Equipe Hépatite C, CNRS-FRE2369, IBL/Institut Pasteur de Lille, 1 rue Calmette, BP 447, 59021 Lille cedex, France1
Author for correspondence: Jean Dubuisson. Fax +33 3 20 87 11 11. e-mail jean.dubuisson{at}ibl.fr
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Synthesis, folding and assembly of E1 and E2 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Folding of proteins in the ER is assisted by chaperone molecules present in this compartment. ER chaperones calnexin, calreticulin and BiP have been shown to interact with HCV envelope glycoproteins (Choukhi et al., 1998 ; Dubuisson & Rice, 1996
). Calnexin and calreticulin are lectin-like chaperones which show an affinity for monoglucosylated N-linked oligosaccharides (Trombetta & Helenius, 1998
). Binding of substrate glycoproteins to and release from calnexin and calreticulin depends on trimming and reglucosylation of the N-linked glycans. The absence of interaction between HCV glycoproteins and calreticulin or calnexin after tunicamycin treatment is consistent with the view that these chaperones act as lectins. In addition, the absence of glycans on E1 and E2 led to misfolding, demonstrating the essential role played by glycosylation for the folding of these proteins. Characterization of HCV envelope glycoproteins associated with ER chaperones indicates that calreticulin and BiP interact preferentially with aggregates of E1 and E2, and calnexin with non-covalently linked complexes (Choukhi et al., 1998
) (Fig. 2
). These observations suggest that folding of the HCV envelope glycoproteins involved in the productive pathway of assembly is assisted by calnexin.
![]() |
Regions involved in heterodimerization |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Deletion of the TM domain of E2 or its replacement by the anchor signal of another protein has been shown to abolish the formation of E1E2 heterodimers (Cocquerel et al., 1998 ; Michalak et al., 1997
; Patel et al., 2001
; Selby et al., 1994
), suggesting that the TM domains of HCV envelope glycoproteins are involved in heterodimerization. Recently, the role of these domains in the assembly of the E1E2 heterodimer has been analysed by alanine scanning insertion mutagenesis (Op De Beeck et al., 2000
). This technique has been shown to be a powerful method for detecting dimerization of TM
-helices (Braun et al., 1997
). Indeed, insertion of a single amino acid into a TM helix displaces the residues on the N-terminal side of the insertion by 110° relative to those on the C-terminal side of the insertion, disrupting a helixhelix packing interface involving residues on both sides of the insertion. Two distinct segments of the TM domain of E1 and one of the TM domain of E2 were very sensitive to alanine insertion (Fig. 3
), demonstrating the essential role played by these domains in the assembly of HCV envelope glycoproteins. Interestingly, the amino-terminal segment of the TM domain of E1 contains a highly conserved GXXXG motif, which has been documented to ensure specific homodimerization of TM domains in TM proteins (Russ & Engelman, 2000
).
|
![]() |
Subcellular localization of the E1E2 heterodimer |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
ER retention of the E1E2 heterodimer suggests that specific signal(s) present in this complex are responsible for this subcellular localization. The presence of a retention signal was first demonstrated for E2 by Cocquerel et al. (1998) . Indeed, E2 expressed in the absence of E1 can fold properly and is retained in the ER, as shown by the lack of complex glycans, its intracellular distribution and the absence of its expression at the cell surface. By using chimeric proteins constructed from E2 and proteins normally expressed at the cell surface, it has been shown that the TM domain of E2 is a signal for ER retention (Cocquerel et al., 1998
; Flint et al., 1999
; Forns et al., 2000
; Patel et al., 2001
; Takikawa et al., 2000
). A similar approach has also been used to show that the TM domain of E1 is another signal for ER retention (Cocquerel et al., 1999
; Flint & McKeating, 1999
; Mottola et al., 2000
). In addition to the TM domain, a second retention signal has also been identified in the ectodomain of E1 (Mottola et al., 2000
). Indeed, a chimeric protein between CD8 and a 44 amino acid sequence of the ectodomain of E1 (located at position 290 to 333 of the polyprotein of the HCV BK strain) has been shown to be retained in the ER. However, data from other laboratories have shown that, when the TM domain of E1 is replaced by the TM domain of a protein normally expressed at the cell surface, E1 can be detected at the plasma membrane by immunofluorescence with anti-E1 antibodies (Forns et al., 2000
; Takikawa et al., 2000
), suggesting that this second retention signal is leaky. Alternatively, this retention signal might be buried in the structure of the ectodomain of E1. Immunolocalization of chimeric proteins containing the TM domain of E1 or E2 and analysis of their glycans have shown that these domains are signals for static retention in the ER (Cocquerel et al., 1999
; Duvet et al., 1998
; Patel et al., 2001
). However, Martire et al. (2001)
have observed that a small fraction of E2 migrates to the intermediate compartment and the cis-Golgi complex region, suggesting that the ER retention signal of E2 might be leaky. These data fit with the observation that a small fraction of E2 is not integrated in the ER membrane and follows the secretory pathway (Cocquerel et al., 2001
).
