Coexpression of hepatitis C virus envelope proteins E1 and E2 in cis improves the stability of membrane insertion of E2

Laurence Cocquerel1, Jean-Christophe Meunier1, Anne Op de Beeck1, Dorine Bonte1, Czeslaw Wychowski1 and Jean Dubuisson1

CNRS-FRE2369, Equipe Hépatite C, IBL/Institut Pasteur de Lille, 1 rue du Professeur Calmette, BP447, 59021 Lille Cedex, France1

Author for correspondence: Jean Dubuisson. Fax +33 3 20 87 11 11. e-mail jean.dubuisson{at}ibl.fr


   Abstract
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Abstract
Introduction
Methods
Results
Discussion
References
 
The hepatitis C virus (HCV) genome encodes two envelope glycoproteins, E1 and E2. These proteins contain a large N-terminal ectodomain, and are anchored into membranes by their C-terminal transmembrane domain (TMD). The TMDs of HCV envelope proteins are multifunctional. In addition to their role as membrane anchors, they possess a signal sequence function in their C-terminal half, and play a major role in subcellular localization and assembly of these envelope proteins. In this work, the expression of full-length E2 led to secretion of a proportion of this protein, which is likely to be due to inefficient membrane insertion of a fraction of E2 expressed alone. However, when E1 and E2 were coexpressed from the same polyprotein, E2 was not secreted and remained tightly associated with membranes, suggesting that an early interaction between the TMDs of HCV envelope proteins improves the stability of membrane insertion of E2. These results reinforce the hypothesis that the TMDs of E1 and E2 are major factors in the assembly of the HCV envelope glycoprotein complex.


   Introduction
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Abstract
Introduction
Methods
Results
Discussion
References
 
Hepatitis C virus (HCV) encodes two membrane-associated envelope glycoproteins, E1 and E2, which are N-glycosylated in their large N-terminal ectodomains and anchored into membranes by their C-terminal hydrophobic sequences (Dubuisson, 2000 ). These two proteins have been shown to assemble as a noncovalent E1E2 heterodimer (Deleersnyder et al., 1997 ). This heterodimer is retained in the endoplasmic reticulum (ER) and is believed to be the prebudding form of HCV envelope protein complex. Expression of chimeric proteins in which HCV envelope protein domains were exchanged with corresponding domains of proteins normally transported to the plasma membrane has shown that the transmembrane domains (TMDs) of E1 and E2 play a major role in the subcellular localization of the native E1E2 complex (Cocquerel et al., 1998 , 1999 ; Flint & McKeating, 1999 ; Flint et al., 1999 ). In addition, the glycans of HCV envelope proteins are not modified by Golgi enzymes, indicating that the TMDs of E1 and E2 are responsible for true retention in the ER without recycling through the Golgi apparatus (Cocquerel et al., 1999 ; Duvet et al., 1998 ).

Besides their role as membrane anchors and ER retention signals, the TMDs of HCV envelope proteins contain a signal sequence function in their C-terminal half, and they are involved in assembly of the noncovalent E1E2 heterodimer (Cocquerel et al., 1998 , 2000 ; Michalak et al., 1997 ; Op De Beeck et al., 2000 ; Selby et al., 1994 ). This multifunctionality highlights the crucial role played by these TMDs in the biogenesis of the prebudding form of HCV envelope glycoprotein complex. These domains are composed of two stretches of hydrophobic residues separated by a short segment containing one (E1) or two (E2) fully conserved charged residues. Recent data from our laboratory have shown that replacement of these charged residues by an alanine leads to an alteration of all the functions played by these TMDs (Cocquerel et al., 2000 ), indicating that these functions are tightly linked together.

In this study, we show that a proportion of HCV envelope protein E2 is secreted when this protein is expressed in the absence of E1. Our data indicate that this is due to inefficient membrane insertion of a fraction of E2 expressed alone. However, when E1 and E2 were coexpressed from the same polyprotein, E2 remained tightly associated with membranes, suggesting that an interaction between the TMDs of HCV envelope proteins helps the insertion of E2 into the ER membrane.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Cell culture and antibodies.
HepG2 and BHK-21 cell lines were obtained from the ATCC. HepG2 and BHK-21 cells were grown in Dulbecco’s modified essential medium and {alpha}-minimal essential medium, respectively. Both media were supplemented with 10% foetal bovine serum.

