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
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Abstract |
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Introduction |
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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 (G1439) lacking the transmembrane and intracytoplasmic domains (residues 440505) (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 G1439.
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Methods |
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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 TrisHCl, 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.
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 ASepharose (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 SDSPAGE (10% polyacrylamide) and autoradiography. Quantification of radioactivity was performed using a phosphorimager (Molecular Dynamics).
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 phosphatidylinositolphospholipase 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.
Endo H treatment.
Immune complexes associated with Protein ASepharose were incubated in phosphatecitrate 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 SDSPAGE (10% polyacrylamide).
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.
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 SDSPAGE (10% polyacrylamide).
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Results |
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All these antibodies were able to recognize G (Fig. 2A, panel G), which migrated as two bands in SDSPAGE. 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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Taken together, these results indicated that Ggpi, although it is anchored in the membrane, behaves like G1439 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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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 G1439 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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The oligomeric status of all the constructs described in this paper was thus analysed on a linear 520% 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 (G1439, 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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Discussion |
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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 G1439 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 G1439 and Ggpi bind calnexin (data not shown) as wild-type G does. Furthermore, G1439 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 G1439 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.
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Acknowledgments |
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References |
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Benmansour, A., Leblois, H., Coulon, P., Tuffereau, C., Gaudin, Y., Flamand, A. & Lafay, F. (1991). Antigenicity of rabies virus glycoprotein. Journal of Virology 65, 4198-4203.[Medline]
Bogdanov, M. & Dowhan, W. (1998). Phospholipid-assisted protein folding: phosphatidylethanolamine is required at a late step of the conformational maturation of the polytopic membrane protein lactose permease. EMBO Journal 17, 5255-5264.
Bogdanov, M. & Dowhan, W. (1999). Lipid-assisted protein folding. Journal of Biological Chemistry 274, 36827-36830.
Cocquerel, L., Meunier, J. C., Pillez, A., Wychowski, C. & Dubuisson, J. (1998). A retention signal necessary and sufficient for endoplasmic reticulum localization maps to the transmembrane domain of hepatitis C virus glycoprotein E2. Journal of Virology 72, 2183-2191.
Cox, J. H., Dietzschold, B. & Schneider, L. G. (1977). Rabies virus glycoprotein. II. Biological and serological characterization. Infection and Immunity 16, 754-759.[Medline]
Crise, B., Ruusala, A., Zagouras, P., Shaw, A. & Rose, J. K. (1989). Oligomerization of glycolipid-anchored and soluble forms of the vesicular stomatitis virus glycoprotein. Journal of Virology 63, 5328-5333.[Medline]
Dietzschold, B., Gore, M., Marchadier, D., Niu, H. S., Bunschoten, H. M., Otvos, L.Jr, Wunner, W. H., Ertl, H. C. J., Osterhaus, A. D. & Koprowski, H. (1990). Structural and immunological characterization of a linear neutralizing epitope of the rabies virus glycoprotein and its possible use in a synthetic vaccine. Journal of Virology 64, 3804-3809.[Medline]
Doms, R. W., Lamb, R. A., Rose, J. K. & Helenius, A. (1993). Folding and assembly of viral membrane proteins. Virology 193, 545-562.[Medline]
Ellgaard, L., Molinari, M. & Helenius, A. (1999). Setting the standards: quality control in the secretory pathway. Science 286, 1882-1888.
Fuerst, T. R., Niles, E. G., Studier, F. W. & Moss, B. (1986). Eukaryotic transient expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proceedings of the National Academy of Sciences, USA 83, 8122-8126.[Abstract]
Gaudin, Y. (1997). Folding of rabies virus glycoprotein: epitope acquisition and interaction with endoplasmic reticulum chaperones. Journal of Virology 71, 3742-3750.[Abstract]
Gaudin, Y., Ruigrok, R. W. H., Tuffereau, C., Knossow, M. & Flamand, A. (1992). Rabies virus glycoprotein is a trimer. Virology 187, 627-632.[Medline]
Gaudin, Y., Ruigrok, R. W. H., Knossow, M. & Flamand, A. (1993). Low-pH conformational changes of rabies virus glycoprotein and their role in membrane fusion. Journal of Virology 67, 1365-1372.[Abstract]
Gaudin, Y., Moreira, S., Bénéjean, J., Blondel, D., Flamand, A. & Tuffereau, C. (1999). Soluble ectodomain of rabies virus glycoprotein expressed in eukaryotic cells folds in a monomeric conformation that is antigenically distinct from the native state of the complete membrane-anchored glycoprotein. Journal of General Virology 80, 1647-1656.[Abstract]
Gething, M. J., McCammon, K. & Sambrook, J. (1986). Expression of wild-type and mutant forms of influenza hemagglutinin: the role of folding in intracellular transport. Cell 46, 939-950.[Medline]
Godet, M., Rasschaert, D. & Laude, H. (1991). Processing and antigenicity of entire and anchor-free spike glycoprotein S of coronavirus TGEV expressed by recombinant baculovirus. Virology 185, 732-740.[Medline]
Hampl, H., Ben-Porat, T., Ehrlicher, L., Habermehl, K. O. & Kaplan, A. S. (1984). Characterization of the envelope proteins of pseudorabies virus. Journal of Virology 52, 583-590.[Medline]
Kemble, G. W., Henis, Y. I. & White, J. M. (1993). GPI- and transmembrane-anchored influenza hemagglutinin differ in structure and receptor binding activity. Journal of Cell Biology 122, 1253-1265.[Abstract]
Kornfeld, R. & Kornfeld, S. (1985). Assembly of asparagine-linked oligosaccharides. Annual Review of Biochemistry 54, 631-664.[Medline]
Lafay, F., Benmansour, A., Chebli, K. & Flamand, A. (1996). Immunodominant epitopes defined by a yeast-expressed library of random fragments of the rabies virus glycoprotein map outside major antigenic sites. Journal of General Virology 77, 339-346.[Abstract]
Lafon, M., Wiktor, T. J. & Macfarlan, R. I. (1983). Antigenic sites of the CVS rabies virus glycoprotein: analysis with monoclonal antibodies. Journal of General Virology 64, 843-851.[Abstract]
Lazarovits, J., Shia, S. P., Ktistakis, N., Lee, M. S., Bird, C. & Roth, M. G. (1990). The effects of foreign transmembrane domains on the biosynthesis of the influenza virus hemagglutinin. Journal of Biological Chemistry 265, 4760-4767.
