Rabies virus glycoprotein can fold in two alternative, antigenically distinct conformations depending on membrane-anchor type

Antoine P. Maillard1 and Yves Gaudin1

Laboratoire de Génétique des Virus du CNRS, 91198 Gif sur Yvette Cedex, France1

Author for correspondence: Yves Gaudin. Fax +33 1 69 82 43 08. e-mail gaudin{at}gv.cnrs-gif.fr


   Abstract
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Abstract
Introduction
Methods
Results
Discussion
References
 
Rabies virus glycoprotein (G) is a trimeric type I transmembrane glycoprotein that mediates both receptor recognition and low pH-induced membrane fusion. We have previously demonstrated that a soluble form of the ectodomain of G (G1–439), although secreted, is folded in an alternative conformation, which is monomeric and antigenically distinct from the native state of the complete, membrane-anchored glycoprotein. This has raised questions concerning the role of the transmembrane domain (TMD) in the correct native folding of the ectodomain. Here, we show that an ectodomain anchored in the membrane by a glycophosphatidylinositol is also folded in an alternative conformation, whereas replacement of the TMD of G by other peptide TMDs results in correct antigenicity of G. However, mutants with an insertion of a hydrophilic linker between the ectodomain and the TMD also fold in an alternative conformation. The influence of the membrane-anchor type on G ectodomain trimerization and folding is discussed.


   Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Glycoproteins are translated on membrane-bound ribosomes and inserted co-translationally into the endoplasmic reticulum (ER) in an unfolded form. In the case of transmembrane proteins, folding occurs in three topologically and biochemically distinct environments: the ER lumen, the ER membrane and the cytosol. For type I glycoproteins, it has generally been supposed that the ectodomain, the transmembrane domain (TMD) and the cytoplasmic domain constitute independent folding domains within the glycoprotein. A survey of the literature indicates that this is not always the case and that the presence or absence of a TMD, as well as its nature, can influence the correct folding of the ectodomain (Roth et al., 1986 ; Paterson & Lamb, 1990 ; Singh et al., 1990 ; Godet et al., 1991 ; Gaudin et al., 1999 ). However, in most cases, except when it is involved in oligomerization, the exact role of the TMD in the folding of the ectodomain has not been investigated.

Rabies virus glycoprotein (RV G) is a type I membrane glycoprotein. It is a trimer (3x65 kDa), which forms a spike extending 8·3 nm from the viral membrane (Whitt et al., 1991 ; Gaudin et al., 1992 ). The native glycoprotein is 505 residues long (Anilionis et al., 1981 ). It is responsible for cellular receptor recognition (Thoulouze et al., 1998 ; Tuffereau et al., 1998 ) and for the low pH-induced fusion of the viral envelope with the endosomal membranes (Whitt et al., 1991 ; Gaudin et al., 1993 ). Rabies virus glycoprotein is also the target of neutralizing antibodies (Cox et al., 1977 ) and its antigenicity has been extensively studied during the past years, leading to the definition of two major antigenic sites (site II and site III), one minor site (site a) and several isolated epitopes (Lafon et al., 1983 ; Seif et al., 1985 ; Préhaud et al., 1988 ; Dietzschold et al., 1990 ; Benmansour et al., 1991 ; Lafay et al., 1996 ). Antigenic site II extends from amino acid 34 to 42 and from amino acid 198 to 200 (Préhaud et al., 1988 ). These peptides are maintained together in the tertiary structure by a disulphide bridge between cysteines 35 and 207 (Walker & Kongsuwan, 1999 ). Antigenic site III extends from amino acid 333 to 338 (Seif et al., 1985 ). Finally, minor site a contains amino acids 342 and 343 (Benmansour et al., 1991 ). It has also been demonstrated that G can assume at least three different states (Gaudin et al., 1993 ): the native (N) state detected at the viral surface, which is responsible for receptor binding, the activated hydrophobic state (A), which interacts with the target membrane as a first step in the fusion process, and the fusion inactive conformation (I). There is a complex pH-dependent equilibrium between these states, the equilibrium being shifted toward the I state at low pH. Using electron microscopy, it has been shown that the I conformation is 3 nm longer than the N state, from which it is also antigenically distinct (Gaudin et al., 1993 ).

