JBC PeproTech; Our Business is Cytokines!

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M111020200 on April 5, 2002

J. Biol. Chem., Vol. 277, Issue 23, 20625-20630, June 7, 2002
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
277/23/20625    most recent
M111020200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lambot, M.
Right arrow Articles by Dubuisson, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lambot, M.
Right arrow Articles by Dubuisson, J.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Reconstitution of Hepatitis C Virus Envelope Glycoproteins into Liposomes as a Surrogate Model to Study Virus Attachment*

Michel LambotDagger §, Stéphanie FrétierDagger , Anne Op De BeeckDagger ||, Brigitte QuatannensDagger , Sophie Lestavel**, Véronique Clavey**, and Jean DubuissonDagger DaggerDagger

From the Dagger  CNRS-Institut de Biologie de Lille & Institut Pasteur de Lille, 59021 Lille Cedex, France and the ** INSERM-U545, Institut Pasteur de Lille, Faculté de Pharmacie - Université de Lille 2, 59019 Lille Cedex, France

Received for publication, November 16, 2001, and in revised form, April 3, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The envelope glycoproteins, E1 and E2, of hepatitis C virus (HCV) assemble intracellularly to form a noncovalent heterodimer that is expected to be essential for viral assembly and entry. However, due to the lack of a cell culture system supporting efficient HCV replication, it is very difficult to obtain relevant information on the functions of this glycoprotein oligomer. To get better insights into its biological and biochemical properties, HCV envelope glycoprotein heterodimer expressed by a vaccinia virus recombinant was purified by immunoaffinity. Purified E1E2 heterodimer was recognized by conformation-dependent monoclonal antibodies, showing that the proteins were properly folded. In addition, it interacted with human CD81, a putative HCV receptor, as well as with human low and very low density lipoproteins, which have been shown to be associated with infectious HCV particles isolated from patients. Purified E1E2 heterodimer was also reconstituted into liposomes. E1E2-liposomes were recognized by a conformation-dependent monoclonal antibody as well as by human CD81. Together, these data indicate that E1E2-liposomes are a valuable tool to study the molecular requirements for HCV binding to target cells.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Hepatitis C virus (HCV)1 is the causal agent of hepatitis C, which is a major health problem worldwide (1). HCV is a positive stranded RNA virus that belongs to the Flaviviridae family (2). Its genome encodes a single polyprotein of ~3000 amino acid residues that is co- and post-translationally cleaved to generate at least 10 polypeptides (3). An additional HCV protein is produced by a ribosomal frameshift in the N-terminal region of the polyprotein (4). The two envelope glycoproteins, E1 and E2, are released from HCV polyprotein precursor after cleavage by host signal peptidase(s) (3). 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. However, indirect evidence, such as virus neutralization by antibodies, supports the idea that HCV envelope glycoproteins are present on the surface of the virion (5). The current knowledge accumulated on HCV envelope glycoproteins is based on cell culture transient expression assays with viral or nonviral expression vectors. These membrane proteins are composed of a large N-terminal ectodomain and a C-terminal hydrophobic anchor. Immunolocalization studies and glycan analyses have shown that HCV envelope glycoproteins are located in an early compartment of the secretory pathway (6, 7).

Studies using transient expression systems have shown that E2 interacts with E1 to form oligomers (8). In the presence of nonionic detergents, two forms of E1E2 complexes are detected: a heterodimer of E1 and E2 stabilized by noncovalent interactions and heterogeneous disulfide-linked aggregates (9). An extensive characterization of the noncovalent heterodimer supports the idea that it is most likely the prebudding form of the functional complex that will play an active role in the entry process into host cells (6). Indeed, this noncovalent heterodimer is homogeneous and resistant to protease digestion. In addition, its components have acquired intramolecular disulfide bonds and are no longer interacting with endoplasmic reticulum chaperones, indicating that they are extensively folded. The disulfide-linked aggregates are probably dead-end products, and their formation might be due to inefficient folding of HCV envelope glycoproteins (8).

When expressed by using heterologous expression systems, large amounts of misfolded HCV envelope glycoproteins are produced. The presence of a mixture of properly folded and misfolded proteins in such preparations can lead to misinterpretation of some biological and biochemical studies. Here, we developed an immunoaffinity purification procedure that allows the selective isolation of the noncovalent E1E2 heterodimer. Characterization of this complex indicates that it binds to several biologically relevant ligands. In addition, E1E2 heterodimer reconstituted into liposomes exhibits expected biological features and represents a valuable tool to study the interactions between HCV envelope glycoproteins and the host cell surface. This is the first attempt to reconstitute HCV envelope glycoproteins to study some of their biological properties.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Igepal CA-630, [3H]cholesterol, beta -D-octylglucopyranoside (octyl glucoside), egg yolk phosphatidylcholine (type V-E), and Galanthus nivalis (GNA)-agarose were from Sigma. Glutathione-Sepharose 4B, CNBr-activated Sepharose 4B, and Protein A-Sepharose were from Amersham Biosciences. Spectrapor-2 dialysis tubings and Spectra/GelTM Absorbent were purchased from Spectrum Laboratories. The anti-mouse IgG-peroxidase conjugate was from DAKO. The anti-mouse IgG-fluorescein isothiocyanate conjugate was from Jackson ImmunoResearch. The CV-1, Daudi, and HepG2 cell lines were obtained from the American Type Culture Collection, Manassas, VA. Cells were grown in Dulbecco's modified essential medium (Invitrogen) supplemented with 10% fetal bovine serum. The vaccinia virus-HCV recombinant expressing HCV envelope proteins E1 and E2 has been described previously (10). vTF7-3 virus, a vaccinia virus recombinant expressing the T7 DNA-dependent RNA polymerase (11), was kindly provided by B. Moss (National Institutes of Health, Bethesda, MD). Stocks of vaccinia virus recombinants were grown and titrated on CV-1 monolayers. Anti-E2 mouse monoclonal antibodies (mAbs) H33, H50, and H53 have been described previously (12, 13) and were produced in vitro by using a MiniPerm apparatus (Heraeus). Recombinant glutathione S-transferase (GST) fusion proteins containing the large extracellular loop of human CD81 (hCD81) or murine CD81 (mCD81) (14) were kindly provided by S. Levy (Stanford University). Goat anti-human apolipoprotein B polyclonal antibody conjugated to horseradish peroxidase was kindly provided by Z. Majd (Institut Pasteur, Lille, France).