The TM domains of E1 and E2 have been proposed to start at polyprotein positions 353 (Cocquerel et al., 1999 ) and 718 (Cocquerel et al., 2000
), respectively (Fig. 3
). These TM domains are composed of two hydrophobic stretches connected by a short hydrophilic segment containing one (Lys for E1) or two (Asp and Arg for E2) fully conserved charged residues (Cocquerel et al., 2000
). In an attempt to identify residues in the TM domains of HCV envelope glycoproteins that are involved in ER retention, these charged residues have been mutated. Indeed, it has been shown for several non-viral proteins that a usual feature of membrane determinants for ER retention is the presence of one or several hydrophilic residues in the middle of the TM domain (Bonifacino et al., 1991
). A mutagenesis study has confirmed that the presence of charged amino acid residues located in the middle of the TM domains of E1 and E2 plays a major role in ER retention of these proteins (Fig. 3
) (Cocquerel et al., 2000
). Indeed, replacement of the lysine residue in the TM domain of E1 by an alanine led to cell surface expression of a chimeric protein made of the ectodomain of CD4 in fusion with the TM domain of E1. Similarly, replacement of the aspartic acid and the arginine by alanine residues in the TM domain of E2 led to cell surface expression of E2. However, these mutations affected other functions of the TM domains of HCV envelope glycoproteins. They led to an alteration of the processing of the polyprotein and disrupted the heterodimerization of E1E2, indicating that these functions are tightly linked. In addition, these observations suggest that the charged residues located in the middle of the TM domains of HCV envelope glycoproteins play a crucial role in the structure of these domains.
![]() |
Topology of the TM domains of E1 and E2 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The topology adopted by the TM domains of HCV envelope glycoproteins is still unclear. The presence of a first hydrophobic stretch and a signal sequence function separated by charged residue(s) in the TM domains of E1 and E2 suggests that these domains are composed of two membrane-spanning segments with the charged residues facing the cytosol (Fig. 3). However, amino acid sequence analysis (Cocquerel et al., 2000
) and recent data from our laboratory suggest that, after E1E2 heterodimerization, these TM domains likely exhibit a single membrane-spanning topology (Op De Beeck et al., 2000
). How can we reconcile a single membrane-spanning topology for the TM domains of E1 and E2 with the signal sequence function present in the C-terminal half of these domains? The most likely explanation is that before signalase cleavage, the TM domains of E1 and E2 adopt a transient hairpin structure in the translocon with both their N- and C-termini facing the ER lumen. After cleavage, a reorientation of the second hydrophobic stretch would occur, leading to interaction of the TM domains of HCV envelope glycoproteins (Fig. 3
). This model is supported by experimental data from our laboratory (L. Cocquerel, A. Op De Beeck, M. Lambot, J. Roussel, D. Delgrange, A. Pillez, C. Wychowski, F. Penin & J. Dubuisson, unpublished data). Indeed, we have shown that, in the absence of signal sequence cleavage, the TM domains of HCV envelope proteins form a hairpin structure. In contrast, when E1 or E2 was expressed alone and tagged at the C terminus with an epitope, the TM domain showed a single membrane-spanning topology. In addition, replacement of the charged residues present in the middle of the TM domains of E1 and E2 by alanine residues led to an alteration in the formation of the hairpin structures, indicating that these charged residues play a crucial role in the dynamic changes occurring in the TM domains of HCV envelope proteins. The dynamic behaviour of these TM domains is unique and it is linked to their multifunctionality. By reorienting their C terminus towards the cytosol and being part of a TM domain, the signal sequences at the C termini of E1 and E2 contribute to new functions: (1) membrane anchoring, (2) E1E2 heterodimerization, and (3) ER retention.
![]() |
Concluding remarks |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Because HCV does not replicate efficiently in cell culture, it is currently impossible to study later events in the maturation of HCV envelope glycoproteins that might occur during budding and maturation of the particle. However, the data that have been accumulated on closely related viruses, e.g. the flaviviruses and the pestiviruses (Rice, 1996 ), can provide information that might help us to understand the later steps of the HCV lifecycle.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Braun, P., Persson, B., Kaback, R. & von Heijne, G. (1997). Alanine insertion scanning mutagenesis of lactose permease transmembrane helices. Journal of Biological Chemistry 272, 29566-29571.
Choukhi, A., Ung, S., Wychowski, C. & Dubuisson, J. (1998). Involvement of endoplasmic reticulum chaperones in folding of hepatitis C virus glycoproteins. Journal of Virology 72, 3851-3858.