Monoclonal antibodies (MAbs) A4 [anti-E1 (Dubuisson et al., 1994 )], H53 [anti-E2 conformation-sensitive MAb (Cocquerel et al., 1998 )] and H47 [anti-E2 conformation-insensitive MAb; (A. Pillez & J. Dubuisson, unpublished data)] were produced in vitro by using a MiniPerm apparatus (Heraeus) as recommended by the manufacturer. Polyclonal anti-HA (HA11) antibody was purchased from Eurogentec.

{blacksquare} Viruses.
The following vaccinia virus recombinants have been described previously: vTF7-3 (expressing the T7 DNA-dependent RNA polymerase) (Fuerst et al., 1986 ), vHCV1-3011 (expressing the entire polyprotein of the HCV-H strain) (Lin et al., 1994 ), vE1E2p7 (expressing the signal sequence of E1, E1, E2 and the p7 polypeptide) (Fournillier-Jacob et al., 1996 ), vE2p7 (expressing the signal sequence of E2, E2 and the p7 polypeptide) (Fournillier-Jacob et al., 1996 ), vE2 (expressing E2 with its signal sequence) (Cocquerel et al., 2000 ), vE2(715) (expressing the signal sequence of E2 and the entire ectodomain of E2 without its TMD) (Michalak et al., 1997 ), vE1 (expressing the C-terminal 60 amino acids of the capsid protein and E1) (Fournillier-Jacob et al., 1996 ) and vE1*E2 (expressing E1E2 polyprotein with an alanine residue inserted at position 358 in the TMD of E1) (Op De Beeck et al., 2000 ). The genes of HCV proteins expressed in this work are under the control of a T7 promoter and expression of the proteins of interest is achieved by coinfection with vTF7-3.

{blacksquare} Metabolic labelling, immunoprecipitation and endoglycosidase digestion.
HepG2 cells expressing HCV proteins were metabolically labelled with 35S-Protein Labelling Mix (3·7x106 Bq/ml) as described previously (Dubuisson et al., 1994 ). Supernatants were harvested and cells were lysed with 0·5% Igepal CA-630 in TBS (50 mM Tris–HCl, pH 7·5, 150 mM NaCl). Immunoprecipitations were carried out as described (Dubuisson & Rice, 1996 ). Gel autoradiographs were exposed in the linear range and analysed by densitometric scanning. Quantification by phosphorimaging was also done. For endoglycosidase digestion, immunoprecipitated proteins were eluted from protein A–Sepharose in 30 µl of dissociation buffer (0·5% SDS and 1% 2-mercaptoethanol) by boiling for 10 min. The protein samples were then divided into equal portions for digestion with endo-{beta}-N-acetylglucosaminidase H (endo H) or peptide:N-glycosidase F (PNGase F) and an undigested control. Digestions were carried out for 1 h at 37 °C in the buffer provided by the manufacturer. Digested samples were mixed with an equal volume of 2x Laemmli sample buffer and analysed by SDS–PAGE.

{blacksquare} Sedimentation through sucrose gradients.
Supernatant from labelled infected cells was layered on a 10 ml gradient of 5–35% sucrose in TBS with or without 0·1% NP-40. Following overnight centrifugation at 4 °C at 36000 r.p.m. in a Beckman SW41 rotor, 13 fractions were collected from the bottom of the gradient and analysed by immunoprecipitation as described above.

{blacksquare} Sodium carbonate extraction of membranes.
At 5 h post-infection, infected cells were washed twice with 0·25 M sucrose, 5 mM HEPES buffer pH 6·8, resuspended in the same buffer and disrupted using a Dounce homogenizer (30 strokes). The homogenates were centrifuged for 10 min at 2000 r.p.m. to remove intact cells and nuclei. Supernatants were spun for 15 min at 65000 r.p.m. in a Beckman TL-100 centrifuge. Membrane pellets were resuspended in 0·5 ml 0·1 M sodium carbonate, pH 11·3, using a Dounce homogenizer (10 strokes) and incubated for 30 min on ice. The extracted proteins were separated from membranes by an additional centrifugation for 15 min at 65000 r.p.m. Membranes were again resuspended in 0·5 ml 0·1M sodium carbonate, pH 11·3. After neutralization to pH 7 by addition of 1 M HCl, samples were treated with 0·5% Triton X-100. Membrane-bound and soluble proteins were analysed by Western blotting.