Protein Production and Purification-- Subconfluent HepG2 cell monolayers were coinfected with vTF7-3 and the vaccinia virus recombinant expressing HCV envelope glycoproteins at a multiplicity of infection of 5 plaque-forming units/cell. At 18 h post-infection, cells were lysed in Tris-buffered saline (TBS; 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA), 0.5% Igepal CA-630. Cell lysates were centrifuged at 20,000 × g for 15 min at 4 °C. In some experiments, metabolic labeling of the proteins was performed as described (9). E1E2 heterodimer was purified by affinity chromatography with mAb H50 covalently coupled either to CNBr-activated Sepharose or to Protein A-Sepharose by using a coupling protocol as described (15). As a prepurification step, cell lysates were successively applied to a Sepharose 4B column and a Sepharose 4B column covalently coupled to an irrelevant mAb. Cell lysates were then applied through the Sepharose H50 column (3.5-ml bed volume) with a flow rate of about 10 ml/h. The column was successively washed with 10 column volumes of lysis buffer, 5 column volumes of a 15 mM triethanolamine (TEA, pH 8.0) solution containing 0.5% deoxycholate, and 5 column volumes of a 15 mM TEA (pH 8.0) solution containing 0.5% octyl glucoside. E1E2 heterodimer was eluted with 15 mM TEA (pH 12) containing 500 mM NaCl and 0.5% octyl glucoside followed by immediate neutralization with 1 M Tris-HCl (pH 6.6). An additional affinity chromatography purification step was performed on GNA-agarose as described (16). Prior to reconstitution, pooled fractions of purified E1E2 were dialyzed against TBS, 60 mM octyl glucoside and concentrated with Spectra/GelTM absorbent beads.

Immunoprecipitation-- SDS-PAGE and immunoprecipitation were carried out as described (17). For quantitative experiments, the gels were analyzed with a PhosphorImager (Molecular Dynamics).

Analysis of E1E2 Binding to CD81 and Lipoproteins-- The CD81 capture enzyme immunoassay was as described (18). For the E1E2-lipoprotein binding assay, low (LDL)(d = 1.030-1.055 kg/liter) and very low density lipoprotein (VLDL)(d < 1.006 kg/liter) were isolated from plasma of normolipidemic subjects by sequential ultracentrifugation as described previously (19). enzyme-linked immunosorbent assay plates were incubated for 18 h at 4 °C with 0.5 µg/well of E1E2 in 100 µl of TBS containing 10 mM octyl glucoside. After being washed in TBS, plates were blocked for 1 h at 37 °C with TBS containing 3% (w/v) essentially free fatty acid bovine serum albumin. Plates were washed twice with TBS followed by the addition of increasing amount of LDL or VLDL in TBS, 1% bovine serum albumin. After 2 h at room temperature, plates were washed three times with TBS, and bound lipoproteins were detected by incubation with a goat anti-human apolipoprotein B polyclonal antibody conjugated to horseradish peroxidase (dilution 1:500) and tetramethylbenzidine substrate.

Incorporation of HCV Envelope Glycoproteins into Lipid Vesicles-- Incorporation of HCV envelope glycoproteins into lipid vesicles was performed as described (20) with some modifications. Briefly, 200 µg of egg yolk phosphatidylcholine (type V-E) as well as a trace amount of [3H]cholesterol were dissolved in chloroform/methanol 2:1(v/v) and mixed with 800 µg of octyl glucoside in a glass tube. After evaporation under N2, the mixture was redissolved twice in diethyl ether and dried. The thin lipid-detergent film was then solubilized in 10 mM Tris-HCl (pH 7.5) containing 140 mM NaCl, 60 mM octyl glucoside, and 50 µg of 35S-labeled E1E2 complexes and incubated for 10 min with gentle agitation at 37 °C to ensure adequate mixing. The resulting clear solution was then extensively dialyzed against 10 mM Tris-HCl (pH 7.5), 140 mM NaCl. Mean diameters of reconstituted vesicles were determined by quasi-elastic light scattering by using a nanosizer apparatus (Coulter counter). Control liposomes were formed in identical experimental conditions but in the absence of glycoproteins.