The folding of G in the ER has been studied by analysing epitope acquisition and interaction with ER chaperones (Gaudin, 1997 ). More recently, we have investigated the folding of a soluble form of G (G1–439) lacking the transmembrane and intracytoplasmic domains (residues 440–505) (Gaudin et al., 1999 ). Although this soluble ectodomain was secreted and was thus able to leave the ER, a characteristic of folded proteins (Gething et al., 1986 ; Ellgaard et al., 1999 ), it had a monomeric conformation that was antigenically distinct from the native state of the complete membrane-anchored glycoprotein. Together with the fact that a mutant G lacking only the cytoplasmic domain was shown to be correctly folded (Gaudin et al., 1999 ), these results have raised questions concerning a possible role for the TMD in the correct native folding of the ectodomain.

To examine the requirements for correct native folding of the ectodomain of G, we have analysed the folding of various chimeric glycoproteins. We have shown that the TMD of G can be replaced by foreign TMDs without affecting the antigenicity of the ectodomain although this resulted in proteins with more stable or less stable quaternary structures. However, replacement of the TMD by a glycophosphatidylinositol (GPI) anchor or insertion of a flexible hydrophilic domain between the ectodomain and the TMD of G resulted in folding of the ectodomain in an alternative conformation similar to G1–439.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Cells, viruses and monoclonal antibodies (mAbs).
BSR cells, a clone of BHK 21 (baby hamster kidney) cells, were grown in minimal essential medium (MEM; Gibco BRL) supplemented with 10% calf serum. Recombinant vaccinia virus (VTF7-3) containing the T7 RNA polymerase gene has been described previously (Fuerst et al., 1986 ). The properties of the mAbs are summarized in Table 1. They were used as mouse ascites preparations.


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Table 1. Properties of the mAbs used to characterize the conformation of G ectodomain

 
{blacksquare} Plasmid constructs.
Plasmids were constructed by insertion of PCR-amplified fragments into the pcDNA I (InvitroGen) mammalian expression vector (Fig. 1). The plasmid encoding Ggpi was assembled from a fragment encoding the full ectodomain of G (PV strain) and a PCR-amplified fragment from pTM1/E2(371-661)DAF (Cocquerel et al., 1998 ) corresponding to the decay accelerating factor (DAF) propeptide, which allowed the addition of a GPI anchor. The MluI site used to ligate both fragments resulted in two additional amino acid residues linking the G ectodomain and the 37 C-terminal residues from the DAF. For TMD substitutions, we used unique BspHI and PvuI sites, respectively, present upstream and downstream of the nucleotide sequence encoding the TMD. The fragment of G between BspHI and the region encoding the TMD was amplified between the pre-existing BspHI site and a new MfeI site and cloned in pGEM-T (Promega). The mutation to an MfeI site concerns the tripeptide Pro-Asn-Trp (residues 436–438) and is silent from the peptide point of view. Fragments encoding the TMD from human nerve growth factor (NGF) receptor p75 or pseudorabies virus gC, flanked by membrane-proximal sequences from G, were amplified between MfeI and PvuI to construct the plasmids encoding Gp75 and GgC, respectively. The plasmid encoding G4G was constructed by insertion of a mutated MfeI–PvuI fragment. For subsequent ease of cloning, the vector encoding G was silently modified to contain an additional unique AflII site in the region encoding the first residues of the TMD. The plasmid encoding GOgd56 was obtained by insertion of the sequence encoding the Ser/Thr-rich domain of human p75 between MfeI and AflII. Plasmids encoding GOgd21 and GOgd35 were obtained from the plasmid encoding GOgd56 by deletion using the Quikchange site-directed mutagenesis kit (Stratagene).



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Fig. 1. (A) Schematic representation of the different glycoproteins characterized in this study. Signal peptide has been omitted. G is the glycoprotein from rabies virus (PV strain). Ggpi is the ectodomain of G (residues 1–439) linked to the 37 C-terminal residues of DAF. The TMD of G has been replaced by human p75 or PrV gC TMD in Gp75 or GgC, respectively. G4G is essentially the same as G except that three glycines have been inserted between residues G438 and K439. In GOgd56, the 56 residue long O-glycosylated Ser/Thr-rich domain of p75 has been inserted between residues G438 and K439. GOg21 and GOg35 contain the first 21 and 35 amino acids of this domain of p75, respectively. (B) Sequence detail of the chimeric junctions. Substituted or inserted elements are underlined. TMD is in bold when present. In Ggpi, the asterisk marks the predicted point of attachment to the GPI anchor (Cocquerel et al., 1993); the two residues in italics correspond to the restriction site used in the cloning procedure. (C) Schematic drawing of the different proteins. The dotted line symbolizes the membrane surface.