{blacksquare} Western blotting.
Proteins bound to nitrocellulose membranes were revealed by enhanced chemiluminescence detection (ECL Plus; Amersham Pharmacia) as recommended by the manufacturer. Briefly, after separation by SDS–PAGE under reducing conditions, proteins were transferred to nitrocellulose membranes by using a Trans-Blot apparatus (Bio-Rad) and revealed with a specific MAb (H47; dilution 1/5000) followed by rabbit anti-mouse immunoglobulin conjugated to peroxidase (Biosys; dilution 1/1000).


   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
A fraction of E2 expressed alone is slowly released into the supernatant of tissue culture
It has been shown by members of our laboratory that E2 expressed in the absence of E1 can fold properly (Michalak et al., 1997 ), and that this protein expressed alone is retained in the ER (Cocquerel et al., 1998 ; Duvet et al., 1998 ). Indeed, pulse–chase experiments indicated that, after a 4 h chase, no E2 molecule had left the ER compartment. Here, we wanted to determine the fate of this protein over longer periods of time.

HepG2 cells infected by a vaccinia virus recombinant expressing E2 were pulse-labelled for 10 min and chased for different times. The presence of E2 in the cellular fraction and the supernatant was analysed by immunoprecipitation with a conformation-sensitive E2-specific MAb (H53). The amount of E2 detected in the intracellular fraction was very low during the pulse and increased during the first 4 h of chase as previously observed (Cocquerel et al., 1998 ; Michalak et al., 1997 ) (Fig. 1, intracellular). This reflects the slow folding of E2. After 8 and 12 h of chase, the intensity of the band corresponding to E2 decreased slightly. It is worth noting that a faint diffuse band was observed above the intracellular form of E2 after 4 to 12 h of chase. This band might contain E2 molecules with modified glycans which have moved through the secretory pathway. However, it cannot be clearly separated from background signals. No secreted form of E2 was detected during the first 4 h of chase as previously reported (Cocquerel et al., 1998 ). However, a diffuse band (E2s) migrating more slowly than the intracellular form of E2 was detected in the supernatant after 8 and 12 h of chase (Fig. 1, supernatant). Similar results were observed when E2 was expressed in BHK-21 cells with a Sindbis virus vector (data not shown), indicating that secretion of a fraction of E2 is independent of the expression system used. It is worth noting that CD4 expressed by a vaccinia virus recombinant was not detected in the tissue culture supernatant (data not shown). Together, these data indicate that secretion of E2 is an intrinsic property of this protein.



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Fig. 1. Expression of E2 analysed in pulse–chase experiments. HepG2 cells were coinfected with vTF7-3 and a vaccinia virus recombinant expressing E2 (vE2) at an m.o.i. of 5 p.f.u. per cell. At 4·5 h post-infection, cells were pulse-labelled for 10 min and chased for the indicated times (in hours). Cells and supernatants were immunoprecipitated separately with MAb H53. Immunoprecipitates were analysed by SDS–PAGE (10% polyacrylamide). The sizes (in kDa) of protein molecular mass markers are indicated on the left. E2s, secreted form of E2.

 
The difference in electrophoretic mobility between intracellular E2 and its secreted form is due to modification of E2-associated glycans during transport in the secretory pathway. Indeed, as shown in Fig. 2, the intracellular form of E2 remained endo H sensitive, due to its retention in the ER (Cocquerel et al., 1998 ). In contrast, similar to what was observed for the secreted form of E2 deleted of its TMD (E2-715), E2s was resistant to endo H digestion, indicating that this protein has moved through the secretory pathway. The E2s protein might have been released into the supernatant of cells expressing full-length E2 after cleavage of its membrane anchor by an intracellular protease. However, an E2 protein with an HA epitope tag at its C terminus and expressed in HepG2 cells was immunoprecipitated from the tissue culture supernatant with an anti-HA antibody (data not shown), confirming that the TMD of E2 is still present in its secreted form.



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Fig. 2. Sensitivity of E2s to endo H treatment. HepG2 cells coinfected with vTF7-3 and a vaccinia virus recombinant expressing the full-length (E2) or a C-terminal truncated form of E2 (E2-715) were labelled from 4 to 20 h post-infection. Cells and supernatants were immunoprecipitated separately with MAb H53, and immunoprecipitates were treated or not with endo H. Samples were analysed by SDS–PAGE (10% polyacrylamide). The intracellular form of E2 is indicated by an ‘i’ and the extracellular form by an ‘s’. Deglycosylated proteins are indicated by an asterisk.