Purification and Characterization of Reconstituted Vesicles-- E1E2-liposomes were analyzed by floatation in 5-40% (w/v) sucrose gradient in TBS (10 ml). Following centrifugation at 170,000 × g at 8 °C for 26 h in a Beckman SW41 rotor, the presence of lipids and proteins was determined by radioactive counting of each fraction. For proteolytic digestion, E1E2-liposomes (5-10 µg of protein) were incubated with 600 µg of Pronase in 250 µl of TBS at 30 °C for 30 min. Salt extraction of proteins associated with liposomes was performed by adding 0.5 M KCl or 1 M KCl in sucrose gradient prior to floatation analysis as described (21). Alkaline extraction (22) was performed by making the sucrose gradient in a carbonate buffer pH 11.5. The binding of reconstituted vesicles to hCD81 was determined by GST pull-down. Radioactive E1E2-liposomes dialyzed against TBS were first incubated for 2 h at 4 °C with soluble GST-hCD81 or GST-mCD81 at a concentration of 10 µg/ml. The liposomes were then incubated for 2 h at 4 °C with glutathione-Sepharose 4B beads. The beads were washed twice with TBS, and the radioactivity associated with them was determined by liquid scintillation counting. The binding of E1E2-liposomes to LDL was determined by a modification of the immunoprecipitation procedure. Radioactive E1E2-liposomes dialyzed against TBS were first incubated for 2 h at 4 °C with (V)LDL (final concentration, 20 µg/ml). The liposomes were then incubated for 2 h at 4 °C with a rabbit anti-human apolipoprotein B polyclonal antibody bound to protein A-Sepharose beads. The beads were washed twice with TBS, and the radioactivity associated with them was determined by liquid scintillation counting.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Purification and Characterization of HCV Envelope E1E2 Heterodimer-- When using heterologous expression systems, large amounts of misfolded HCV envelope glycoproteins are produced, and crude cellular extract preparations of these proteins cannot be used for biological and biochemical characterization (6). Our idea was therefore to develop a purification procedure that allows the selective isolation of the noncovalent E1E2 heterodimer. To develop this procedure, we analyzed a panel of E2-specific conformation-dependent mAbs that specifically recognize noncovalent E1E2 heterodimers when the two HCV envelope glycoproteins are coexpressed (13). To select antibodies with an appropriate affinity for E1E2 heterodimers, we compared the relative affinity of our mAbs in a quantitative immunoprecipitation assay. mAbs H53 and H50 showed the highest relative affinity for E1E2 heterodimer (data not shown) and were tested for their potential use in immunoaffinity purification. Elution of E1E2 heterodimer from the H50 column required the mildest conditions, and this antibody was therefore used throughout this work to purify HCV envelope glycoproteins. The proteins were eluted from the column by high pH (pH 12) and immediately neutralized. This was followed by an affinity chromatography on GNA-agarose. SDS-PAGE analysis of purified proteins showed two major bands corresponding to the sizes of E1 and E2 (Fig. 1). The heterodimer was purified to greater than 85% purity. Radiolabeled E1E2 complex purified by this protocol was analyzed by immunoprecipitation with conformation-sensitive mAbs (Fig. 1, H33, H50, and H53) and compared with E1E2 heterodimer immunoprecipitated from crude cell lysates. The profiles of HCV envelope glycoproteins were very similar, indicating that no major conformational change had occurred and that E1 and E2 remained associated. It is worth noting that an additional band with an apparent molecular mass of ~95 kDa was systematically observed. It likely represents residual heterodimers that were not dissociated in the Laemmli buffer probably due to interactions between their transmembrane domains (10), as observed for some other transmembrane proteins analyzed by SDS-PAGE (23). We usually recovered between 1 and 5 µg of purified proteins per 107 cells.


View larger version (70K):
[in this window]
[in a new window]
  		Fig. 1.   Analysis of HCV envelope glycoproteins purified by immunoaffinity chromatography. Purified HCV envelope glycoproteins (20 µg) were analyzed by SDS-PAGE (10% acrylamide) and revealed by Coomassie Brilliant Blue staining (Coomassie). Radiolabeled HCV envelope glycoproteins obtained before purification (Cell lysate) or after purification (Purified E1E2) were analyzed by immunoprecipitation with conformation-sensitive mAbs H33, H50, or H53 and separated by SDS-PAGE (10% acrylamide). The sizes (in kilodaltons) of molecular mass markers (MW) are indicated on the right.

Recently, a truncated form of E2 glycoprotein has been shown to interact with human CD81 (hCD81), suggesting that this protein might be a receptor for HCV (24). Since HCV proteins are likely present on virions as oligomers involving both E1 and E2, we were interested to know whether E1E2 heterodimer would also specifically interact with this putative receptor. In addition, interaction with hCD81 is a good indicator of the protein quality because it needs proper folding of E2 to occur (25). To examine the interaction of purified E1E2 heterodimer with hCD81, we used a capture enzyme immunoassay that has been developed previously to characterize the interaction between a truncated form of E2 and hCD81 (18). As shown in Fig. 2, E1E2 heterodimer interacted with GST-hCD81 in a dose-dependent manner. In addition, no interaction was observed between E1E2 heterodimer and mouse CD81 (GST-mCD81), indicating that the binding is specific for human CD81. These data are very similar to those reported for the interactions between a truncated form of E2 and CD81 (14, 18, 25-28). In addition, these observations indicate that the presence of E1 in the heterodimer does not dramatically modify E2-CD81 interaction. Together, these data indicate that we have purified E1E2 heterodimer in a conformation that might be similar to its native intracellular form.


View larger version (16K):
[in this window]
[in a new window]
  		Fig. 2.   Interaction of purified E1E2 heterodimer with human CD81. Increasing amounts of purified E1E2 glycoproteins were assessed for their ability to bind a recombinant fusion protein consisting of GST and the large extra cellular loop of human CD81 (solid circle) or mouse CD81 (open circle) coated in enzyme-linked immunosorbent assay plates at a final concentration of 1.0 µg/ml. Specific interaction was detected with anti-E2 mAb H53 and an anti-mouse IgG horseradish peroxidase-conjugated followed by tetramethylbenzidine substrate; error bars represent the standard error of triplicate samples.

