Glycosylation of the hepatitis C virus envelope protein E1 occurs posttranslationally in a mannosylphosphoryldolichol-deficient CHO mutant cell line

Sandrine Duvet1,3, Anne Op De Beeck1,4, Laurence Cocquerel4, Czeslaw Wychowski4, René Cacan3 and Jean Dubuisson2,4

3CNRS-UMR 8576/USTL, 59655 Villeneuve d’Ascq Cedex, France; 4Unité Hépatite C, CNRS-FRE2369, Institut de Biologie de Lille/Institut Pasteur de Lille, BP447, 59021 Lille Cedex, France

Received on July 4, 2001; revised on July 30, 2001; accepted on August 15, 2001.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The addition of N-linked glycans to a protein is catalyzed by oligosaccharyltransferase, an enzyme closely associated with the translocon. N-glycans are believed to be transferred as the protein is being synthesized and cotranslationally translocated in the lumen of the endoplasmic reticulum. We used a mannosylphosphoryldolichol-deficient Chinese hamster ovary mutant cell line (B3F7 cells) to study the temporal regulation of N-linked core glycosylation of hepatitis C virus envelope protein E1. In this cell line, truncated Glc3Man5GlcNAc2 oligosaccharides are transferred onto nascent proteins. Pulse-chase analyses of E1 expressed in B3F7 cells show that the N-glycosylation sites of E1 are slowly occupied until up to 1 h after protein translation is completed. This posttranslational glycosylation of E1 indicates that the oligosaccharyltransferase has access to this protein in the lumen of the endoplasmic reticulum for at least 1 h after translation is completed. Comparisons with the N-glycosylation of other proteins expressed in B3F7 cells indicate that the posttranslational glycosylation of E1 is likely due to specific folding features of this acceptor protein.

Key words: endoplasmic reticulum/N glycosylation/posttranslational glycosylation/posttranslational modification


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
N-linked glycosylation is the major modification of a nascent protein targeted to the secretory pathway. It is now clearly established that the key reaction of N-glycosylation is the transfer en bloc of a Glc3Man9GlcNAc2 oligosaccharide from a lipid intermediate to an Asn residue in the consensus sequence Asn-X-Thr/Ser of a nascent protein. The addition of this glycan precursor is catalyzed by oligosaccharyltransferase (OST), an enzyme closely associated with the translocon and thought to have access only to nascent chains as they emerge from the ribosome at the lumenal face of the rough endoplasmic reticulum (ER) (Silberstein and Gilmore, 1996Go). The biosynthesis of an N-glycosylprotein is dependent on several events: the translation of the protein backbone, its translocation inside the ER lumen, and the transfer of the glycan(s) onto the protein backbone. For this latter process, the nature of the oligosaccharide donor and the quality of the nascent protein acceptor seem to be critical. In yeast, the relationship between these parameters are well defined. Indeed, many N-linked glycosylation-mutant strains that are unable to complete the synthesis of lipid-linked oligosaccharides produce underglycosylated glycoproteins (Knauer and Lehle, 1999Go). In addition, it is well known that translation can be uncoupled from translocation (Hansen et al., 1986Go; Waters and Blobel, 1986Go). In contrast, less is known about the temporal and structural controls of N-glycosylation in mammalian cells. N-glycans are believed to be linked as the protein is being synthesized and cotranslationally translocated (Kornfeld and Kornfeld, 1985Go; Kaplan et al., 1987Go). However, it is unclear what dictates the usage efficiency of a consensus site because not all consensus sequences in every protein are used (Gavel and von Heijne, 1990Go; Shakin-Eshleman et al., 1992Go). It has been shown that the type of amino acid present at the X and Y positions of the sequon can modulate the efficiency of core glycosylation in vitro (Shakin-Eshleman et al., 1996Go; Mellquist et al., 1998Go); in some instances, folding has been shown to compete with glycosylation (Allen et al., 1995Go; Holst et al., 1996Go; Capellari et al., 1999Go).

