CNRS-UMR 85261 and CNRS-UMR 85252, Institut de Biologie de Lille/Institut Pasteur de Lille, BP447, 59021 Lille cedex, France
Author for correspondence: Jean Dubuisson.Fax +33 3 20 87 11 11. e-mail jean.dubuisson{at}ibl.fr
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Abstract |
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Introduction |
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There are several possible fates for newly synthesized proteins inside cells. The major distinction between these fates is whether a protein succeeds in folding correctly, or whether it aggregates. Aggregation has been commonly regarded as a nuisance which affects in vitro protein refolding studies. It is now apparent that aggregation is also a problem within cells (Dobson & Ellis, 1998 ; Thomas et al., 1995
). In the intracellular environment, the competition between folding, aggregation and degradation determines whether a polypeptide can achieve its functional state with the efficiency required for successful cell growth, or whether it aggregates into a state that causes cellular damage. Evidence is accumulating that many disease-causing mutations and modifications exert their effects by altering protein folding. Cystic fibrosis is an example of a genetic disease where a variant protein is unable to fold correctly to a stable state in the ER and fails to reach the plasma membrane, eventually being degraded (Cheng et al., 1990
; Denning et al., 1992
; Thomas et al., 1992
). Interestingly, even the wild-type chains do not fold with high efficiency; only about 30% of wild-type chains survive the quality-control mechanisms of the ER. Aggregation is currently seen as a specific process, which may be amplified by the high concentration of identical nascent chains emerging from polysomes (Dobson & Ellis, 1998
).
Studies of reactivity with conformation-sensitive MAbs and analysis of protease sensitivity indicate that the production of properly assembled E1E2 oligomers is inefficient (Deleersnyder et al., 1997 ). This does not seem to be due to mutations introduced during cDNA synthesis or PCR amplification of the original clone used in these studies. Indeed, similar results are obtained with a vaccinia virus recombinant expressing the sequence of the structural proteins of a recently characterized infectious cDNA clone (Kolykhalov et al., 1997
; J. Dubuisson & C. M. Rice, unpublished data). Aggregates of HCV glycoproteins were first observed when expressed by viral vectors such as vaccinia virus and Sindbis virus which can induce a high level of protein synthesis (Dubuisson et al., 1994
; Grakoui et al., 1993
). However, such aggregates have also been reported when HCV proteins are expressed in a non-viral vector which drives a lower level of protein synthesis (Duvet et al., 1998
). This tendency to aggregate is therefore probably an intrinsic property of HCV glycoproteins. Indeed, slow folding of HCV glycoproteins may increase the fraction of these proteins shunted into a competing non-productive pathway, such as aggregation (Fischer & Schmid, 1990
). In this study, we produced a MAb which specifically reacts with HCV glycoprotein aggregates but not with non-covalently associated glycoproteins. Characterization of these aggregates indicates that they share a common epitope with a cellular protein.
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Methods |
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Viruses.
vTF7-3 (a vaccinia virus recombinant expressing the T7 DNA-dependent RNA polymerase) (Fuerst et al., 1986 ), vaccinia virusHCV recombinants vHCV1-1488 (expressing CE1E2p7NS2NS31488), vHCV170-809 (E1E2p7), vHCV371-809 (E2p7) and vHCV1-383 (CE1) (Fournillier-Jacob et al., 1996
; Grakoui et al., 1993
; Michalak et al., 1997
), and vaccinia virus recombinants expressing truncated forms of HCV glycoproteins [vHCV170-311 (E1311), vHCV170-361 (E1361), vHCV371-715 (E2715) and vHCV371-661 (E2661)] (Michalak et al., 1997
) were used in this work. The Sindbis virus recombinant expressing a truncated form of HCV polyprotein (amino acid residues 11207: SINrep/HCV-H1-1207) has been described previously (Dubuisson et al., 1994
).
Antibodies.
Anti-HCV E1 (A4) and E2 (H2 and H47) MAbs have been described previously (Deleersnyder et al., 1997 ; Dubuisson et al., 1994
; A. Pillez & J. Dubuisson, unpublished data) and were produced in vitro by using a MiniPerm apparatus (Heraeus) as recommended by the manufacturer. To produce other MAbs, HepG2 cells co-infected with vTF7-3 and vHCV1-1488 were lysed with 0·5% NP-40 in TBS (20 mM TrisHCl, pH 7·4; 137 mM NaCl). HCV glycoproteins were purified by immunoaffinity on Protein ASepharose (Amersham Pharmacia Biotech) with anti-E1 MAb A4. HCV glycoprotein complexes bound to the immunobeads were injected into BALB/c mice to produce HCV-specific, antibody-secreting hybridomas as described (Harlow & Lane, 1988
). Screening was performed in 96-well plates containing HepG2 cells that had been co-infected with vTF7-3 and vHCV1-1488, and fixed with isopropanol as described (Dubuisson et al., 1994
). Anti-Mac-2 binding protein (M2BP) antibody was kindly provided by R. Timpl (Max Planck Institute für Biochemie, Martinsried, Germany).