Reconstitution of E1E2 Heterodimer into Liposomes-- One of the major difficulties in studying HCV interactions with the surface of its host cells is the current impossibility of obtaining sufficient amounts of homogeneous viral particles produced in tissue culture. An alternative approach would be to obtain biologically active purified HCV envelope glycoproteins reconstituted into liposomes. We therefore tried to incorporate our immunoaffinity-purified E1E2 heterodimer into liposomes. The products of reconstitution were analyzed by centrifugation on sucrose density gradients, and the insertion of E1E2 heterodimer into liposomes was determined by measuring the radioactivity associated with the lipids (3H-labeled cholesterol tracer) and the proteins (35S-labeled E1E2). As shown in Fig. 3B, a substantial proportion of HCV envelope glycoproteins (57%) was associated with the lipid fraction. Increasing the solubilization of the viral proteins with higher concentrations of octyl glucoside did not improve protein incorporation. We cannot exclude that a fraction of E1E2 proteins has been altered during elution at pH 12. However, if this is the case, reconstitution would selectively eliminate misfolded proteins. Alternatively, due to the potential difficulties in reconstituting membrane proteins, it is also possible that our procedure is not optimal. In the absence of lipids, HCV glycoproteins were found at the bottom of the gradient (Fig. 3E), indicating that the low density of HCV envelope glycoproteins reconstituted in the presence of lipids is due to their association with lipids. To determine whether HCV envelope proteins associated with lipids are integrated into the vesicles or peripherally associated with the surface of these vesicles, a salt extraction with 0.5 M KCl was performed on the vesicles prior to centrifugation. As shown in Fig. 3, B and C, the distributions of lipids and proteins in the gradients were very similar, indicating that HCV envelope glycoproteins are likely integrated into the membranes of the vesicles. Similar results were observed after incubation of E1E2 heterodimer-lipid complexes with 1 M KCl or by alkaline (pH 11.5) extraction (data not shown). To determine the orientation of HCV envelope glycoproteins associated with reconstituted vesicles, we tested the sensitivity of HCV glycoproteins to protease digestion (Fig. 3D). After treatment of reconstituted proteins with Pronase, between 70 and 80% of the 35S signal remained in the bottom of the gradient, indicating that the ectodomains of HCV envelope glycoproteins are accessible from the surface of the vesicles. The residual 35S radioactivity associated with the vesicles is likely due to the presence of methionine and cysteine residues in the transmembrane domains of HCV envelope glycoproteins. SDS-PAGE analysis of the liposomes after Pronase digestion revealed the presence of a diffuse low molecular band of ~6-8 kDa, which might correspond to the transmembrane domains of E1 and E2 (data not shown). The orientation of HCV envelope glycoproteins was also confirmed by a capture assay with mAb H53 or GNA lectin (data not shown). The presence of intact E1E2 heterodimer reconstituted into liposomes was confirmed by SDS-PAGE (Fig. 4). The dynamic light scattering measurements of vesicles prepared with HCV glycoproteins and lipids were similar to protein-free vesicles, but the former had a broader size distribution. Indeed, their average diameter was 170 ± 85 nm, as compared with 131 ± 59 nm in the absence of glycoproteins. Together, these data indicate that E1E2 heterodimer is integrated in the right side-out orientation into the vesicles.


View larger version (21K):
[in this window]
[in a new window]
  		Fig. 3.   Association of purified E1E2 glycoprotein complexes with lipid vesicles. Reconstitution was performed with 200 µg of lipids containing egg phosphatidylcholine, trace amounts of [3H]cholesterol, and 50 µg of 35S-labeled E1E2 proteins. The products of the reconstitution were analyzed by centrifugation on a 5-40% linear sucrose gradients. After fractionation, positions of lipids and proteins were determined by analyzing the radioactivity associated with each fraction. A, analysis of liposomes formed in the absence of proteins. B, reconstitution of E1E2 into liposomes and analysis by floatation in a sucrose gradient. C, HCV envelope proteins reconstituted into vesicles overlaid with sucrose gradient containing 0.5 M KCl prior to centrifugation. D, HCV envelope proteins reconstituted into vesicles digested with Pronase prior to centrifugation. E, control-purified HCV envelope proteins treated as for reconstitution but without lipids.


View larger version (53K):
[in this window]
[in a new window]
  		Fig. 4.   Electrophoresis analysis of the glycoproteins reconstituted into lipid vesicles. After centrifugation in a sucrose gradient, fractions containing liposomes carrying 35S-labeled E1E2 proteins were pooled, dialyzed, and acetone-precipitated. The 35S material associated with the liposomes (lane B) was analyzed by SDS-PAGE (10% acrylamide) and compared with purified HCV envelope glycoproteins obtained before reconstitution (lane A).

Characterization of E1E2 Heterodimer Reconstituted into Liposomes-- To verify whether E1E2 heterodimer was incorporated into liposomes in a functional configuration, we tested the ability of radiolabeled liposomes to specifically interact with human CD81. E1E2-liposomes purified on sucrose gradient were incubated with soluble GST-hCD81 fusion protein, and CD81-E1E2-liposomes complexes were recovered by pull-down with glutathione-Sepharose. The CD81-E1E2-liposomes interaction was analyzed by measuring the amount of radioactivity associated with glutathione-Sepharose beads. As shown in Fig. 5, a strong radioactive signal (3H) was detected when GST-hCD81 was incubated with E1E2-liposomes. In contrast, only a background signal was observed when E1E2-liposomes were incubated with GST-mCD81 or when CD81 was incubated with liposomes devoid of proteins. Similar results were obtained when 35S label was measured (data not shown). Together, these data show that E1E2-liposomes have conserved their capacity to bind human CD81.


View larger version (20K):
[in this window]
[in a new window]
  		Fig. 5.   Interaction between E1E2-liposomes and human CD81. 3H-labeled E1E2-liposomes were purified by floatation in a density gradient, dialyzed to remove the sucrose, and incubated for 2 h at 4 °C with soluble recombinant fusion protein consisting of GST-hCD81 (filled columns) or GST-mCD81 (open columns). 3H-labeled liposomes devoid of proteins were used as negative control. The CD81-liposome complexes were recovered by pull-down with glutathione-Sepharose 4B beads, and the 3H radioactivity associated with the beads was measured. The data represent the means ± S.E. of duplicate measurements within two independent experiments.