In this work, we used a mannosylphosphoryldolichol-deficient Chinese hamster ovary (CHO) mutant cell line (B3F7 cells) (Stoll, 1986Go) to study N-linked core glycosylation in mammalian cells. This cell line has a defect in mannosylphosphoryldolichol synthase, which leads to the transfer of truncated Glc3Man5GlcNAc2 oligosaccharides onto nascent proteins (Cacan et al., 1992Go; Duvet et al., 1998Go). Analysis of glycosylation in such a mutant cell line is interesting because it shows the influence of the quality of oligosaccharide donors on core glycosylation. We used hepatitis C virus (HCV) envelope protein E1 as a model to study N-linked glycosylation in B3F7 cell line. E1 is a type I transmembrane protein with a large N-terminal ectodomain and a C-terminal hydrophobic anchor that determines a static retention in the ER without retrieval from the Golgi apparatus (Cocquerel et al., 1999Go). E1 possesses four potential N-glycosylation sites (Figure 1) (Meunier et al., 1999Go). However, only approximately 50% of this protein is fully glycosylated when expressed alone (Dubuisson et al., 2000Go). The inefficient glycosylation that can be observed in E1 makes this protein an interesting tool to study N-linked glycosylation in the context of a modified donor. Here, we show in pulse-chase experiments that the N-glycosylation sites of E1 are slowly occupied in B3F7 cells until up to 1 h after protein translation is completed. This delayed glycosylation indicates that the OST has access to this protein in the lumen of the ER for at least 1 h after translation is completed.



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Fig. 1. Schematic representation of HCV glycoprotein E1. The signal sequence is indicated by a solid box, the ectodomain by an open box, and the transmembrane domain by a shaded box. The positions indicated on E1 refer to the position on HCV polyprotein. The positions of the N-linked glycosylation sites (N1, N2, N3, and N4) are indicated. The positions of the first and the last amino acid residues of E1 are indicated by the arrows.

 

    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
N-glycosylation of E1 glycoprotein expressed in B3F7 cells
Previous reports have indicated that expression of glycoproteins in glycosylation mutant cell lines of yeast lead to underglycosylation of potential N-glycosylation sites (Burda et al., 1999Go). The synthesis of these glycoforms suggests that the efficiency of transfer by the OST onto a nascent protein can be reduced for truncated oligosaccharide-lipid donors. To determine the influence of truncated oligosaccharides on the N-glycosylation process in mammalian cells, we used HCV envelope glycoprotein E1 as a model (Figure 1). Expression of HCV envelope glycoprotein E1 was analyzed in B3F7 cells which synthesize Glc3Man5GlcNAc2 oligosaccharide lipids. Recombinant vaccinia viruses previously used to study the glycosylation of E1 (Dubuisson et al., 2000Go; Meunier et al., 1999Go) could not be used in B3F7 cells because, like the parental CHO cells, they are resistant to infection by vaccinia virus. We therefore used the Sindbis virus expression system, which has been shown to be working in B3F7 cells (Duvet et al., 2000Go). Cells were infected with a Sindbis virus recombinant expressing E1 and were metabolically labeled for 25 min. Cell lysates were then used for immunoprecipitation with an anti-E1 Mab (A4). When analyzed by sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE), five bands were detected (Figure 2). However, in the presence of tunicamycin, a drug that blocks core glycosylation of nascent glycoprotein precursors, E1 was resolved as a single band that comigrated with the fastest-migrating band observed in nontreated cells. These data support the conclusion that the four slowest migrating bands correspond to distinct glycoforms of the glycoprotein bearing one (1G), two (2G), three (3G), or four (4G) glycans. It has to be noted that an additional band that migrated more slowly than the nonglycosylated form of E1 was also observed (Figure 2, asterisk). This protein might correspond to a nonglycosylated form of E1 with its uncleaved signal peptide. However, it was not detected by western blotting with the anti-E1 antibody (data not shown). More likely, this protein might be the capsid protein of the Sindbis virus vector because it has the expected size, and we have previously observed that it is precipitated by protein A–Sepharose even in the absence of specific antibodies (unpublished data).



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Fig. 2. Glycosylation of E1 expressed in B3F7 cells and in the CHO parental cell line (Pro-5). Cells were infected with Sin//E1 at a multiplicity of 5 plaque forming units/cell. At 4.5 h postinfection, cells were pulse-labeled for 25 min with 35S-protein labeling mix. For tunicamycin treatment, cells were incubated with the inhibitor (10 µg/ml) for 45 min before as well as during metabolic labeling. Treated and control cell lysates were immunoprecipitated with mAb A4 and samples were analyzed by SDS–PAGE (13% acrylamide). Bands corresponding to nonglycosylated E1 (0G) or mon-, di-, tri-, and tetraglycosylated E1 (1G, 2G, 3G, and 4G) as well as an additional band (asterisk) are indicated.

