305–310. [PubMed: 12734408]
  87. Mottola G, Jourdan N, Castaldo G, Malagolini N, Lahm A, Serafini-Cessi F, Migliaccio G, Bonatti S. A new determinant of endoplasmic reticulum localization is contained in the juxtamembrane region of the ectodomain of hepatitis C virus glycoprotein E1. J Biol Chem. 2000;275:24070–24079. [PubMed: 10783397]
  88. Mukhopadhyay S, Kuhn RJ, Rossmann MG. A structural perspective of the flavivirus life cycle. Nat Rev Microbiol. 2005;3:13–22. [PubMed: 15608696]
  89. Nakano I, Fukuda Y, Katano Y, Hayakawa T. Conformational epitopes detected by cross-reactive antibodies to envelope 2 glycoprotein of the hepatitis C virus. J Infect Dis. 1999;180:1328–1333. [PubMed: 10479166]
  90. Negre D, Duisit G, Mangeot PE, Moullier P, Darlix JL, Cosset FL. Lentiviral vectors derived from simian immunodeficiency virus. Curr Top Microbiol Immunol. 2002;261:53–74. [PubMed: 11892253]
  91. Ohuchi M, Ohuchi R, Feldmann A, Klenk HD. Regulation of receptor binding affinity of influenza virus hemagglutinin by its carbohydrate moiety. J Virol. 1997a;71:8377–8384. [PMC free article: PMC192299] [PubMed: 9343193]
  92. Ohuchi R, Ohuchi M, Garten W, Klenk HD. Oligosaccharides in the stem region maintain the influenza virus hemagglutinin in the metastable form required for fusion activity. J Virol. 1997b;71:3719–3725. [PMC free article: PMC191521] [PubMed: 9094646]
  93. Op De Beeck A, Cocquerel L, Dubuisson J. Biogenesis of hepatitis C virus envelope glycoproteins. J Gen Virol. 2001;82:2589–2595. [PubMed: 11602769]
  94. Op De Beeck A, Dubuisson J. Another putative receptor for hepatitis C virus. Hepatology. 2003;37:705–707. [PubMed: 12627595]
  95. Op De Beeck A, Molenkamp R, Caron M, Ben Younes A, Bredenbeek P, Dubuisson J. Role of the transmembrane domains of prM and E proteins in the formation of yellow fever virus envelope. J Virol. 2003;77:813–820. [PMC free article: PMC140810] [PubMed: 12502797]
  96. Op De Beeck A, Montserret R, Duvet S, Cocquerel L, Cacan R, Barberot B, Le Maire M, Penin F, Dubuisson J. Role of the transmembrane domains of hepatitis C virus envelope proteins E1 and E2 in the assembly of the noncovalent E1E2 heterodimer. J Biol Chem. 2000;275:31428–31437. [PubMed: 10807921]
  97. Op De Beeck A, Voisset C, Bartosch B, Ciczora Y, Cocquerel L, Keck Z, Foung S, Cosset FL, Dubuisson J. Characterization of functional hepatitis C virus envelope glycoproteins. J Virol. 2004;78:2994–3002. [PMC free article: PMC353750] [PubMed: 14990718]
  98. Ott DE. Cellular proteins in HIV virions. Rev Med Virol. 1997;7:167–180. [PubMed: 10398481]
  99. Owsianka A, Clayton RF, Loomis-Price LD, McKeating JA, Patel AH. Functional analysis of hepatitis C virus E2 glycoproteins and viruslike particles reveals structural dissimilarities between different forms of E2. J Gen Virol. 2001;82:1877–1883. [PubMed: 11457993]
  100. Patel J, Patel AH, McLauchlan J. The transmembrane domain of the hepatitis C virus E2 glycoprotein is required for correct folding of the E1 glycoprotein and native complex formation. Virology. 2001;279:58–68. [PubMed: 11145889]
  101. Pavlovic D, Neville DC, Argaud O, Blumberg B, Dwek RA, Fischer WB, Zitzmann N. The hepatitis C virus p7 protein forms an ion channel that is inhibited by long-alkyl-chain iminosugar derivatives. Proc Natl Acad Sci USA. 2003;100:6104–6108. [PMC free article: PMC156333] [PubMed: 12719519]
  102. Penin F, Combet C, Germanidis G, Frainais PO, Deléage G, Pawlotsky JM. Conservation of the conformation and positive charges of hepatitis C virus E2 envelope glycoprotein hypervariable region 1 points to a role in cell attachment. J Virol. 2001;75:5703–5710. [PMC free article: PMC114285] [PubMed: 11356980]
  103. Penin F, Dubuisson J, Rey FA, Moradpour D, Pawlotsky JM. Structural biology of hepatitis C virus. Hepatology. 2004;39:5–19. [PubMed: 14752815]