 
{blacksquare} DNA transfection.
Proteins were transiently expressed using a T7 vaccinia virus expression system according to the method of Fuerst et al. (1986 ). Depending on subsequent use, BSR cells were grown to about half confluency on 60 mm (for immunoprecipitations and Endo H resistance experiments) or 35 mm diameter (for sedimentation analysis experiment) Petri dishes. They were infected with VTF7-3 at an m.o.i. of 5 p.f.u. per cell. After adsorption for 45 min at room temperature, the cells were transfected with 8 µg (5 µg for 35 mm diameter dish) of supercoiled plasmid DNA pre-incubated with TransfectAce (Rose et al., 1991 ) at room temperature and diluted in MEM. After 3 h at 37 °C, the medium was supplemented to a final concentration of 3% calf serum.

{blacksquare} Metabolic labelling and cell lysis.
At 20 h post-transfection, the culture medium was replaced with methionine- and cysteine-free medium (ICN) and the cells were starved for 1 h at 37 °C. The cells were then labelled with 600 µl (300 µl for 35 mm diameter dishes) of pre-warmed methionine- and cysteine-free medium supplemented with 200 µCi of [35S]methionine and [35S]cysteine (Promix, Amersham) for 10 min. Pre-warmed MEM containing 5 mM cold methionine and 1 mM cold cysteine was then added for the chase time. The cells were lysed on ice in 1 ml (500 µl for 35 mm diameter dishes) of TD buffer (137 mM NaCl, 5 mM KCl, 0·7 mM Na2HPO4, 25 mM Tris–HCl, pH 7·5) containing 1% CHAPS or 0·5% Triton X-100 and an anti-protease cocktail (CLAPA: 2 µg/ml leupeptin, 2 µg/ml antipain, 2 µg/ml pepstatin, 2 µg/ml chymostatin and 16 µg/ml aprotinin). The lysates were spun in a microcentrifuge at 4 °C for 5 min at 12000 g before immunoprecipitation.

{blacksquare} Immunoprecipitations.
The lysates were aliquoted and each sample was incubated with one of the anti-G mAbs used in the study for 45 min at 4 °C. Protein A–Sepharose (Sigma) was then added and the mixture was incubated for 45 min at 4 °C. The resulting immune complexes were centrifuged and washed twice with TD containing 1% CHAPS and CLAPA. The complexes were then boiled for 5 min in reducing Laemmli buffer before analysis by SDS–PAGE (10% polyacrylamide) and autoradiography. Quantification of radioactivity was performed using a phosphorimager (Molecular Dynamics).

{blacksquare} PI-PLC treatment.
Cells were metabolically labelled for 30 min and chased for 150 min. Cell monolayers were then washed with TD buffer and incubated at 37 °C for 60 min in 900 µl TD either with or without the addition of 0·5 U of phosphatidylinositol–phospholipase C (PI-PLC; Roche). After this treatment, the supernatant and the wash were pooled while the cells were lysed in TD containing 1% CHAPS and CLAPA. In each sample, G was then immunoprecipitated by mAb 21H8.

{blacksquare} Endo H treatment.
Immune complexes associated with Protein A–Sepharose were incubated in phosphate–citrate buffer, pH 5·6, containing 1% SDS for 5 min at 100 °C. The supernatant was split into two aliquots of 30 µl. One aliquot was then treated with 8 mU Endo H (Roche) overnight at 37 °C. The second aliquot was incubated at 37 °C, in the absence of Endo H, as a control. Both samples were then analysed by SDS–PAGE (10% polyacrylamide).

{blacksquare} Immunofluorescence experiments.
At 24 h post-transfection, BSR cells were fixed for 15 min at room temperature in 4% paraformaldehyde in PBS (150 mM NaCl, 10 mM phosphate, pH 7·4). Each of the following steps was performed at room temperature and followed by extensive washing with PBS. For immunodetection of G at the cell surface, cells were incubated for 45 min with the indicated mAb (diluted 1/100 from ascitic fluid). For immunodetection of intracellular G, in parallel experiments, the cells were permeabilized by adding 0·1% Triton X-100 to the mAb solution. G was then labelled either with a goat anti-mouse antibody conjugated to fluorescein when cells were not permeabilized or with a goat anti-mouse antibody conjugated to rhodamine when cells were permeabilized.