 
We have previously shown that E1 is not efficiently glycosylated when expressed alone in HepG2 cells (Dubuisson et al., 2000Go), indicating that this protein is also underglycosylated in cells synthesizing full-length glycans. We therefore compared the migration profiles of E1 expressed in B3F7 cells and in the parental CHO cell line (Pro-5 cells), which synthesizes the complete Glc3Man9GlcNAc2 donor. As shown in Figure 2, several glycoforms of E1 were also detected in the parental CHO cell line. However, a difference in the ratio of the glycoforms was observed. The tri- and tetraglycosylated forms were predominantly observed in the Pro-5 cells, whereas in B3F7 cells, the intensity of the tetraglycosylated form was faint and the band with the highest intensity was the diglycosylated form. It is worth noting that the glycoforms of E1 migrated slightly faster when expressed in B3F7 cells compared with those expressed in the parental cell line. This is likely due to the smaller size of the glycans that are transferred in B3F7 cells.

Glycosylation of E1 occurs posttranslationally in B3F7 cells
Because a difference in the ratios of the glycoforms was observed when the expression of E1 was analyzed in B3F7 and Pro-5 cells, we wondered whether this would reflect differences in the efficiency of glycosylation or some change(s) in the kinetics of degradation and/or glycosylation of this protein. To answer these questions, E1 was analyzed in pulse-chase experiments. Surprisingly, at the end of a 2-min pulse, tri- and tetraglycosylated forms (3G and 4G) were barely detectable in B3F7 cells (Figure 3a, time 0). The major bands detected in B3F7 cells at the end of the pulse were the nonglycosylated and the mono- and diglycosylated forms of E1 (0G, 1G, and 2G) (Figure 3a, time 0). However, during the chase the tri- and tetraglycosylated forms (3G and 4G) appeared sequentially to finally represent the major bands detected at the end of the chase (1 h). At that time, 50% of E1 was totally glycosylated. The ratio of fully glycosylated E1 observed after a 2-h chase was similar to the one obtained after 1 h of chase (data not shown).



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Fig. 3. Posttranslational N-glycosylation of E1 in B3F7 cells. B3F7 and Pro-5 cells were infected with Sin//E1 at a multiplicity of 5 plaque forming units/cell. At 4.5 h postinfection, cells were pulse-labeled for 2 min with 35S-protein labeling mix and chased for 5, 15, 30, 45, and 60 min. Cell lysates were immunoprecipitated with mAb A4, and samples were analyzed by SDS–PAGE (13% acrylamide) (a and c). Bands corresponding to nonglycosylated E1 (0G) or mon-, di-, tri-, and tetraglycosylated E1 (1G, 2G, 3G, and 4G) are indicated. For each time of chase, the various glycoforms of E1 were quantified by phosphorimaging and expressed as % of total E1 (b and d).

 
Quantitative analyses revealed a precursor/product relationship between underglycosylated and fully glycosylated forms (Figure 3b). Similar results were observed when cycloheximide was added during the chase or in the presence of lactacystin, an inhibitor of the proteasome (data not shown), indicating that glycosylation occurred after E1 was completely translated and that the partially glycosylated species have not been degraded during the chase. This indicates that posttranslational glycosylation of E1 occurs in B3F7 cells.

When E1 was expressed in Pro-5 cells, the tri- and tetraglycosylated forms were already predominantly detected during the pulse (Figure 3c). In addition, the underglycosylated species (< 3G) disappeared progressively during the chase (Figures 3c and d). The relative intensity of the tetraglycosylated form of E1 slightly increased during the chase, whereas the relative intensity of the triglycosylated form slightly decreased during that time (Figure 3d). These data are very similar to what we have previously observed in HepG2 cells (Dubuisson et al., 2000Go). It should be noted that the total amount of radioactivity recovered during the pulse was slightly lower compared with that recovered at the different chase times. This is likely due to completion of protein synthesis initiated during the short pulse.

Altogether, these data indicate that, in B3F7 cells, glycosylation of E1 is essentially a posttranslational phenomenon that occurs slowly.