  104. Pileri P, Uematsu Y, Campagnoli S, Galli G, Falugi F, Petracca R, Weiner AJ, Houghton M, Rosa D, Grandi G, Abrignani S. Binding of hepatitis C virus to CD81. Science. 1998;282:938–941. [PubMed: 9794763]
  105. Pohlmann S, Zhang J, Baribaud F, Chen Z, Leslie GJ, Lin G, Granelli-Piperno A, Doms RW, Rice CM, McKeating JA. Hepatitis C virus glycoproteins interact with DC-SIGN and DC-SIGNR. J Virol. 2003;77:4070–4080. [PMC free article: PMC150620] [PubMed: 12634366]
  106. Premkumar A, Wilson L, Ewart GD, Gage PW. Cation-selective ion channels formed by p7 of hepatitis C virus are blocked by hexamethylene amiloride. FEBS Lett. 2004;557:99–103. [PubMed: 14741348]
  107. Ralston R, Thudium K, Berger K, Kuo C, Gervase B, Hall J, Selby M, Kuo G, Houghton M, Choo Q-L. Characterization of hepatitis C virus envelope glycoprotein complexes expressed by recombinant vaccinia viruses. J Virol. 1993;67:6753–6761. [PMC free article: PMC238116] [PubMed: 8411378]
  108. Reed KE, Rice CM. Overview of hepatitis C virus genome structure, polyprotein processing, and protein properties. Curr Top Microbiol Immunol. 2000;242:55–84. [PubMed: 10592656]
  109. Rey FA, Heinz FX, Mandl C, Kunz C, Harrison SC. The envelope glycoprotein from tick-borne encephalitis virus at 2 A resolution. Nature. 1995;375:291–298. [PubMed: 7753193]
  110. Rosa D, Campagnoli S, Moretto C, Guenzi E, Cousens L, Chin M, Dong C, Weiner A, Lau JYN, Choo Q-L, et al. A quantitative test to estimate neutralizing antibodies to the hepatitis C virus: cytofluorimetric assessment of the envelope glycoprotein 2 binding to target cells. Proc Natl Acad Sci USA. 1996;93:1759–1763. [PMC free article: PMC39854] [PubMed: 8700831]
  111. Sandrin V, Boson B, Salmon P, Gay W, Nègre D, Le Grand R, Trono D, Cosset F-L. Lentiviral vectors pseudotyped with a modified RD114 envelope glycoprotein show increased stability in sera and augmented transduction of primary lymphocytes and CD34+ cells derived from human and non-human primates. Blood. 2002;100:823–832. [PubMed: 12130492]
  112. Santolini E, Migliaccio G, La Monica N. Biosynthesis and biochemical properties of the hepatitis C virus core protein. J Virol. 1994;68:3631–3641. [PMC free article: PMC236867] [PubMed: 8189501]
  113. Saunier B, Triyatni M, Ulianich L, Maruvada P, Yen P, Kohn LD. Role of the asialoglycoprotein receptor in binding and entry of hepatitis C virus structural proteins in cultured human hepatocytes. J Virol. 2003;77:546–559. [PMC free article: PMC140572] [PubMed: 12477859]
  114. Scarselli E, Ansuini H, Cerino R, Roccasecca RM, Acali S, Filocamo G, Traboni C, Nicosia A, Cortese R, Vitelli A. The human scavenger receptor class B type I is a novel candidate receptor for the hepatitis C virus. EMBO J. 2002;21:5017–5025. [PMC free article: PMC129051] [PubMed: 12356718]
  115. Selby MJ, Glazer E, Masiarz F, Houghton M. Complex processing and protein:protein interactions in the E2:NS2 region of HCV. Virology. 1994;204:114–122. [PubMed: 8091646]
  116. Shimizu YK, Hijikata M, Iwamoto A, Alter HJ, Purcell RH, Yoshikura H. Neutralizing antibodies against hepatitis C virus and the emergence of neutralization escape mutant viruses. J Virol. 1994;68:1494–1500. [PMC free article: PMC236605] [PubMed: 8107212]
  117. Shimizu YK, Igarashi H, Kiyohara T, Cabezon T, Farci P, Purcell RH, Yoshikura H. A hyperimmune serum against a synthetic peptide corresponding to the hypervariable region 1 of hepatitis C virus can prevent viral infection in cell cultures. Virology. 1996;223:409–412. [PubMed: 8806581]
  118. Silberstein S, Gilmore R. Biochemistry, molecular biology, and genetics of the oligosaccharyltransferase. FASEB J. 1996;10:849–858. [PubMed: 8666161]