Characterization of HCV particles isolated from patient sera has shown that HCV associates with low and very low density lipoproteins (LDL and VLDL) (29-31). Thus, it is possible that the envelope proteins of HCV directly interact with (V)LDL to form HCV-(V)LDL complexes. To test this hypothesis, E1E2-liposomes were incubated with LDL or VLDL, and the interaction was analyzed by immunoprecipitation. However, no specific interaction was observed (data not shown). To determine whether our purified E1E2 heterodimer can interact with lipoproteins, we analyzed its capacity to recognize LDL or VLDL in a capture immunoenzymatic assay. Purified HCV envelope proteins were coated onto the solid phase and incubated with increasing concentrations of LDL or VLDL. Bound lipoproteins were revealed with an HRP-conjugated anti-human apolipoprotein B antibody. LDL and VLDL were shown to bind to E1E2 heterodimer (Fig. 6). This interaction was saturable and was detected at lipoprotein concentrations as low as 6.25 ng/ml. Interestingly, the binding curves of LDL and VLDL were very similar. However, in these experiments, we cannot exclude that, due to the removal of detergent, the transmembrane domains of E1 and E2 are available for interactions with the phospholipids contained in the LDL and VLDL. This would explain why no interaction was detected between E1E2-liposomes and the lipoproteins. Alternatively, we cannot exclude a technical problem in our analyses of the interactions between E1E2-liposomes and the lipoproteins.


View larger version (12K):
[in this window]
[in a new window]
  		Fig. 6.   Interaction of purified E1E2 heterodimer with LDL (open square) and VLDL (solid square). Increasing amounts of purified LDL or VLDL were assessed for their ability to bind purified HCV envelope proteins (5 µg/ml) coated in enzyme-linked immunosorbent assay plates. Specific interaction was detected with a goat anti-human apolipoprotein B polyclonal antibody conjugated to horseradish peroxidase followed by tetramethylbenzidine substrate. Buffer alone (circle) served as a negative control. The data presented are the mean value of two samples from a single experiment; comparable results were obtained in additional experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The envelope proteins of a virus play an essential role in its lifecycle. They participate in the assembly of the infectious particle and also play a crucial role in virus entry by binding to a receptor present on the host cell and inducing fusion between the viral envelope and a host cell membrane. To fulfil these functions, viral envelope proteins have to adopt dramatically different conformations as well as different oligomeric states during the virus lifecycle. Biological and structural studies of such proteins are therefore essential not only to understand how a virus assembles or enters into a cell but also to gain insights into the structural dynamics of proteins. Here, we developed an immunoaffinity purification procedure to isolate the HCV envelope glycoprotein heterodimer. Characterization of this purified protein complex indicates that it binds to several biologically relevant ligands. In addition, E1E2 heterodimer was reconstituted into liposomes, and these virus-like particles exhibit interesting biological features.

HCV envelope proteins have been successfully reconstituted into liposomes. These particles have indeed incorporated HCV envelope proteins in an asymmetric orientation with the ectodomain of the proteins being accessible from the surface of the liposomes. HCV particles isolated from patient sera have been shown to be associated with LDL and VLDL (29-31), and it has been proposed that HCV-(V)LDL complexes might enter cells by using the LDL receptor (32-34). Although purified E1E2 heterodimer interacted with lipoproteins, E1E2-liposomes did not show any interaction with LDL or VLDL in our experiments, suggesting that the region(s) involved in interactions with lipoproteins are not accessible when the proteins are incorporated into liposomes. By using a solid-phase immunoassay, Wünschmann et al. (34) did not find any interaction between the ectodomain of E2 and LDL, suggesting that other region(s) of E1E2 are involved in these interactions. Other data are in favor of interactions with the C-terminal part of E2 and the central domain of E1 (35). Since these regions are hydrophobic, it is possible that they interact with the lipids contained in the LDL. Therefore, a possible interpretation of the observation that E1E2-liposomes do not interact with LDL and VLDL is that these lipoproteins might not be able to interact with HCV particles when the virions are already assembled. This suggests that the lipoproteins might interact before or during the assembly of HCV particles and potentially help in this process. In this regard, it is interesting to note that the budding of HCV particles is thought to occur in the endoplasmic reticulum (36), the compartment where VLDL assembles (37). Further investigation will be necessary to understand how VLDL interacts with HCV particles.

Pronase digestion shows that HCV envelope glycoproteins are oriented mainly with their ectodomain facing outward. This asymmetric orientation has often been obtained in classical reconstitution experiments using large glycoproteins (38-40). It is thought to be due to steric constraints imposed by large glycoproteins on a vesicle of small radius of curvature. Another possible explanation is that, during detergent removal, vesicles formation precedes the association of proteins with lipids (38, 41, 42).

Studying HCV interactions with the cell surface is very difficult. In the absence of a tissue culture system to replicate HCV, it is currently not possible to obtain homogeneous preparations of HCV particles that would be useful to decipher the early steps of HCV lifecycle (binding and entry). Alternative approaches are therefore sorely needed to study these crucial events of the infectious cycle of this major human pathogen. As discussed above, a soluble truncated form of the envelope glycoprotein E2 has first been used to analyze the interactions between HCV envelope and the host cell surface. This has allowed the identification of CD81 as a putative receptor for HCV (24). Although E2-CD81 interaction is specific, the use of a soluble truncated E2 in cell binding experiments is not truly representative of HCV in terms of interaction with the surface of target cells (43) as shown in experiments comparing the binding of E2 with that of HCV isolated from patients (34). In addition, alternative HCV receptors have been proposed (32-34,44). It is indeed very difficult to demonstrate that a cell surface molecule is a virus receptor in the absence of a cell culture replication system. Because HCV envelope glycoproteins form a noncovalent heterodimer (6), it would be preferable to use this protein complex rather than a truncated E2 to study HCV attachment. It is indeed possible that the E1 protein present in the heterodimer plays a role in HCV attachment either by binding directly to a ligand or by modulating receptor binding by E2. Interactions of HCV envelope glycoproteins with the cell surface could also be influenced by the exposure of these proteins on the viral particle. This is the reason why some investigators are trying to obtain HCV virus-like particles. Production of such virus-like particles has already been reported (45, 46). These particles were obtained by expressing HCV structural proteins in insect cells, using a baculovirus expression system. However, they are not secreted, and when harvested from intracellular compartments, they are highly contaminated by cellular proteins. Pseudotyping vesicular stomatitis virus with chimeric HCV envelope glycoproteins that have been modified to be exported to the cell surface has also been used as an alternative approach to obtain HCV virus-like particles (47, 48). Replacement of the transmembrane domains of HCV envelope proteins by the transmembrane domain of vesicular stomatitis virus G protein leads, however, to an absence of HCV envelope glycoprotein oligomerization (48). Pseudotyped viruses containing these proteins are therefore probably not the best candidates to mimic HCV particles.