Choukhi, A., Pillez, A., Drobecq, H., Sergheraert, C., Wychowski, C. & Dubuisson, J. (1999). Characterization of aggregates of hepatitis C virus glycoproteins. Journal of General Virology 80, 3099-3107.
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.
Cocquerel, L., Duvet, S., Meunier, J.-C., Pillez, A., Cacan, R., Wychowski, C. & Dubuisson, J. (1999). The transmembrane domain of hepatitis C virus glycoprotein E1 is a signal for static retention in the endoplasmic reticulum. Journal of Virology 73, 2641-2649.
Cocquerel, L., Wychowski, C., Minner, F., Penin, F. & Dubuisson, J. (2000). Charged residues in the transmembrane domains of hepatitis C virus glycoproteins play a key role in the processing, subcellular localization and assembly of these envelope proteins. Journal of Virology 74, 3623-3633.
Cocquerel, L., Meunier, J.-C., Op de Beeck, A., Bonte, D., Wychowski, C. & Dubuisson, J. (2001). Coexpression of hepatitis C virus envelope proteins E1 and E2 in cis improves the stability of membrane insertion of E2. Journal of General Virology 82, 1629-1635.
Deleersnyder, V., Pillez, A., Wychowski, C., Blight, K., Xu, J., Hahn, Y. S., Rice, C. M. & Dubuisson, J. (1997). Formation of native hepatitis C virus glycoprotein complexes. Journal of Virology 71, 697-704.[Abstract]
Doms, R. W., Lamb, R. A., Rose, J. K. & Helenius, A. (1993). Folding and assembly of viral membrane proteins. Virology 193, 545-562.[Medline]
Dubuisson, J. (2000). Folding, assembly and subcellular localization of HCV glycoproteins. Current Topics in Microbiology and Immunology 242, 135-148.[Medline]
Dubuisson, J. & Rice, C. M. (1996). Hepatitis C virus glycoprotein folding: disulfide bond formation and association with calnexin. Journal of Virology 70, 778-786.[Abstract]
Dubuisson, J., Hsu, H. H., Cheung, R. C., Greenberg, H. B., Russell, D. G. & Rice, C. M. (1994). Formation and intracellular localization of hepatitis C virus envelope glycoprotein complexes expressed by recombinant vaccinia and Sindbis viruses. Journal of Virology 68, 6147-6160.[Abstract]
Dubuisson, J., Duvet, S., Meunier, J. C., Op De Beeck, A., Cacan, R., Wychowski, C. & Cocquerel, L. (2000). Glycosylation of the hepatitis C virus envelope protein E1 is dependent on the presence of a downstream sequence on the viral polyprotein. Journal of Biological Chemistry 275, 30605-30609.
Duvet, S., Cocquerel, L., Pillez, A., Cacan, R., Verbert, A., Moradpour, D., Wychowski, C. & Dubuisson, J. (1998). Hepatitis C virus glycoprotein complex localization in the endoplasmic reticulum involves a determinant for retention and not retrieval. Journal of Biological Chemistry 273, 32088-32095.
Flint, M. & McKeating, J. A. (1999). The C-terminal region of the hepatitis C virus E1 glycoprotein confers localization within the endoplasmic reticulum. Journal of General Virology 80, 1943-1947.
Flint, M. & McKeating, J. A. (2000). The role of hepatitis C virus glycoproteins in infection. Reviews in Medical Virology 10, 101-117.[Medline]
Flint, M., Thomas, J. M., Maidens, C. M., Shotton, C., Levy, S., Barclay, W. S. & McKeating, J. A. (1999). Functional analysis of cell surface-expressed hepatitis C virus E2 glycoprotein. Journal of Virology 73, 6782-6790.
Forns, X., Allander, T., Rohwer-Nutter, P. & Bukh, J. (2000). Characterization of modified hepatitis C virus E2 proteins expressed on the cell surface. Virology 274, 75-85.[Medline]
Grakoui, A., Wychowski, C., Lin, C., Feinstone, S. M. & Rice, C. M. (1993). Expression and identification of hepatitis C virus polyprotein cleavage products. Journal of Virology 67, 1385-1395.[Abstract]
Habersetzer, F., Fournillier, A., Dubuisson, J., Rosa, D., Abrigniani, S., Wychowski, C., Nakano, I., Trépo, C., Desgranges, C. & Inchauspé, G. (1998). Characterization of human monoclonal antibodies specific to the hepatitis C virus glycoprotein E2 with in vitro binding neutralization properties. Virology 249, 32-41.[Medline]
Hernandez, L. D., Hoffman, L. R., Wolfsberg, T. G. & White, J. M. (1996). Viruscell and cellcell fusion. Annual Review of Cell and Developmental Biology 12, 627-661.[Medline]
Lanford, R. E., Notvall, L., Chavez, D., White, R., Frenzel, G., Simonsen, C. & Kim, J. (1993). Analysis of hepatitis C virus capsid, E1, and E2/NS1 proteins expressed in insect cells. Virology 197, 225-235.[Medline]
Liberman, E., Fong, Y. L., Selby, M. J., Choo, Q. L., Cousens, L., Houghton, M. & Yen, T. S. (1999). Activation of the grp78 and grp94 promoters by hepatitis C virus E2 envelope protein. Journal of Virology 73, 3718-3722.