Kinetics of posttranslational glycosylation in mutants lacking one glycosylation site
It has been shown previously in in vitro experiments that the N4 site was less efficiently glycosylated (Meunier et al., 1999Go). In addition, in vivo expression of glycosylation mutants of E1 has shown that both N1 and N4 sites are less efficiently glycosylated (Dubuisson et al., 2000Go). Therefore, we wondered whether these observations might explain the posttranslational glycosylation of E1. We chose to extend our studies by examining the kinetics of glycosylation of mutants lacking one glycosylation site at position N1 (aminoacid position 196), N2 (position 209), N3 (position 234), or N4 (position 305). Sindbis virus recombinants expressing these mutated proteins were constructed and used to infect B3F7 cells. Expression of the glycosylation mutants of E1 was analyzed by pulse-chase analyses and immunoprecipitation. During the pulse, the underglycosylated forms of E1 (< 3G) were the major bands detected for all the mutants (Figure 4). During the chase, the intensity of the fully glycosylated form (3G) increased progressively. This was rather similar to what was observed for wild-type E1 expressed in B3F7 cells, indicating that the mutations did not dramatically alter the posttranslational glycosylation of E1. However, slight differences in the kinetics of detection of fully glycosylated species (3G) were repeatedly observed between the four glycosylation mutants (Figure 4). Indeed, there was a slight increase in the efficiency of glycosylation in relation to the position of the mutation toward the C-terminus of the protein. The higher percentage of fully glycosylated molecules observed after a 1-h chase for E1 having a mutation at the N4 site is in agreement with previous observations indicating that this site is less efficiently glycosylated when expressed in vivo (Dubuisson et al., 2000Go) or in vitro (Meunier et al., 1999Go).



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Fig. 4. Kinetics of glycosylation of E1 mutated at glycosylation sites N1, N2, N3, or N4 and expressed in B3F7 cells. B3F7 cells were infected with the appropriate Sindbis virus recombinants at a multiplicity of 5 plaque forming units/cell. At 4.5 h postinfection, cells were pulse-labeled for 2 min with 35S-protein labeling mix and chased for 5, 15, 30, 45, and 60 min. Cell lysates were immunoprecipitated with mAb A4 and samples analyzed by SDS–PAGE (13% acrylamide). For each chase-time, the various glycoforms of E1 (< 3G and 3G) were quantified by phosphorimaging and expressed as % of total E1.

 
Altogether, these data indicate that the posttranslational glycosylation of E1 is not due to the presence of glycosylation sites that are less efficiently glycosylated.

Posttranslational glycosylation is due to specific features of E1
We wondered whether the posttranslational glycosylation observed in B3F7 cell line is due to specific features of E1 or whether this phenomenon would also apply to other glycoproteins expressed in this cell line. To answer this question, we analyzed the glycosylation of two other glycoproteins. The first one is the human CD4 glycoprotein, which is a type I transmembrane protein normally exported to the cell surface. To analyze the glycosylation of CD4, a Sindbis virus recombinant was used to infect B3F7 cells. The second protein, the human placental alkaline phosphatase (SeAP) lacking the GPI-anchoring signal, is stably expressed in B3F7 cells (Ermonval et al., 2000Go). These glycoproteins have two potential N-linked glycosylation sites. B3F7 cells were infected with a recombinant Sindbis virus expressing CD4 and metabolically labeled. Cell lysates were then used for immunoprecipitation with an anti-CD4 monoclonal antibody (mAb) (OKT4). When analyzed by SDS–PAGE, CD4 was resolved as a fully diglycosylated band (Figure 5). Indeed, partial digestion of CD4 with peptide:N-glycosidase (PNGase F) produced two bands that migrated faster. Similar results were observed for CD4 expressed in the control parental cell line (Pro-5). The secreted form of SeAP expressed in B3F7 cells was also resolved as a fully diglycosylated band (data not shown). Together, these data indicate that the transfer of the Glc3Man5GlcNAc2 donor onto other proteins is not delayed and the posttranslational glycosylation of E1 is due to specific features of this acceptor protein.



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Fig. 5. Profile of glycosylation of CD4 during its synthesis in B3F7 cells. B3F7 cells were infected with CD4 at a multiplicity of 5 plaque forming units/cell. At 4.5 h postinfection, cells were pulse-labeled for 10 min with 35S-protein labeling mix. Cell lysates were immunoprecipitated with mab OKT4 and then partially digested (+) or not (–) with PNGase F. Proteins were separated by SDS–PAGE (10% polyacrylamide) under reducing conditions.