 
Since expression of the envelope proteins prM and E of some flaviviruses can lead to secretion of pseudoparticles composed of the envelope proteins embedded in membranes (Heinz & Allison, 2000 ), we wondered whether this would also be the case for E2 expressed alone. For this purpose, E2s was analysed by sedimentation through a sucrose gradient in the presence or absence of detergent to see whether the presence of detergent would lead to a change in the sedimentation of E2s as described for prME of Japanese encephalitis virus (Konishi et al., 1992 ). As shown in Fig. 3, no change in the sedimentation behaviour of E2s was observed in the presence of NP-40. Additional experiments indicated that E2s sedimented as a monomer (data not shown). Together, these data suggest that E2s is not associated with lipids in the form of pseudoparticles.



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Fig. 3. Sedimentation of E2s through sucrose gradients. HepG2 cells coinfected with vTF7-3 and a vaccinia virus recombinant expressing E2 (vE2) were labelled from 4 to 20 h post-infection. Supernatants were sedimented through sucrose gradients (5–35%) with or without NP-40 as described in Methods. Fractions were collected from the bottom and immunoprecipitated with MAb H53. Immunoprecipitates were analysed by SDS–PAGE (10% polyacrylamide). Molecular mass markers are indicated on the right.

 
Altogether, these data indicate that a fraction of a presumably properly folded form of E2 is secreted, suggesting that a fraction of this protein might not be inserted into membranes.

Membrane insertion of E2 is assisted by the presence of E1
Since E2 is produced after cleavage of HCV polyprotein and interacts with E1 in the ER, we wanted to know whether E2 expressed from the polyprotein would also be secreted. To test this hypothesis, we monitored the release of E2 into the supernatants of cells infected with vaccinia virus recombinants expressing full-length or truncated forms (E1E2p7, E2p7 or E2) of HCV polyprotein. The vaccinia virus recombinant expressing E2-715 was used as a control for secretion. As shown in Fig. 4, the intensities of secreted E2 were similar in the presence or absence of p7, suggesting that the presence of p7 does not interfere with the secretion of E2. Interestingly, E2 was barely detectable in the supernatants of cells infected with vaccinia virus recombinants expressing the full-length HCV polyprotein or E1E2p7. Quantitative analyses indicated that 12 to 14% of E2 was secreted in the supernatant when expressed alone or as an E2p7 polyprotein, whereas only 4% of E2 was detected when HCV envelope proteins were coexpressed (Fig. 4, compare E1E2p7 with E2 and E2p7). These data indicate that coexpression with E1 reduces the secretion of E2.



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Fig. 4. Effect of coexpression of E1 on secretion of E2. HepG2 cells coinfected with vTF7-3 and the appropriate vaccinia virus recombinant were labelled from 4 to 20 h post-infection. Cells and supernatants were immunoprecipitated separately with MAb H53. Immunoprecipitates were analysed by SDS–PAGE (10% polyacrylamide). For each recombinant vaccinia virus, the percentages of intracellular and secreted E2 were evaluated by phosphorimaging (bottom panel). Each column represents the mean value of three different experiments (with error bar).

 
Secretion of a proportion of E2 expressed alone suggests that, in the absence of E1, the nature of the association of E2 with ER membranes may be altered. This possibility was examined by treatment of membranes with 0·1 M sodium carbonate, pH 11·3, as described by Howell & Palade (1982) and Fujiki et al. (1982) . This procedure results in the extraction of lumenal and peripheral proteins, while integral membrane proteins remain bound to the membranes. For this purpose, HepG2 cells were infected with vaccinia virus recombinants expressing E2 alone or in association with E1 and treated as described in Methods. After separation of membrane-bound (M) and soluble (S) proteins by SDS–PAGE under reducing conditions, E2 was revealed by Western blotting with a conformation-insensitive E2-specific MAb (H47) (Fig. 5). When expressed as an E1E2 polyprotein, only 6·2% of E2 was detected in the soluble fraction, confirming previous observations (Ralston et al., 1993 ). However, when expressed in the absence of E1, 22·6% of E2 was isolated in the soluble fraction (Fig. 5). These data suggest that a proportion of E2 expressed alone is loosely associated with the lipid bilayer. It is worth noting that coexpression of E1 and E2 in trans did not lead to the same stability of membrane insertion as when expressed in cis. Similarly, the insertion of an alanine residue in the TMD of E1, which impairs the assembly of the heterodimer (Op De Beeck et al., 2000 ), reduced the efficiency of membrane insertion of E2 (Fig. 5; E1*E2), indicating that the interaction between the TMDs is essential to improve the membrane insertion of E2.