Martire, G., Viola, A., Iodice, L., Lotti, L. V., Gradini, R. & Bonatti, S. (2001). Hepatitis C virus structural proteins reside in the endoplasmic reticulum as well as in the intermediate compartment/cis-Golgi complex region of stably transfected cells. Virology 280, 176-182.[Medline]
Matsuura, Y., Suzuki, T., Suzuki, R., Sato, M., Aizaki, H., Saito, I. & Miyamura, T. (1994). Processing of E1 and E2 glycoproteins of hepatitis C virus expressed in mammalian and insect cells. Virology 205, 141-150.[Medline]
Meunier, J.-C., Fournillier, A., Choukhi, A., Cahour, A., Cocquerel, L., Dubuisson, J. & Wychowski, C. (1999). Analysis of the glycosylation sites of hepatitis C virus (HCV) glycoprotein E1 and the influence of E1 glycans on the formation of the HCV glycoprotein complex. Journal of General Virology 80, 887-896.[Abstract]
Michalak, J.-P., Wychowski, C., Choukhi, A., Meunier, J.-C., Ung, S., Rice, C. M. & Dubuisson, J. (1997). Characterization of truncated forms of hepatitis C virus glycoproteins. Journal of General Virology 78, 2299-2306.[Abstract]
Mottola, G., Jourdan, N., Castaldo, G., Malagolini, N., Lahm, A., Serafini-Cessi, F., Migliaccio, G. & Bonatti, S. (2000). A new determinant of endoplasmic reticulum localization is contained in the juxtamembrane region of the ectodomain of hepatitis C virus glycoprotein E1. Journal of Biological Chemistry 275, 24070-24079.
Op De Beeck, A., Montserret, R., Duvet, S., Cocquerel, L., Cacan, R., Barberot, B., Le Maire, M., Penin, F. & Dubuisson, J. (2000). Role of the transmembrane domains of hepatitis C virus envelope proteins E1 and E2 in the assembly of the noncovalent E1E2 heterodimer. Journal of Biological Chemistry 275, 31428-31437.
Patel, J., Patel, A. H. & McLauchlan, J. (1999). Covalent interactions are not required to permit or stabilize the non-covalent association of hepatitis C virus glycoproteins E1 and E2. Journal of General Virology 80, 1681-1690.[Abstract]
Patel, J., Patel, A. H. & McLauchlan, J. (2001). The transmembrane domain of the hepatitis C virus E2 glycoprotein is required for correct folding of the E1 glycoprotein and native complex formation. Virology 279, 58-68.[Medline]
Pettersson, R. F. (1991). Protein localization and virus assembly at intracellular membranes. Current Topics in Microbiology and Immunology 170, 67-104.[Medline]
Reed, K. E. & Rice, C. M. (2000). Overview of hepatitis C virus genome structure, polyprotein processing, and protein properties. Current Topics in Microbiology and Immunology 242, 55-84.[Medline]
Rice, C. M. (1996). Flaviviridae: the viruses and their replication. In Fields Virology , pp. 931-959. Edited by B. N. Fields, D. M. Knipe & P. M. Howley. Philadelphia:LippincottRaven.
Russ, W. P. & Engelman, D. M. (2000). The GxxxG motif: a framework for transmembrane helixhelix association. Journal of Molecular Biology 296, 911-919.[Medline]
Selby, M. J., Glazer, E., Masiarz, F. & Houghton, M. (1994). Complex processing and protein:protein interactions in the E2:NS2 region of HCV. Virology 204, 114-122.[Medline]
Shimizu, Y. K., Feinstone, S. M., Kohara, M., Purcell, R. H. & Yoshikura, H. (1996). Hepatitis C virus: detection of intracellular virus particles by electron microscopy. Hepatology 23, 205-209.[Medline]
Takikawa, S., Ishii, K., Aizaki, H., Suzuki, T., Asakura, H., Matsuura, Y. & Miyamura, T. (2000). Cell fusion activity of hepatitis C virus envelope proteins. Journal of Virology 74, 5066-5074.
Trombetta, E. S. & Helenius, A. (1998). Lectins as chaperones in glycoprotein folding. Current Opinion in Structural Biology 8, 587-592.[Medline]
Yi, M., Nakamoto, Y., Kaneko, S., Yamashita, T. & Murakami, S. (1997). Delineation of regions important for heteromeric association of hepatitis C virus E1 and E2. Virology 231, 119-129.[Medline]