 
Coexpression with HCV glycoprotein E2 improves the temporal control of glycosylation of E1
Folding has been shown to compete with glycosylation (Allen et al., 1995Go; Holst et al., 1996Go; Capellari et al., 1999Go). Indeed, the presence of mild concentrations of the reducing agent dithiothreitol in cell culture prevents the formation of disulfide bonds in the ER and leads to complete glycosylation of sequons that otherwise undergo variable glycosylation in untreated cells (Allen et al., 1995Go; Capellari et al., 1999Go). However, treatment of B3F7 cells expressing E1 with dithiothreitol did not lead to any change in the kinetics of glycosylation of E1 (data not shown), suggesting that disulfide bond formation has no effect on the posttranslational glycosylation of E1. E1 has been shown to form a noncovalent heterodimer with the other HCV envelope protein, E2 (Deleersnyder et al., 1997Go), and coexpression of E1 with E2 has also been shown to be necessary for the proper folding of E1 (Michalak et al., 1997Go). We therefore wondered whether the presence of E2 could improve the kinetics of glycosylation of E1. Interestingly, when coexpressed with E2 in B3F7 cells, E1 was rapidly glycosylated (Figure 6). Indeed, the tri- and tetraglycosylated forms were the major glycoforms of E1 at the end of a 5-min pulse, indicating that the presence of E2 rapidly stabilizes the structure of E1 in a conformation that facilitates its glycosylation in B3F7 cells.



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Fig. 6. Effect of the coexpression of E2 on the glycosylation of E1 in B3F7 cells. For the coexpression of E1 and E2, a Sindbis virus recombinant (SINrep/HCV-H1-1207) which contains the sequence of a truncated polyprotein containing the N-terminal 1207 amino acid residues of HCV was used. When using this vector, the envelope proteins E1 and E2 are produced after cleavage of the polyprotein by a cellular signal peptidase (Dubuisson et al., 1994Go). B3F7 cells were infected with SINrep/HCV-H1-1207 at a multiplicity of 5 plaque forming units/cell. At 4.5 h postinfection, cells were pulse-labeled for 5 min with 35S-protein labeling mix and chased for 15, 30, and 60 min. Cell lysates were immunoprecipitated with mAb A4 and samples were analyzed by SDS-PAGE (13% acrylamide). Bands corresponding to di-, tri-, and tetraglycosylated E1 (2G, 3G, and 4G) are indicated.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The glycosylation process of HCV envelope protein E1 in the Man-P-Dol-deficient cell line B3F7 occurs essentially as a slow posttranslational event. In mammalian cells, core glycosylation usually occurs cotranslationally, as the glycosylation site of a nascent protein enters the ER lumen. But posttranslational N-glycosylation has been observed in unusual circumstances, such as on small acceptor tripeptides (Geetha-Habib et al., 1990Go), on aglycoinsulin receptor generated by tunicamycin treatment (Ronnett and Lane, 1981Go), or on a truncated form of peptidylglycine {alpha}-amidating monooxygenase (Kolhekar et al., 1998Go).

Here, we have clearly demonstrated the posttranslational N-glycosylation of the full-length HCV envelope protein E1, which is a type I membrane protein. The OST is supposed to be associated with the translocon (Johnson and van Waes, 1999Go), and the posttranslational glycosylation observed for E1 suggests that this protein likely remains in the environment of the translocon machinery for at least 1 h. It should also be pointed out that the C-terminal hydrophobic anchor of E1 is responsible for a strict retention in the ER without recycling through the Golgi apparatus (Cocquerel et al., 1999Go). The ER retention of E1 likely plays a role in its posttranslational glycosylation because the active site of the OST is exposed at the luminal face of the ER membrane (Fu et al., 1997Go).

Does glycosylation of E1 occur posttranslationally in the presence of a normal donor? Our data indicate that in the context of a normal donor (this work and Dubuisson et al., 2000Go), glycosylation of E1 occurs very rapidly and our experimental conditions do not allow to clearly conclude whether glycosylation of E1 is co- or posttranslational. However, previous data from our group indicate that the efficiency of glycosylation of E1 is improved by the presence of a polypeptide sequence located downstream of E1 on HCV polyprotein (Dubuisson et al., 2000Go). This observation is in favor of a slightly delayed glycosylation in the context of a normal donor. Indeed, the glycosylation sites are located 188, 175, 150, and 79 residues from the C-terminal residue of E1. The fact that a sequence far downstream of E1 glycosylation sites has some influence on the efficiency of transfer of N-glycans on some of these sites suggests that the glycosylation of this protein is improved when its synthesis is completed. The OST is therefore likely able to transfer oligosaccharides on fully synthesized E1 in the context of a normal donor.