Metabolic labelling and immunoprecipitation.
Subconfluent monolayers in 35 mm dishes were infected with the appropriate recombinant at an m.o.i. of 5 p.f.u. per cell. After 1 h, medium containing 5% FBS was added. Between 4 and 4·5 h post-infection, monolayers were washed once with pre-warmed medium lacking methionine and cysteine, and incubated in the same medium for an additional half hour. Infected cells were then pulse-labelled for 5 min with 100 µCi/ml 35S-Protein Labelling Mix (NEN Life Science Products). Cells were washed twice with pre-warmed medium containing 10-fold excess methionine and cysteine, followed by a chase for various times. Cells were then lysed with 0·5 % TBS. Iodoacetamide (20 mM) was included in the lysis buffer for experiments in which disulfide bond formation was determined. Cell lysates were clarified by centrifugation in an Eppendorf centrifuge for 5 min at 4 °C. In steady-state labelling, cells were labelled at 4 h post-infection with 50 µCi/ml 35S-Protein Labelling Mix in medium lacking methionine and cysteine. Immunoprecipitations were carried out as described previously (Dubuisson & Rice, 1996 ). The precipitates were boiled for 5 min in SDSPAGE sample buffer (under non-reducing conditions;
-mercaptoethanol was omitted) and run on a 10% polyacrylamide gel (Laemmli, 1970
). After electrophoresis, gels were treated with sodium salicylate (Chamberlain, 1979
), dried and exposed at -70 °C to pre-flashed Hyperfilm-MP (Amersham Pharmacia Biotech). 14C-Methylated protein molecular mass markers were purchased from Amersham Pharmacia Biotech.
Western blotting.
For Western blotting studies, proteins were separated by SDSPAGE, transferred to nitrocellulose membranes (Hybond-ECL; Amersham Pharmacia Biotech) by using Trans-Blot apparatus (Bio-Rad). After transfer, nitrocellulose membranes were incubated with specific antibodies (MAb or polyclonal antibody) followed by goat anti-mouse or swine anti-rabbit immunoglobulin conjugated to horseradish peroxidase or alkaline phosphatase (DAKO) as described previously (Harlow & Lane, 1988 ). The presence of proteins specifically recognized by the primary antibody was revealed by chemiluminescence (ECL; Amersham Pharmacia Biotech) as recommended by the manufacturer or with BCIP/NBT alkaline phosphatase substrate (Sigma).
Immunofluorescence and alkaline phosphatase detection.
Cells grown on cover-slips were infected with the appropriate recombinant virus expressing HCV glycoproteins. At 8 h post-infection, cells were fixed for 10 min at 4 °C with isopropanol. For immunofluorescence detection, cells were stained for 2 h at room temperature with MAb H14 (diluted 1/200), followed by incubation for 1 h at room temperature with donkey anti-mouse (Rhodamine-linked) immunoglobulins (diluted 1/100; Jackson Immunoresearch). For alkaline phosphatase detection, cells were stained for 2 h at room temperature with MAb H14 (diluted 1/200) or A4 (diluted 1/400), followed by incubation for 1 h at room temperature with rabbit anti-mouse (alkaline phosphatase-linked) immunoglobulins (diluted 1/200; DAKO) and revealed with BCIP/NBT alkaline phosphatase substrate.
Amino acid sequencing.
Internal amino acid sequences were determined as described previously (Rosenfeld et al., 1992 ). Briefly, the cellular protein recognized by MAb H14 was purified by immunoprecipitation as described (Harlow & Lane, 1988
) and separated by SDSPAGE. After Coomassie blue staining, the band of interest was cut and treated with acetonitrile and ammonium carbonate to clear the sample of impurity. After a Speedvac dehydration, the protein was digested with endoproteinase Lys-C (Boehringer Mannheim) at 37 °C for 18 h. Peptides of interest were purified by reverse-phase HPLC, eluted with acetonitrile in 0·1% trifluoroacetic acid, lyophilized and sequenced in an automated microsequencer.
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Results |
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Characterization of HCV glycoprotein aggregates
HCV glycoproteins expressed in different cell lines (chick cells, insect cells, BHK-21, CV-1 and HepG2) were precipitated by MAb H14 suggesting that aggregates are formed in these different cell lines (data not shown). In addition, MAb H14 precipitated HCV glycoproteins expressed by viral (vaccinia and Sindbis viruses) and non-viral vectors (data not shown), indicating that the formation of HCV glycoprotein aggregates is not influenced by the expression system. Similar ratios of aggregates were observed even when the expression level was 1020 times lower.