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Fig. 5. Membrane integration of E2. HepG2 cells were coinfected with vTF7-3 and the appropriate vaccinia virus recombinant(s). At 5 h post-infection, membrane fractions were prepared as described in Methods and treated with 0·1 M sodium carbonate, pH 11·3. After separation of membrane-bound (M) and soluble (S) proteins, the presence of E2 in these fractions was revealed by Western blotting with the anti-E2 MAb H47. Quantitative analyses were performed with the Personal Densitometer SI and Image Quant 5.0 (Molecular Dynamics) (bottom panel). For each recombinant vaccinia virus, the percentages of membrane-bound and soluble E2 were evaluated. Each column represents the mean value of three different experiments (with error bar).

 
Heterodimerization of E1 and E2 occurs preferentially in cis
As shown above, expression of E1 and E2 from different translation products (expression in trans) leads to a less efficient integration of E2 into the lipid bilayer, and we wanted to know whether this might have some consequences for the assembly of the noncovalent heterodimer. We have previously shown that the noncovalent heterodimer can be formed when HCV envelope proteins are expressed in trans (Cocquerel et al., 2000 ). However, the efficiency of assembly of these proteins expressed in trans has not been compared to the efficiency of oligomerization of proteins expressed from the same translation product (expression in cis). To analyse the association of these envelope proteins, HepG2 cells infected with vaccinia virus recombinants expressing E1E2, or E1 and E2 were pulse-labelled for 10 min and chased for 4 h. These conditions have been previously shown to correspond to the peak of detection of the noncovalent heterodimer (Deleersnyder et al., 1997 ). The oligomerization of HCV envelope proteins was analysed by immunoprecipitation with the conformation-sensitive E2-specific MAb H53 (Fig. 6). To be sure that all the cells would express both E1 and E2 when produced by different vaccinia virus recombinants, they were infected with an m.o.i. of 5 p.f.u. per cell. The expression of E1 and E2 was confirmed by immunoprecipitation with conformation-insensitive MAbs to E1 (A4) and E2 (H47) (Fig. 6, A4 and H47). Since MAb H53 is E2-specific, and because E2 can fold independently of E1 (Michalak et al., 1997 ), the amount of E1 coprecipitated by MAb H53 is a good indicator of the assembly of the noncovalent heterodimer. It has to be noted that the intensity of E2 was lower when expressed in trans. This is likely to be due to a difference in expression levels between the vaccinia virus recombinants expressing E1E2 and E2 alone. However, immunoprecipitation with MAb A4 revealed similar levels of expression of E1 expressed in cis and in trans, indicating that the amount of E1 molecules available for assembly is not limiting when E1 and E2 are expressed in trans. As previously observed with MAb H53 (Cocquerel et al., 2000 ; Duvet et al., 1998 ), E1 was coprecipitated with E2 when HCV envelope proteins were expressed in cis or in trans (Fig. 6, H53). To evaluate the percentage of heterodimerization, E1/E2 ratios were measured. As shown in Fig. 6, this ratio was repeatedly reduced by about 40% when E1 and E2 were expressed in trans. Together, these results suggest that the formation of native E1E2 heterodimer occurs preferentially between E1 and E2 that originate from the same translation product.



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Fig. 6. Comparison of the heterodimerization of E1 and E2 expressed in cis or in trans. HepG2 cells coinfected with vTF7-3 and the appropriate vaccinia virus recombinant at an m.o.i. of 5 p.f.u. per cell were pulse-labelled for 10 min and chased for 4 h. Cells were used for immunoprecipitation with MAbs H47 (conformation-insensitive anti-E2 MAb), A4 (conformation-insensitive anti-E1 MAb) or H53 (conformation-sensitive anti-E2 MAb). Immunoprecipitates were analysed by SDS–PAGE (10% polyacrylamide) under reducing (MAbs H47 and A4) or nonreducing conditions (MAb H53). To compare the heterodimerization reaction of E1 and E2 expressed in cis or in trans, the intensities of E1 and E2 precipitated by MAb H53 were measured by phosphorimaging and the E1/E2 ratios were calculated in each case (bottom panel). The percentage of noncovalent complex formed was calculated as follows: (E1/E2 ratio for the proteins of interest)/(E1/E2 ratio for E1 and E2 expressed in cis).