Because N-glycosylation involves two separate metabolic pathways localized in the rough ER—that is, the synthesis of oligosaccharide precursors (the donor) and the protein translation (the acceptor)—specific features of the donor or the acceptor should modulate the first step of the N-glycosylation process. Thus the question arises as to know whether the posttranslational glycosylation of E1 depends on specific features of the truncated donor (Glc3Man5GlcNAc2 oligosaccharide) or the E1 acceptor protein. The use of glycosylation mutant cell lines allows to point out the influence of the quality of oligosaccharide donors on the efficiency of core glycosylation. It has been demonstrated in vitro (Murphy and Spiro, 1981Go) and in vivo (Burda and Aebi, 1998Go) that glucosylated lipid-linked oligosaccharides—in particular the presence of the {alpha}1,2-linked glucose—is necessary for efficient N-glycosylation. The improvement of N-glycosylation by the presence of a glucosyl residue has also been observed with mutant cell lines synthesizing truncated oligomannosides (Man5GlcNAc2) (Ermonval et al., 2000Go).

When the SeAP was expressed in the Man5-synthesizing CHO mutant cell line B3F7, the secreted form of this protein was fully glycosylated. However, an underglycosylated glycoform of the SeAP was detected in the supernatant of tissue culture when this protein was expressed in MadIA214 cells, another Man5-synthesizing CHO mutant cell line. The difference between these two cell lines is that the oligosaccharide donor is not glucosylated in MadIA214 cells (Ermonval et al., 1997Go, 2000), whereas it is fully glucosylated in B3F7 cells. As observed in the present study, the expression of SeAP or CD4 glycoproteins in B3F7 cells does not lead to hypoglycosylation because the presence of a fully diglycosylated form of both glycoproteins is observed even after a short pulse. These data indicate that the glycosylation process occurring in B3F7 cells is not impaired when the fully glucosylated oligomannoside (Glc3Man5GlcNAc2) is used as a donor and SeAP and CD4 as acceptors. In contrast, when E1 was used as an acceptor protein in B3F7 cells, a severe hypoglycosylation was observed very early after its synthesis, which was followed by a slow posttranslational glycosylation.

Together, these data indicate that the posttranslational glycosylation process observed in E1 involves specific features of this acceptor protein. However, the differences observed between the kinetics of glycosylation of E1 expressed in B3F7 cells and the parental cell line indicate that glycosylation of this acceptor protein also depends on specific features of the truncated donor (Glc3Man5GlcNAc2 oligosaccharide).

The posttranslational glycosylation observed for E1 in B3F7 cells is likely due to a problem of folding of this protein. Coexpression of E1 with the other HCV envelope protein, E2, has been shown to be necessary for the proper folding of E1 (Michalak et al., 1997Go). When coexpressed with E2, E1 was rapidly glycosylated, indicating that the presence of E2 stabilizes the structure of E1 in a conformation that facilitates its glycosylation in B3F7 cells. This prevention of underglycosylation of E1 by E2 likely occurs posttranslationally because HCV envelope proteins E1 and E2 are synthesized as a polyprotein and the synthesis of E1 precedes the synthesis of E2 (Op De Beeck et al., 2001Go). The folding of E1 is slow (Dubuisson and Rice, 1996Go) and it is likely that some state of folding dependent on the presence of E2 might be needed to expose to acceptor sites in a conformation optimal for recognition by the OST. In vitro studies have indeed shown that the acceptor peptide has likely to adopt a specific conformation, the AsnX turn, to become glycosylated (Imperiali et al., 1992Go). Such a conformation might even be more important in the context of a modified donor. In conclusion, HCV envelope protein E1 provides a very interesting model to analyze the temporal regulation of N-linked core glycosylation in mammalian cells.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Reagents
Mutant cell line B3F7 isolated from B 4-2-1, Lec15.1 which does not synthesize mannosylphosphoryldolichol (Stoll, 1986Go) was a gift of Sharon S. Krag (Johns Hopkins University, Baltimore, MD). 35S-protein labeling mix was purchased from DuPont NEN. Cycloheximide, Igepal CA-630, and tunicamycin were obtained from Sigma (St.Louis, MO). Lactacystin was obtained from Calbiochem. PNGase F was purchased from New England Biolabs. mAbs A4 (anti-E1) (Dubuisson et al., 1994Go) and OKT4 (anti-CD4) (Reinherz et al., 1979Go) were produced in vitro by using a MiniPerm apparatus (Heraeus) as recommended by the manufacturer. Protein A–Sepharose CL-4B was from Amersham Pharmacia Biotech.