{blacksquare} Oligomerization analysis.
Cellular lysates (300 µl out of 500 µl) were loaded on linear sucrose gradients. Gradients were prepared from stocks of 5 and 20% sucrose in TD supplemented with CLAPA and containing 1% CHAPS or 0·5% Triton X-100. The gradients were then centrifuged at 35000 r.p.m. for 16 h at 4 °C using an SW 41 rotor (Beckman). Fractions (12x1 ml) were collected from the bottom of each gradient. In each fraction, the glycoprotein was immunoprecipitated by mAb 21H8 before analysis by SDS–PAGE (10% polyacrylamide).


   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
G1–439 and Ggpi fold in an alternative conformation antigenically distinct from the native ectodomain of the wild-type, complete, membrane-anchored glycoprotein
We have previously shown that G1–439 folds in a monomeric, alternative conformation, which is antigenically distinct from the N conformation (Gaudin et al., 1999 ). As a first step to investigate the role of the TMD in the correct native folding of the glycoprotein, the mutant Ggpi, in which the G TMD was replaced by a GPI anchor, was constructed (Fig. 1). Cells were transfected with pcDNA I encoding either G, Ggpi or G1–439. They were then labelled with [35S]methionine and [35S]cysteine for 10 min and chased for periods of 60 min. The cells were then lysed and the lysates were immunoprecipitated using a battery of mAbs. The characteristics of these mAbs are summarized in Table 1.

All these antibodies were able to recognize G (Fig. 2A, panel G), which migrated as two bands in SDS–PAGE. The faster migrating band (labelled NT) corresponded to the untrimmed glycoprotein with one sugar chain. The slower migrating one (labelled GT) corresponded to the glycoprotein that had already moved to the Golgi complex, where the high-mannose carbohydrates were trimmed and new sugars were added. This form is resistant to Endo H treatment (Gaudin, 1997 ; Gaudin et al., 1999 ).



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Fig. 2. Characterization of Ggpi. (A) Antigenicity of G, Ggpi and G1–439. Transfected BSR cells were pulse-labelled for 10 min, chased for 60 min then lysed in cold TD buffer containing 1% CHAPS. Aliquots of lysates were incubated with the mAb specified. Immunoprecipitated material was analysed by SDS–PAGE (10% polyacrylamide). NT, untrimmed G; GT, fully trimmed G. (B) Kinetics of Endo H resistance acquisition by Ggpi. Transfected cells were pulse-labelled for 10 min and chased for the times specified. Cell lysates were immunoprecipitated with mAb 21H8. Immunoprecipitated material was incubated overnight in the presence (+) or absence (-) of Endo H. Immunoprecipitated material was analysed by SDS–PAGE (10% polyacrylamide) and autoradiography. D, Endo H-deglycosylated G; NT, untrimmed G; GT, fully trimmed G. (C) Sensitivity of Ggpi to PI-PLC treatment. Transfected cells were metabolically labelled for 10 min and chased for 60 min. Then medium was replaced by TD buffer either with (+) or without (-) PI-PLC. After 60 min, the supernatant was recovered and cells were lysed in cold TD buffer containing 1% CHAPS. Supernatant (S) and lysates (C) were subjected to immunoprecipitation with 21H8. Immunoprecipitated material was analysed by SDS–PAGE and autoradiography.

 
G1–439 and Ggpi were both immunoprecipitated by monoclonal antibody 21H8. Both the untrimmed glycoprotein (NT) and the mature one (GT) were found in the immunoprecipitates (Fig. 2A, panels G1–439 and Ggpi). Only GT was resistant to Endo H treatment [see Fig. 2B for Ggpi and Fig. 4 in Gaudin et al. (1999 ) for G1–439]. The presence of GT showed that both G1–439 and Ggpi were able to leave the ER (Kornfeld & Kornfeld, 1985 ) indicating that they have passed the ER quality control and thus have the characteristics of folded proteins (Gething et al., 1986 ; Ellgaard et al., 1999 ). However, neither G1–439 nor Ggpi were recognized by monoclonal antibodies directed against site II, site III or antigenic site a, indicating that their antigenic structure was different from the native ectodomain. Finally, mAb 17D2, which recognizes G both in its unfolded and native structure but not in its fusion inactive conformation (Gaudin, 1997 ), immunoprecipitated the NT form of G1–439 and Ggpi but not (or with a poor efficiency) the GT form indicating that this antibody only recognized unfolded forms retained in the ER, as previously demonstrated for G1–439 (Gaudin et al., 1999 ).