E1E2-liposomes described here are a new tool to study HCV interactions with the cell surface. Several investigators have successfully incorporated envelope proteins of other viruses into liposomes to investigate early interactions between these viruses and their host cells. Functional proteoliposomes displaying biological activities such as receptor binding or fusion have been described in several cases (49-55). Reconstituted envelope proteins represent therefore an advantageous alternative approach to study virus entry. In the absence of a cell culture system supporting efficient HCV replication, E1E2-liposomes represent a valuable surrogate model to study the molecular requirements for HCV binding to target cells. In addition, such liposomes will be an essential tool to study the humoral immune response against HCV.

    ACKNOWLEDGEMENTS

We thank François Penin and Françoise Jacob-Dubuisson for critical reading of the manuscript, M.-A. Benoit of the Louvain Catholic University for help with the Coulter counter, and André Pillez and Sophana Ung for excellent technical assistance. We are grateful to S. Levy and Z. Majd for providing us with fusion CD81 proteins and anti-apolipoprotein B antibody, respectively.

    FOOTNOTES

* This work was supported by the CNRS, the Institut Pasteur de Lille, the "Réseau National Hépatite" from the French Ministry of Research, a European Regional Development Fund (ERDF), European Union Grant QLK2-1999-00356, and Grant 5651 from the Association pour la Recherche sur le Cancer.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Successively supported by an Association pour la Recherche sur le Cancer and a CNRS fellowship.

Supported by an Agence Nationale de Recherches sur le SIDA fellowship.

|| Successively supported by an Association pour la Recherche sur le Cancer and an Agence Nationale de Recherches sur le SIDA fellowship.

Dagger Dagger To whom correspondence should be addressed: Unité Hépatite C, CNRS-UPR2511, Institut de Biologie de Lille, 1 rue Calmette, BP447, 59021 Lille Cedex, France. Tel.: 33-3-20-87-11-60; Fax: 33-3-20-87-11-11; E-mail: jean.dubuisson@ibl.fr.