Generation of Sindbis virus recombinants
The Sindbis virus recombinants expressing HCV glycoprotein E1 (Sin//E1) or a truncated polyprotein containing the N-terminal 1207 amino acid residues of HCV that contains the envelope proteins E1 and E2 (SINrep/HCV-H1-1207) have been described previously (Dubuisson et al., 1994Go; Duvet et al., 2000Go). Sindbis virus recombinants expressing glycosylation mutants of E1 (N1, N2, N3, and N4) have been obtained by inserting the sequences of the mutated proteins (Meunier et al., 1999Go) into XbaI site of plasmid pTE3'2J (Hahn et al., 1992Go). Sindbis virus recombinant expressing CD4 was obtained by inserting the sequence of CD4 into XbaI site of plasmid pTE3'2J. 5'-Capped RNA transcripts were synthesized by using SP6 polymerase and the plasmid DNA template that had been linearized with NotI or PvuI (Rice et al., 1987Go). Stocks of Sindbis virus recombinants were generated by electroporation of BHK-21 cells with in vitro transcribed RNA as described (Bredenbeek et al., 1993Go) and harvested 24 h after electroporation. Virus titers were determined by plaque immunostaining with anti-E1 mAb A4 as described (Dubuisson and Rice, 1993Go).

Metabolic labeling and immunoprecipitation
Cells were routinely cultured in monolayers in {alpha}-minimal essential medium with 10% (v/v) fetal calf serum at 34°C in 10-cm petri dishes under air/CO2 (19:1). Cells expressing the recombinant glycoproteins were metabolically labeled with 35S-protein labeling mix (100 µCi/ml) as previously described (Dubuisson et al., 1994Go). In pulse-chase experiments, cells were washed twice after the pulse and then incubated in culture medium containing a 10-fold excess of methionine and cysteine. Cells were lyzed with 0.5% Igepal CA-630 in Tris-buffered saline (50 mM Tris-Cl, pH 7.5, 150 mM NaCl). Immunoprecipitations were carried out as described (Dubuisson and Rice, 1996Go).

When tunicamycin was used, cells were pretreated for 45 min in {alpha}-minimal essential medium containing 10 µg/ml of the inhibitor. The presence of tunicamycin was maintained throughout the incubation. For PNGase F treatment, immunoprecipitated proteins were eluted from protein A–Sepharose in 30 µl of dissociation buffer (0.5% SDS, 1% 2-mercaptoethanol) by boiling for 10 min. Digestions were carried out for 1 h at 37°C in the buffer provided by the manufacturer. An undigested control was simultaneously prepared. Samples were mixed with an equal volume of 2x Laemmli sample buffer and analyzed by SDS–PAGE. Quantifications were performed by Phosphor imaging.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We thank Françoise Jacob-Dubuisson for critical reading of the manuscript and André Pillez, Sophana Ung, and Anne-Marie Mir for excellent technical assistance. We are grateful to Charles M. Rice (Washington University School of Medicine, St Louis, MO) for providing plasmid pTE3'2J and to Sharon S. Krag from the school of Hygiene and Public Health (John Hopkins University, Baltimore, MD) for her generous gift of B3F7 cells. This work was supported by the CNRS, the University of Lille, the Institut Pasteur of Lille, a European Regional Development Fund, and grant 5651 from the ARC. A. Op De Beeck was successively supported by an ARC and an ANRS fellowship. L. Cocquerel was successively supported by an MENRT and a FRM fellowship.


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
CHO, Chinese hamster ovary; HCV, hepatitis C virus; ER, endoplasmic reticulum; PNGase F, peptide:N-glycosidase F; mAb, monoclonal antibody; OST, oligosaccharyltransferase; SeAP, human placental alkaline phosphatase; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis.


    Footnotes
 
1 These authors contributed equally to the results of this study. Back

2 To whom correspondence should be addressed Back


    References
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
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