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Fig. 4. (A) Recognition of G4G and GOgd56 by mAbs 21H8, 30AA5 and 41BC2. Transfected BSR cells were pulse-labelled for 10 min, chased for 60 min, then lysed in cold TD buffer containing 1% CHAPS. Cell lysates were aliquoted and immunoprecipitated with selected mAbs. NT, untrimmed G; GT, fully trimmed G. (B) Kinetics of Endo H resistance acquisition by GOgd56. Transfected cells were pulse-labelled for 10 min and chased for the times specified. Cell lysates were immunoprecipitated with mAb 21H8. Immunoprecipitated material was incubated overnight in the presence (+) or absence (-) of Endo H and analysed by SDS–PAGE (10% polyacrylamide). D, Endo H-deglycosylated G; NT, untrimmed G; GT, fully trimmed G.

 
We checked that Ggpi was indeed anchored in the membrane. Fig. 2(C) shows that no Ggpi was found in the supernatant of transfected cells in the absence of phospholipase C (PI-PLC) treatment indicating that it was anchored in the membrane by its glycolipidic tail.

Taken together, these results indicated that Ggpi, although it is anchored in the membrane, behaves like G1–439 and folds in an alternative conformation. This indicates that a glycolipid anchor is not sufficient for correct native folding of G.

A peptide TMD is sufficient for correct native folding of the G ectodomain
Chimeric glycoproteins in which the G TMD was replaced by either the TMD of p75, the human low affinity receptor for NGF (Gp75), or the TMD of pseudorabies virus glycoprotein gC (GgC) were then constructed (Fig. 1). Neither p75 nor gC are trimeric (Hampl et al., 1984 ; Zhu & Courtney, 1988 ; Yano & Chao, 2000 ). The folding of these chimeric glycoproteins was then analysed (Fig. 3) using mAbs 21H8, 30AA5 and 41BC2, which were representative of our panel of monoclonal antibodies.



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Fig. 3. Recognition of Gp75 and GgC by mAbs 21H8, 30AA5 and 41BC2. Transfected BSR cells were pulse-labelled for 10 min, chased for 60 min, then lysed in cold TD containing 1% CHAPS. Cell lysates were aliquoted and immunoprecipitated with selected mAbs. NT, untrimmed G; GT, fully trimmed G.

 
After a 10 min labelling pulse followed by a 60 min chase, both NT and GT were detected by mAb 21H8 for both chimeras. Furthermore, in contrast with the results obtained with G1–439 and Ggpi, the GT form of GgC and Gp75 was also efficiently immunoprecipitated by mAbs 30AA5 and 41BC2. These results indicated that the presence of a peptide TMD was sufficient to allow folding of G with its native antigenicity. This ruled out the possibility that the G TMD could assist folding by self-trimerizing because Gp75 and GgC appear to be correctly folded although p75 and gC are not trimeric molecules.

Analysis of the role of a peptide TMD in correct folding of G
Another possibility could be that a peptide TMD would form an alpha helix, which would extend into the carboxy-terminal part of the ectodomain. In the absence of any TMD, the carboxy-terminal part of the ectodomain would not be able to adopt a helical conformation and the complete ectodomain would fold in an alternative conformation. This hypothesis was challenged by inserting three glycines between glycine 438 and lysine 439 (Fig. 1): a putative alpha-helical structure extending from the TMD would be broken by this cluster of four glycines. However, the corresponding mutant glycoprotein (G4G) had the same antigenicity as wild-type G; as shown in Fig. 4(A), the GT form was efficiently immunoprecipitated by mAbs 30AA5 and 41BC2. Thus, a short flexible linker located between the ectodomain and the TMD did not affect the folding of G and the role of the TMD is not to nucleate an alpha-helical structure.

