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
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Abstract |
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Introduction |
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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.
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Methods |
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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)] and H47 [anti-E2 conformation-insensitive MAb; (A. Pillez & J. Dubuisson, unpublished data)] were produced in vitro by using a MiniPerm apparatus (Heraeus) as recommended by the manufacturer.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.
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.
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 TrisHCl, 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 ASepharose 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-
-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 SDSPAGE.
Sedimentation through sucrose gradients.
Supernatant from labelled infected cells was layered on a 10 ml gradient of 535% 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.
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.
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 SDSPAGE 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).
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Results |
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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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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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Discussion |
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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.
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Acknowledgments |
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References |
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Received 23 January 2001;
accepted 8 March 2001.