Published, JBC Papers in Press, April 5, 2002, DOI 10.1074/jbc.M111020200

    ABBREVIATIONS

The abbreviations used are: HCV, hepatitis C virus; mAb, monoclonal antibody; LDL, low density lipoprotein; VLDL very low density lipoprotein, GNA, G. nivalis; GST, glutathione S-transferase; TBS, Tris-buffered saline; h, human; m, murine.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Houghton, M. (1996) in Fields Virology (Fields, B. N. , Knipe, D. M. , and Howley, P. M., eds), 3rd Ed. , pp. 1035-1058, Lippincott-Raven, Philadelphia, PA
2. Francki, R. I. B., Fauquet, C. M., Knudson, D. L., and Brown, F. (1991) Arch. Virol. 2 (suppl.), 223
3. Reed, K. E., and Rice, C. M. (2000) Curr. Top. Microbiol. Immunol. 242, 55-84[Medline] [Order article via Infotrieve]
4. Xu, Z., Choi, J., Yen, T. S., Lu, W., Strohecker, A., Govindarajan, S., Chien, D., Selby, M. J., and Ou, J. H. (2001) EMBO J. 20, 3840-3848[CrossRef][Medline] [Order article via Infotrieve]
5. Flint, M., and McKeating, J. A. (2000) Rev. Med. Virol. 10, 101-117[CrossRef][Medline] [Order article via Infotrieve]
6. Deleersnyder, V., Pillez, A., Wychowski, C., Blight, K., Xu, J., Hahn, Y. S., Rice, C. M., and Dubuisson, J. (1997) J. Virol. 71, 697-704[Abstract]
7. Duvet, S., Cocquerel, L., Pillez, A., Cacan, R., Verbert, A., Moradpour, D., Wychowski, C., and Dubuisson, J. (1998) J. Biol. Chem. 273, 32088-32095[Abstract/Free Full Text]
8. Dubuisson, J. (2000) Curr. Top. Microbiol. Immunol. 242, 135-148[Medline] [Order article via Infotrieve]
9. Dubuisson, J., Hsu, H. H., Cheung, R. C., Greenberg, H. B., Russell, D. G., and Rice, C. M. (1994) J. Virol. 68, 6147-6160[Abstract/Free Full Text]
10. Op De Beeck, A., Montserret, R., Duvet, S., Cocquerel, L., Cacan, R., Barberot, B., Le, Maire, M., Penin, F., and Dubuisson, J. (2000) J. Biol. Chem. 275, 31428-31437[Abstract/Free Full Text]
11. Fuerst, T. R., Niles, E. G., Studier, F. W., and Moss, B. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 8122-8126[Abstract/Free Full Text]
12. Cocquerel, L., Meunier, J.-C., Pillez, A., Wychowski, C., and Dubuisson, J. (1998) J. Virol. 72, 2183-2191[Abstract/Free Full Text]
13. Patel, A. H., Wood, J., Penin, F., Dubuisson, J., and McKeating, J. A. (2000) J. Gen. Virol. 81, 2873-2883[Abstract/Free Full Text]
14. Higginbottom, A., Quinn, E. R., Kuo, C. C., Flint, M., Wilson, L. H., Bianchi, E., Nicosia, A., Monk, P. N., McKeating, J. A., and Levy, S. (2000) J. Virol. 74, 3642-3649[Abstract/Free Full Text]
15. Harlow, E., and Lane, D. P. (1988) Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
16. Ralston, R., Thudium, K., Berger, K., Kuo, C., Gervase, B., Hall, J., Selby, M., Kuo, G., Houghton, M., and Choo, Q.-L. (1993) J. Virol. 67, 6753-6761[Abstract/Free Full Text]
17. Dubuisson, J., and Rice, C. M. (1996) J. Virol. 70, 778-786[Abstract]
18. Flint, M., Dubuisson, J., Maidens, C., Harrop, R., Guile, G. R., Borrow, P., and McKeating, J. A. (2000) J. Virol. 74, 702-709[Abstract/Free Full Text]
19. Havel, R. J., Eders, H. S., and Bradgon, J. H. (1955) J. Clin. Invest. 34, 1345-1353[Medline] [Order article via Infotrieve]
20. Helenius, A., Fries, E., and Kartenbeck, J. (1977) J. Cell Biol. 75, 866-880[Abstract/Free Full Text]
21. Curman, B., Klareskog, L., and Peterson, P. A. (1980) J. Biol. Chem. 255, 7820-7826[Abstract/Free Full Text]
22. Fujiki, Y., Hubbard, A. L., Fowler, S., and Lazarow, P. B. (1982) J. Cell Biol. 93, 97-102[Abstract/Free Full Text]
23. Lemmon, M. A., Flanagan, J. M., Hunt, J. F., Adair, B., Bormann, B. J., Dempsey, C., and Engelman, D. (1992) J. Biol. Chem. 267, 7683-7689[Abstract/Free Full Text]
24. Pileri, P., Uematsu, Y., Campagnoli, S., Galli, G., Falugi, F., Petracca, R., Weiner, A. J., Houghton, M., Rosa, D., Grandi, G., and Abrignani, S. (1998) Science 282, 938-941[Abstract/Free Full Text]
25. Flint, M., Maidens, C., Loomis-Price, L. D., Shotton, C., Dubuisson, J., Monk, P., Higginbottom, A., Levy, S., and McKeating, J. A. (1999) J. Virol. 73, 6235-6244[Abstract/Free Full Text]
26. Heile, J. M., Fong, Y. L., Rosa, D., Berger, K., Saletti, G., Campagnoli, S., Bensi, G., Capo, S., Coates, S., Crawford, K., Dong, C., Wininger, M., Baker, G., Cousens, L., Chien, D., Ng, P., Archangel, P., Grandi, G., Houghton, M., and Abrignani, S. (2000) J. Virol. 74, 6885-6892[Abstract/Free Full Text]
27. Petracca, R., Falugi, F., Galli, G., Norais, N., Rosa, D., Campagnoli, S., Burgio, V., Di, Stasio, E., Giardina, B., Houghton, M., Abrignani, S., and Grandi, G. (2000) J. Virol. 74, 4824-4830[Abstract/Free Full Text]
28. Allander, T., Forns, X., Emerson, S. U., Purcell, R. H., and Bukh, J. (2000) Virology 277, 358-367[CrossRef][Medline] [Order article via Infotrieve]
29. Prince, A. M., Huima-Byron, T., Parker, T. S., and Levine, M. M. (1996) J. Viral Hepat. 3, 11-17[Medline] [Order article via Infotrieve]
30. Thomssen, R., Bonk, S., Propfe, C., Heermann, K. H., Kochel, H. G., and Uy, A. (1992) Med. Microbiol. Immunol. 181, 293-300[CrossRef][Medline] [Order article via Infotrieve]
31. Thomssen, R., Bonk, S., and Thiele, A. (1993) Med. Microbiol. Immunol. 182, 329-334[Medline] [Order article via Infotrieve]
32. Agnello, V., Abel, G., Elfahal, M., Knight, G. B., and Zhang, Q.-X. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 12766-12771[Abstract/Free Full Text]
33. Monazahian, M., Bohme, I., Bonk, S., Koch, A., Scholz, C., Grethe, S., and Thomssen, R. (1999) J. Med. Virol. 57, 223-229[CrossRef][Medline] [Order article via Infotrieve]
34. Wünschmann, S., Medh, J. D., Klinzmann, D., Schmidt, W. N., and Stapleton, J. T. (2000) J. Virol. 74, 10055-10062[Abstract/Free Full Text]
35. Monazahian, M., Kippenberger, S., Muller, A., Seitz, H., Bohme, I., Grethe, S., and Thomssen, R. (2000) Med. Microbiol. Immunol. 188, 177-184[CrossRef][Medline] [Order article via Infotrieve]
36. Op De Beeck, A., Cocquerel, L., and Dubuisson, J. (2001) J. Gen. Virol. 82, 2589-2595[Free Full Text]
37. Shelness, G. S., and Sellers, J. A. (2001) Curr. Opin. Lipidol. 12, 151-157[CrossRef][Medline] [Order article via Infotrieve]
38. Cornet, B., Decroly, E., Thines-Sempoux, D., Ruysschaert, J. M., and Vandenbranden, M. (1992) AIDS Res. Hum. Retroviruses 8, 1823-1831[Medline] [Order article via Infotrieve]
39. Helenius, A., Sarvas, M., and Simons, K. (1981) Eur. J. Biochem. 116, 27-35[Medline] [Order article via Infotrieve]
40. Petri, W. A., Jr., and Wagner, R. R. (1979) J. Biol. Chem. 254, 4313-4316[Abstract/Free Full Text]
41. Madden, T. D. (1986) Chem. Phys. Lipids 40, 207-222[CrossRef][Medline] [Order article via Infotrieve]
42. Madden, T. D. (1988) Int. J. Biochem. 20, 889-895[CrossRef][Medline] [Order article via Infotrieve]
43. Hamaia, S., Li, C., and Allain, J. P. (2001) Blood 98, 2293-2300
44. Flint, M., Quinn, E. R., and Levy, S. (2001) Clin. Liver Dis. 5, 873-893[CrossRef][Medline] [Order article via Infotrieve]
45. Baumert, T. F., Ito, S., Wong, D. T., and Liang, T. J. (1998) J. Virol. 72, 3827-3836[Abstract/Free Full Text]
46. Owsianka, A., Clayton, R. F., Loomis-Price, L. D., McKeating, J. A., and Patel, A. H. (2001) J. Gen. Virol. 82, 1877-1883[Abstract/Free Full Text]
47. Lagging, L. M., Meyer, K., Owens, R. J., and Ray, R. (1998) J. Virol. 72, 3539-3546[Abstract/Free Full Text]
48. Matsuura, Y., Tani, H., Suzuki, K., Kimura-Someya, T., Suzuki, R., Aizaki, H., Ishii, K., Moriishi, K., Robison, C. S., Whitt, M. A., and Miyamura, T. (2001) Virology 286, 263-275[CrossRef][Medline] [Order article via Infotrieve]
49. Eidelman, O., Schlegel, R., Tralka, T. S., and Blumenthal, R. (1984) J. Biol. Chem. 259, 4622-4628[Abstract/Free Full Text]
50. Harmsen, M. C., Wilschut, J., Scherphof, G., Hulstaert, C., and Hoekstra, D. (1985) Eur. J. Biochem. 149, 591-599[Medline] [Order article via Infotrieve]
51. Huang, R. T., Wahn, K., Klenk, H. D., and Rott, R. (1980) Virology 104, 294-302[CrossRef][Medline] [Order article via Infotrieve]
52. Metsikko, K., van Meer, G., and Simons, K. (1986) EMBO J. 5, 3429-3435[Medline] [Order article via Infotrieve]
53. Nussbaum, O., Lapidot, M., and Loyter, A. (1987) J. Virol. 61, 2245-2252[Abstract/Free Full Text]
54. Scheule, R. K. (1986) Biochemistry 25, 4223-4232[CrossRef][Medline] [Order article via Infotrieve]
55. Stegmann, T., Morselt, H. W., Booy, F. P., van Breemen, J. F., Scherphof, G., and Wilschut, J. (1987) EMBO J. 6, 2651-2659[Medline] [Order article via Infotrieve]