We then asked whether the difference in conformation between Ggpi and G anchored by a peptide TMD could be due to the additional undecapeptide and the oligoglycosidic chain between the G ectodomain and its lipid anchor. First, we inserted the undecapeptide mentioned above between the ectodomain and the TMD. This chimera appeared to have the native antigenicity of wild-type G (not shown). As this construct lacked the oligosaccharide linker of the GPI anchor, we hypothesized that the insertion of a highly flexible, hydrophilic linker between the G ectodomain and the G TMD could drive folding of the ectodomain into its alternative conformation. Thus, a mutant (GOgd56) was constructed, in which the 56 amino acid long O-glycosylated domain of the low affinity receptor for NGF (p75) was inserted between the ectodomain and the TMD of G as a hydrophilic spacer (Van den Steen et al., 1998 ). Both the untrimmed GOgd56 (NT) and the mature one (GT) were immunoprecipitated by 21H8 (Fig. 4A). GT was resistant to Endo H treatment (Fig. 4B). The increased difference in migration between the NT and GT forms of GOgd56, when compared with the other constructs, was due to O-glycosylation acquisition in the trans-Golgi compartments. However, GT was not recognized by mAbs 30AA5 and 41BC2 indicating that GOgd56 behaved as Ggpi and G1–439 and was folded in an alternative conformation.

Finally, in order to investigate the influence of the length of the hydrophilic insertion, mutants GOgd21 and GOgd35 were constructed, in which a shorter O-glycosylated stem (corresponding to the first 21 and the first 35 amino acids of p75 O-glycosylated domain) was inserted between the ectodomain and the TMD. For both mutants, the untrimmed (NT) and the mature (GT) forms were immunoprecipitated by 21H8 (Fig. 5A). The GT forms of GOgd21 and GOgd35 were very poorly recognized by mAbs 30AA5 and 41BC2 (Fig. 5A), yet significantly more than the GT form of Gogd56 (Fig. 5B). This indicated that part of the mature GOgd21 and GOgd35 was correctly folded although most of the protein acquired an alternative conformation.



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Fig. 5. (A) Recognition of GOgd35 and GOgd21 by mAbs 21H8, 30AA5 and 41BC2. Transfected BSR cells were pulse-labelled for 10 min, chased for 60 min, then lysed in cold TD containing 1% CHAPS. Cell lysates were aliquoted and immunoprecipitated with selected mAbs. Immunoprecipitated material was analysed by SDS–PAGE (10% polyacrylamide) and autoradiography. NT, untrimmed G; GT, fully trimmed G. (B) Quantification of the amount of GT form immunoprecipitated by mAbs 30AA5 and 41BC2. The amount of radioactivity contained in the band corresponding to GT was quantified using a phosphorimager. The amount of radioactivity found in the band corresponding to GT immunoprecipitated by 21H8 was defined as 100%. The graph shows the mean of two experiments for GOgd21, GOgd35 and G1–439, three experiments for GOgd56 and nine experiments for G. Error bars indicate standard deviation.

 
Analysis of the antigenicity of the constructs by immunofluorescence
In the immunoprecipitation experiments, it was possible that, although folded in a correct native conformation, Ggpi, GOgd21, GOgd35 and GOgd56 were denatured by CHAPS because their native structure would have been less stable than wild-type G. We thus performed immunofluorescence on cells fixed with 4% paraformaldehyde with or without permeabilization with 0·1% Triton X-100 (Fig. 6). All the constructs were recognized by mAb 21H8 at the cell surface. This was a new indication that they were transported to the cell surface and, thus, that they fulfilled the criteria to escape the ER quality control because they had the characteristics of folded proteins. Finally, in agreement with the results of the immunoprecipitation experiments, neither Ggpi nor GOgd56 were recognized by either mAbs 30AA5 or 41BC2, either at the cell surface (Fig. 6, shown for 30AA5) or intracellularly (Fig. 6, shown for 41BC2) (whereas G and the other constructs were recognized by both antibodies). These experiments ruled out a possible effect of the solubilization on the antigenicity of the constructs and confirmed that GOgd56 and Ggpi ectodomains were indeed folded in an alternative conformation.



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Fig. 6. Immunodetection of the constructs. BSR cells were transfected with G or the indicated mutants (cells labelled GOgd were transfected by the plasmid encoding GOgd56). Cells were fixed 24 h post-infection in 4% paraformaldehyde and were either permeabilized with 0·1% Triton X-100 (41BC2) or not permeabilized (30AA5 and 21H8) before incubation with the indicated mAb. G was stained with a goat anti-mouse antibody conjugated to fluorescein (21H8 and 30AA5) or rhodamine (41BC2). The side of each square is 100 µm long.