Since MAb H14 recognizes an epitope on both E1 and E2 (Fig. 5), we wanted to know whether the H14 epitope would be accessible on these proteins expressed alone, in the absence of any denaturation. For this purpose, HCV glycoproteins were analysed by immunoprecipitation under non-denaturing conditions. As shown in Fig. 6
, MAb H14 precipitated E1 or E2 when expressed alone, suggesting that the H14 epitope is not buried in these glycoproteins. It is worth noting that these proteins were no longer recognized by MAb H14 when deleted at their C-terminus, suggesting that the H14 epitope might be present in close proximity to their transmembrane domain. However, the same truncated forms of E2 were recognized by MAb H14 in Western blotting experiments (data not shown), indicating that the H14 epitope is present on these truncated proteins but is not accessible unless the proteins are denatured. Since truncated forms of E2 also have a tendency to aggregate (Michalak et al., 1997
), these data suggest that the H14 epitope is masked in these aggregates.
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Discussion |
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Aggregates of HCV glycoproteins share an epitope with M2BP. Viruses are major candidates for the induction of autoimmune diseases. Autoimmunity, in the form of autoantibodies, is common after many virus infections and may well result from the mimicking of host proteins by viral antigens. HCV induces a number of diseases of presumed autoimmune background, like mixed cryoglobulinaemia, glomerulonephritis, panarthritis, arthritis, thyroiditis and skin lesions (Houghton, 1996 ). On the other hand, a number of autoantibodies are observed during the course of HCV infection (Manns & Obermayer-Straub, 1997
). Of particular interest are liver/kidney microsomal (LKM) antibodies. LKM antibodies in chronic hepatitis C recognize several autoepitopes that differ from those of autoimmune hepatitis. Hepatitis C-associated LKM antibodies are more heterogeneous. They recognize either conformational or several distinct linear epitopes on cytochrome P4502D6 (Manns & Obermayer-Straub, 1997
). They may also react with other microsomal proteins. Apart from their molecular masses of 59 and 70 kDa, these microsomal antigens have not yet been identified (Durrazzo et al., 1995
). The identification of a common epitope between HCV glycoprotein aggregates and M2BP suggests that M2BP might be a target for autoantibodies. This suggestion is reinforced by the fact that the microsomal form of M2BP is a protein of about 70 kDa and by the observation that among 14 HCV-positive sera tested, one reacted against M2BP (J. Dubuisson, unpublished data). Future studies should help us to evaluate the prevalence of such autoantibodies in HCV-infected patients.
HCV glycoprotein aggregates recognized by MAb H14 are expressed in a small proportion of the infected cells. Indeed, a large number of cells expressing HCV glycoproteins did not show a signal above background when analysed by immunofluorescence with MAb H14. These data suggest that, in some physiological conditions, aggregates of HCV glycoproteins are less likely to be formed. We can expect that an appropriate concentration of the chaperones and/or foldases involved in HCV glycoprotein folding could reduce the formation of aggregates. Recently, we have identified ER chaperones potentially involved in the folding and assembly of HCV glycoproteins (Choukhi et al., 1998 ). However, overexpression of these chaperones did not improve the folding of these glycoproteins. Since several chaperones can be involved in assisted folding of proteins in the ER, it is likely that a proper balance of chaperone activities is required for optimal folding. It is also likely that other chaperone(s) and/or foldase(s), which have not been identified yet, are necessary to assist in HCV glycoprotein folding. The use of MAb H14 should help us to determine the conditions in which cells produce these aggregates at a lower level.
Why do some proteins aggregate in vivo? Since protein folding in the cell is so complex, there is a significant likelihood of defects arising in the process. Potentially, thermodynamic destabilization of the native or an intermediate state, alteration of the folding kinetics, prolonged or inappropriate associations with chaperones or foldases, preferential formation of off-pathway or toxic conformations could all lead to loss of functional protein. Inability of an essential protein to form its native structure under physiological conditions may be the basis of a variety of human diseases (for review, see Thomas et al., 1995 ). In the case of HCV glycoproteins, it is hard to prove that in the course of an HCV infection, aggregates such as those characterized in this work can be formed. This is due to the absence of a tissue culture system which allows efficient replication of HCV. However, the use of different expression systems (viral or non-viral) to study the assembly of HCV glycoproteins allows us to conclude that aggregation of HCV glycoproteins is not an artifact linked to the expression system. In the context of HCV infection, inefficient folding of the HCV glycoproteins might downregulate particle formation and virus replication to minimize exposure of viral antigens to the immune system and/or reduce pathogenicity. Alternatively, the production of HCV glycoprotein aggregates could provide these glycoproteins with additional functions in infected cells or on the particle. The non-covalent HCV glycoprotein complex previously characterized (Deleersnyder et al., 1997
) is most likely the pre-budding form of the functional complex which will play an active role in the entry process in infected cells. However, it cannot be excluded that a portion of HCV glycoprotein aggregates makes up the envelope of the mature particle, providing the viral particle with additional functions. More likely, in infected cells, aggregates of HCV glycoproteins could interact with host proteins in the ER compartment whose transport or function could be altered as a consequence of these interactions. Further work will be necessary to evaluate the potential role of HCV glycoprotein aggregates in the physiopathology of HCV infection.
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Acknowledgments |
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References |
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Received 7 May 1999;
accepted 17 August 1999.