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 GutHome page
M. Jones and J. S Owen
How does interferon inhibit HCV cell entry?
Gut, May 1, 2008; 57(5): 573 - 574.
[Full Text] [PDF]

Home page
 J. Virol.Home page
H. Barth, E. K. Schnober, F. Zhang, R. J. Linhardt, E. Depla, B. Boson, F.-L. Cosset, A. H. Patel, H. E. Blum, and T. F. Baumert
Viral and Cellular Determinants of the Hepatitis C Virus Envelope-Heparan Sulfate Interaction
J. Virol., November 1, 2006; 80(21): 10579 - 10590.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
H. Barth, R. Cerino, M. Arcuri, M. Hoffmann, P. Schurmann, M. I. Adah, B. Gissler, X. Zhao, V. Ghisetti, B. Lavezzo, et al.
Scavenger Receptor Class B Type I and Hepatitis C Virus Infection of Primary Tupaia Hepatocytes
J. Virol., May 1, 2005; 79(9): 5774 - 5785.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
J. A. McKeating, L. Q. Zhang, C. Logvinoff, M. Flint, J. Zhang, J. Yu, D. Butera, D. D. Ho, L. B. Dustin, C. M. Rice, et al.
Diverse Hepatitis C Virus Glycoproteins Mediate Viral Infection in a CD81-Dependent Manner
J. Virol., August 15, 2004; 78(16): 8496 - 8505.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
A. Op De Beeck, C. Voisset, B. Bartosch, Y. Ciczora, L. Cocquerel, Z. Keck, S. Foung, F.-L. Cosset, and J. Dubuisson
Characterization of Functional Hepatitis C Virus Envelope Glycoproteins
J. Virol., March 15, 2004; 78(6): 2994 - 3002.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
J. Zhang, G. Randall, A. Higginbottom, P. Monk, C. M. Rice, and J. A. McKeating
CD81 Is Required for Hepatitis C Virus Glycoprotein-Mediated Viral Infection
J. Virol., February 1, 2004; 78(3): 1448 - 1455.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
L. Cocquerel, C.-C. Kuo, J. Dubuisson, and S. Levy
CD81-Dependent Binding of Hepatitis C Virus E1E2 Heterodimers
J. Virol., October 1, 2003; 77(19): 10677 - 10683.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
M. Hsu, J. Zhang, M. Flint, C. Logvinoff, C. Cheng-Mayer, C. M. Rice, and J. A. McKeating
Hepatitis C virus glycoproteins mediate pH-dependent cell entry of pseudotyped retroviral particles
PNAS, June 10, 2003; 100(12): 7271 - 7276.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
L. Cocquerel, E. R. Quinn, M. Flint, K. G. Hadlock, S. K. H. Foung, and S. Levy
Recognition of Native Hepatitis C Virus E1E2 Heterodimers by a Human Monoclonal Antibody
J. Virol., December 20, 2002; 77(2): 1604 - 1609.
[Abstract] [Full Text] [PDF]

This Article