 
Analysis of the quaternary structure of the constructs
We have previously demonstrated that G1–439 has a sedimentation coefficient of about 4S and is monomeric (Gaudin et al., 1999 ). By comparison, when solubilized in the presence of 1% CHAPS, the major part of the complete glycoprotein solubilized from the virion sedimented with a sedimentation coefficient of about 9S (Gaudin et al., 1992 , 1999 ). This form has been shown to be a trimer. However, the quaternary structure of RV G is not very stable and G solubilization using 0·5% Triton X-100 results in G monomerization.

The oligomeric status of all the constructs described in this paper was thus analysed on a linear 5–20% sucrose gradient containing 1% CHAPS or 0·5% Triton X-100 (Fig. 7). Only G and GgC were found to be trimeric in the presence of CHAPS. These trimers were not very stable: even in presence of CHAPS, monomers were also detected in the gradient in the case of G and in the presence of Triton X-100, GgC and G were both found to be monomeric. All the other constructs were found to be monomeric. Therefore, all the constructs that were folded in an alternative conformation (G1–439, Ggpi, GOgd21, GOgd35, GOgd56) were monomeric. However, constructs such as Gp75 or G4G, whose antigenicity is similar to the antigenicity of G, did not form stable trimers as judged by their sedimentation in a sucrose gradient.



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Fig. 7. Oligomerization state analysis of the different constructs presented in this study. Metabolically labelled proteins were loaded on to 5–20% sucrose gradients containing either 1% CHAPS or 0·5% Triton X-100. The samples were centrifuged and fractions (1–12) were collected. Proteins were immunoprecipitated by 21H8 before analysis by SDS–PAGE under reducing conditions; b and t indicate the bottom and top of the gradient. The positions of the standards [catalase (cat.) and BSA (bsa)] as well as those of trimers and monomers are indicated.

 

   Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
In this paper, we have demonstrated that the ectodomain of rabies virus glycoprotein is able to fold in an alternative conformation, which is monomeric and antigenically distinct from the native state of the complete membrane-anchored ectodomain. In particular, mAbs directed against major antigenic sites of G are unable to bind to the ectodomain when it is folded in this alternative conformation. This alternative conformation is different from the fusion inactive conformation of G because it is not recognized by mAb 9B4, directed against minor antigenic site a, which still binds G in the fusion inactive conformation (Table 1). However, it is not simply the ectodomain in an unfolded state: G1–439, Ggpi, GOgd21, GOgd35 and GOgd56 were able to leave the ER and thus have the characteristics of correctly folded proteins (Gething et al., 1986 ; Ellgaard et al., 1999 ). Therefore, this study demonstrates that the ‘ER quality control’ is based on general criteria that can be fulfilled by different conformations of a protein.

It has generally been supposed that the ectodomain, the TMD and the cytoplasmic part constitute independent folding domains within glycoproteins. This view is well supported by the efficient surface expression of many chimeric proteins when these domains are exchanged between different glycoproteins and also by the successful expression and secretion of soluble ectodomains of cellular proteins and viral glycoproteins (Doms et al., 1993 ). However, in a growing number of cases, more subtle or less subtle structural differences have been detected between the wild-type glycoprotein and some soluble or chimeric constructs (Paterson & Lamb, 1990 ; Godet et al., 1991 ). In the case of influenza HA, the best-characterized viral glycoprotein, it has been shown that anchor-free HA leaves the ER as a monomer and forms aggregates in the late compartment of the Golgi pathway (Singh et al., 1990 ; Vanlandschoot et al., 1996 ). It has been proposed that soluble HA adopts a folded monomeric structure that is different from the ectodomain structure in complete trimeric HA and also undergoes a low-pH induced conformational change in the acidic Golgi compartments leading to its aggregation (Vanlandschoot et al., 1998 ). Furthermore, it has also been demonstrated that GPI- and transmembrane-anchored HA slightly differ in structure and receptor-binding activity (Kemble et al., 1993 ) and that chimeric HAs, having heterologous transmembrane domains, differ in their antigenicities from wild-type HA (Roth et al., 1986 ; Lazarovits et al., 1990 ).

Our results indicate that insertion of flexible, heavily O-glycosylated peptides between the ectodomain and the TMD induces folding of G in an alternative conformation, which is not recognized by mAbs 30AA5 and 41BC2. A reasonable interpretation (also consistent with the results obtained for Ggpi) could be that the presence of additional sugars interferes with folding of the ectodomain (e.g. by steric hindrance). However, the alternative conformation of G1–439 cannot be explained in this way.