 

   Discussion
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Abstract
Introduction
Methods
Results
Discussion
References
 
The TMDs of E1 and E2 are major determinants for heterodimerization (Op De Beeck et al., 2000 ). However, it is likely that they are not the only determinants for E1E2 assembly. Indeed, for many viral envelope proteins the ectodomains have been shown to be involved in oligomerization (Doms, 1990 ), and this is important for regulation of the fusogenic function of these proteins (Hernandez et al., 1996 ). It is possible that a rapid contact between E1 and E2, probably initiated by their TMDs, is necessary to bring their ectodomains into contact. Here, we show that a fraction of E2 expressed alone is not efficiently integrated into membrane. However, when E1 and E2 are coexpressed from the same polyprotein, the stability of membrane insertion of E2 is improved. Although only a fraction of E2 (22·6%) is not integrated into membranes, the improvement of membrane integration of E2 by E1 gives some clues as to the early events occurring during the biogenesis of HCV envelope proteins. Together with the observation that the TMDs of HCV envelope proteins are involved in heterodimerization, the data presented in this work suggest that an early interaction between the TMDs of E1 and E2 might occur before their transfer into the lipid bilayer.

The TMD of E2 is not an efficient stop-transfer sequence. Natural stop-transfer sequences consist of more than 18 mainly hydrophobic amino acid residues and are followed by positive charges (Sakaguchi, 1997 ). They have two functions: interrupt the ongoing protein translocation and anchor the final protein into the lipid bilayer. The sequences of the TMDs of HCV envelope proteins do not have the classical composition of stop-transfer sequences, and this might explain the inefficiency of membrane integration of E2 expressed alone. The TMDs of HCV envelope proteins are approximately 30 residues long and are composed of two stretches of hydrophobic residues separated by a short segment containing one (E1) or two (E2) fully conserved charged residues (Cocquerel et al., 2000 ). In addition, they do not possess positively charged residues in their C termini and their topology remains controversial (see below). These distinctive features are probably not optimal for membrane integration and are likely to be the consequence of the constraints linked to the multifunctionality of these TMDs. Indeed, they are involved in ER retention and assembly of the heterodimer, and they possess a signal sequence function in their C-terminal half (Cocquerel et al., 1998 , 2000 ; Michalak et al., 1997 ; Selby et al., 1994 ).

How can E1 help in membrane integration of E2? Since HCV envelope proteins are synthesized as a polyprotein, it is likely that the same translocon is used for the translocation of both E1 and E2 translated from the same RNA molecule. Therefore, it is possible that E1, which is translated first, is kept in the translocon via its TMD until all of the E2 chain has been made. After synthesis of the TMD of E2, heterodimerization might start by contacts between the TMDs. Thus this would keep the two proteins together and thereby explain the E1-dependent membrane insertion of E2 and the efficiency of cis heterodimerization.

It has previously been shown by members of our laboratory that the folding of E1 is helped by the coexpression of E2 (Michalak et al., 1997 ). More recently, we have shown that glycosylation of E1 is improved by the presence of E2 downstream of E1 on the polyprotein (Dubuisson et al., 2000 ). Here, we show that the coexpression of E1 and E2 in cis improves the efficiency of membrane integration of E2 and the efficiency of assembly of the heterodimer. Together, these observations indicate that HCV envelope proteins cooperate in the formation of a functional complex.


   Acknowledgments
 
We thank Françoise Jacob-Dubuisson for critical reading of the manuscript, C. M. Rice for the vaccinia virus recombinant vHCV1-3011 and André Pillez and Sophana Ung for excellent technical assistance. This work was supported by the CNRS, the Institut Pasteur de Lille, the University of Lille, a European Regional Development Fund (ERDF), EU grant QLK2-1999-00356 and grant 5651 from the ARC. Laurence Cocquerel was successively supported by an MENRT and a FRM fellowship. Anne Op De Beeck was successively supported by an ARC and an ANRS fellowship.


   References
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
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Received 23 January 2001; accepted 8 March 2001.