  119. Slater-Handshy T, Droll DA, Fan X, Di Bisceglie AM, Chambers TJ. HCV E2 glycoprotein: mutagenesis of N-linked glycosylation sites and its effects on E2 expression and processing. Virology. 2004;319:36–48. [PubMed: 14967486]
  120. Soldaini E, Wack A, D’Oro U, Nuti S, Ulivieri C, Baldari CT, Abrignani S. T cell co-stimulation by the hepatitis C virus envelope protein E2 binding to CD81 is mediated by Lck. Eur J Immunol. 2003;33:455–464. [PubMed: 12645944]
  121. Stadler K, Allison SL, Schalich J, Heinz FX. Proteolytic activation of tick-borne encephalitis virus by furin. J Virol. 1997;71:8475–8481. [PMC free article: PMC192310] [PubMed: 9343204]
  122. Strauss JH, Strauss EG. The Alphaviruses: gene expression, replication, and evolution. Microbiol Rev. 1994;58:491–562. [PMC free article: PMC372977] [PubMed: 7968923]
  123. Takikawa S, Ishii K, Aizaki H, Suzuki T, Asakura H, Matsuura Y, Miyamura T. Cell fusion activity of hepatitis C virus envelope proteins. J Virol. 2000;74:5066–5074. [PMC free article: PMC110858] [PubMed: 10799580]
  124. Trombetta ES, Helenius A. Lectins as chaperones in glycoprotein folding. Curr Opin Struct Biol. 1998;8:587–592. [PubMed: 9818262]
  125. Tseng CT, Klimpel GR. Binding of the hepatitis C virus envelope protein E2 to CD81 inhibits natural killer cell functions. J Exp Med. 2002;195:43–49. [PMC free article: PMC2196015] [PubMed: 11781364]
  126. van Kooyk Y, Geijtenbeek TB. DC-SIGN: escape mechanism for pathogens. Nat Rev Immunol. 2003;3:697–709. [PubMed: 12949494]
  127. Voisset C, Callens N, Blanchard E, Op De Beeck A, Dubuisson J, Vu-Dac N. High density lipoproteins facilitate hepatitis C virus entry through the scavenger receptor class B type I. J Biol Chem. 2005;280:7793–7799. [PubMed: 15632171]
  128. von Messling V, Cattaneo R. N-linked glycans with similar location in the fusion protein head modulate paramyxovirus fusion. J Virol. 2003;77:10202–10212. [PMC free article: PMC228470] [PubMed: 12970405]
  129. Wack A, Soldaini E, Tseng C, Nuti S, Klimpel G, Abrignani S. Binding of the hepatitis C virus envelope protein E2 to CD81 provides a costimulatory signal for human T cells. Eur J Immunol. 2001;31:166–175. [PubMed: 11169450]
  130. Wei X, Decker JM, Wang S, Hui H, Kappes JC, Wu X, Salazar-Gonzalez JF, Salazar MG, Kilby JM, Saag MS, et al. Antibody neutralization and escape by HIV-1. Nature. 2003;422:307–312. [PubMed: 12646921]
  131. Weiner AJ, Brauer R, Rosenblatt J, Richman KH, Tung J, Crarford K, Bonino F, Saracco G, Choo Q-L, Houghton M, Han JH. Variable and hypervariable domains are found in the regions of HCV corresponding to the flavivirus envelope and NS1 proteins and the pestivirus envelop glycoproteins. Virology. 1991;180:842–848. [PubMed: 1846505]
  132. Wormald MR, Dwek RA. Glycoproteins: glycan presentation and protein-fold stability. Structure Fold Des. 1999;7:R155–160. [PubMed: 10425673]
  133. Yagnik AT, Lahm A, Meola A, Roccasecca RM, Ercole BB, Nicosia A, Tramontano A. A model for the hepatitis C virus envelope glycoprotein E2. Proteins. 2000;40:355–366. [PubMed: 10861927]
  134. Zhang J, Randall G, Higginbottom A, Monk P, Rice CM, McKeating JA. CD81 is required for hepatitis C virus glycoprotein-mediated viral infection. J Virol. 2004a;78:1448–1455. [PMC free article: PMC321402] [PubMed: 14722300]
  135. Zhang M, Gaschen B, Blay W, Foley B, Haigwood N, Kuiken C, Korber B. Tracking global patterns of N-linked glycosylation site variation in highly variable viral glycoproteins: HIV, SIV, and HCV envelopes and influenza hemagglutinin. Glycobiology. 2004b;14:1229–1246. [PubMed: 15175256]
  136. Zhang W, Chipman PR, Corver J, Johnson PR, Zhang Y, Mukhopadhyay S, Baker TS, Strauss JH, Rossmann MG, Kuhn RJ. Visualization of membrane protein domains by cryo-electron microscopy of dengue virus. Nat Struct Biol. 2003;10:907–912. [PMC free article: PMC4148076] [PubMed: 14528291]
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