An alternative explanation might be that correct native folding of the G ectodomain requires membrane proximity. Indeed, the amount of the mature form (GT) correctly folded (i.e. recognized by mAbs 30AA5 and 41BC2) decreased when the length of the hydrophilic insertion between the ectodomain and the TMD increased. This suggests that the membrane environment somehow assists G ectodomain folding. This assistance could be due to factors close to the membrane or from the membrane itself. For example, membrane proximity might be necessary for interactions between G folding intermediates and ER chaperones. However, it should be noted that both G1–439 and Ggpi bind calnexin (data not shown) as wild-type G does. Furthermore, G1–439 interacts with BiP (Wojczyk et al., 1998 ) as does wild-type G (Gaudin, 1997 ). Although it is conceivable that other unidentified chaperones are involved in G folding, these data suggest that interactions between G and chaperones are probably not involved in the discrimination between the folding pathways of the ectodomain and that the membrane itself plays a direct role in correct native folding of G.

Membrane-assisted protein folding has already been demonstrated for the bacterial polytopic membrane protein lactose permease (Bogdanov & Dowhan, 1998 , 1999 ). In this case, it was demonstrated that phosphatidylethanolamine acts as a molecular chaperone, which is transiently required at a late step of the folding pathway. In the case of RV G, it is not known whether a specific lipid is involved in the folding process, nor whether membrane interaction is required only transiently for correct folding or if this interaction is still present in the native structure of full-length G. Interestingly, in the case of the glycoprotein of vesicular stomatitis virus (VSV), another member of the rhabdovirus family, it has been demonstrated that the 12 membrane-proximal residues of VSV G were necessary for efficient budding of neosynthesized virions. One possible explanation is that this domain interacts with the cell membrane to modify the lipid environment and/or the membrane curvature at the bud site (Robison & Whitt, 2000 ).

This work also provides information about the requirements for G trimerization and its influence on antigenicity. First, mutant GgC appeared to fold in a trimeric structure, which, as with wild-type G, was sensitive to Triton X-100. As gC is not trimeric (Hampl et al., 1984 ; Zhu & Courtney, 1988 ), this demonstrated that the G TMD was not required for the correct oligomerization of G. Secondly, replacement of the G TMD by the p75 TMD resulted in a chimeric glycoprotein (Gp75), which was monomeric. Thus, the nature of the TMD influences the stability of the oligomeric structure. Finally, as G4G and Gp75 were found to be monomeric although they have the same antigenicity as wild-type G, the stability of the trimeric structure is not necessary for the presence of major antigenic sites II and III at the surface of the glycoprotein.

In the case of VSV G, a soluble and a GPI-anchored ectodomain (equivalent to G1–439 and Ggpi, respectively) were both found to be trimeric (Crise et al., 1989 ). This suggests that trimerization determinants in the ectodomain of VSV G are stronger than in RV G. However, it has been demonstrated that, unlike an engineered HA ectodomain anchored into the membrane by a glycophosphatidylinositol, VSV Ggpi is unable to induce lipid mixing (Odell et al., 1997 ). This could be explained by a non-functional alternative folding of the ectodomain of Ggpi, suggesting that VSV G behaves in the same way as RV G.

Finally, our results stress the importance of taking into consideration the whole folding environment when studying the structure of a protein. For instance, in the case of RV G, it appears that obtaining a soluble fragment in the native trimeric conformation, for the purpose of structural studies, cannot be as simple as recombinant expression of a soluble ectodomain.


   Acknowledgments
 
We thank Anne Flamand, Danielle Blondel and Rob Ruigrok for helpful discussion and careful reading of the manuscript, Christine Tuffereau for the plasmid encoding p75, Sybille Dezélée for the plasmid encoding gC and Jean Dubuisson for providing the plasmid pTM1/E2(371-661)DAF. This work was supported by CNRS (UPR 9053) and by funds from the ‘ACI blanche’ and the ‘Programme Physique et Chimie du Vivant (1997)’. Antoine Maillard is a pre-doctoral fellow on a grant from the French ‘Ministère de l’éducation nationale et de la recherche’.


   References
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Abstract
Introduction
Methods
Results
Discussion
References
 
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Received 11 December 2001; accepted 31 January 2002.