Odell, D., Wanas, E., Yan, J. & Ghosh, H. P. (1997). Influence of membrane anchoring and cytoplasmic domains on the fusogenic activity of vesicular stomatitis virus glycoprotein G. Journal of Virology 71, 7996-8000.[Abstract]
Paterson, R. G. & Lamb, R. A. (1990). Conversion of a class II integral membrane protein into a soluble and efficiently secreted protein: multiple intracellular and extracellular oligomeric and conformational forms. Journal of Cell Biology 110, 999-1011.[Abstract]
Préhaud, C., Coulon, P., Lafay, F., Thiers, C. & Flamand, A. (1988). Antigenic site II of the rabies virus glycoprotein: structure and role in viral virulence. Journal of Virology 62, 1-7.[Medline]
Robison, C. S. & Whitt, M. A. (2000). The membrane-proximal stem region of vesicular stomatitis virus G protein confers efficient virus assembly. Journal of Virology 74, 2239-2246.
Rose, J. K., Buonocore, L. & Whitt, M. A. (1991). A new cationic liposome reagent mediating nearly quantitative transfection of animal cells. Biotechniques 10, 520-525.[Medline]
Roth, M. G., Doyle, C., Sambrook, J. & Gething, M. J. (1986). Heterologous transmembrane and cytoplasmic domains direct functional chimeric influenza virus hemagglutinins into the endocytic pathway. Journal of Cell Biology 102, 1271-1283.[Abstract]
Seif, I., Coulon, P., Rollin, P. E. & Flamand, A. (1985). Rabies virulence: effect on pathogenicity and sequence characterization of rabies virus mutations affecting antigenic site III of the glycoprotein. Journal of Virology 51, 505-514.
Singh, I., Doms, R. W., Wagner, K. R. & Helenius, A. (1990). Intracellular transport of soluble and membrane-bound glycoproteins: folding, assembly and secretion of anchor-free influenza hemagglutinin. EMBO Journal 9, 631-639.[Abstract]
Thoulouze, M. I., Lafage, M., Schachner, M., Hartmann, U., Cremer, H. & Lafon, M. (1998). The neural cell adhesion molecule is a receptor for rabies virus. Journal of Virology 72, 7181-7190.
Tuffereau, C., Bénéjean, J., Blondel, D., Kieffer, B. & Flamand, A. (1998). Low-affinity nerve growth factor receptor (P75NTR) can serve as a receptor for rabies virus. EMBO Journal 17, 7250-7259.
Van den Steen, P., Rudd, P. M., Dwek, D. A. & Opdenakker, G. (1998). Concepts and principles of O-linked glycosylation. Critical Reviews in Biochemistry and Molecular Biology 33, 151-208.[Abstract]
Vanlandschoot, P., Beinaert, E., Neirynck, S., Saelens, X., Jou, W. M. & Fiers, W. (1996). Molecular and immunological characterization of soluble aggregated A/Victoria/3/75 (H3N2) influenza haemagglutinin expressed in insect cells. Archives of Virology 141, 1715-1726.[Medline]
Vanlandschoot, P., Beinaert, E., Grooten, J., Jou, W. M. & Fiers, W. (1998). pH-dependent aggregation and secretion of soluble monomeric influenza hemagglutinin. Archives of Virology 143, 227-239.[Medline]
Walker, P. J. & Kongsuwan, K. (1999). Deduced structural model for animal rhabdovirus glycoproteins. Journal of General Virology 80, 1211-1220.[Abstract]
Whitt, M. A., Buonocore, L., Prehaud, C. & Rose, J. K. (1991). Membrane fusion activity, oligomerization, and assembly of the rabies virus glycoprotein. Virology 185, 681-688.[Medline]
Wojczyk, B. S., Stwora-Wojczyk, M., Shakin-Eshleman, S., Wunner, W. H. & Spitalnik, S. L. (1998). The role of site-specific N-glycosylation in secretion of soluble forms of rabies virus glycoprotein. Glycobiology 8, 121-130.
Yano, H. & Chao, M. V. (2000). Neurotrophin receptor structure and interactions. Pharmaceutica Acta Helvetiae 74, 253-260.[Medline]
Zhu, Q. & Courtney, R. J. (1988). Chemical crosslinking of glycoproteins on the envelope of herpes simplex virus. Virology 167, 377-384.[Medline]
Received 11 December 2001;
accepted 31 January 2002.