1 CNRS-UPR2511, Unité Hépatite C, Institut de Biologie de Lille Institut Pasteur de Lille, 1 rue Calmette, BP 447, 59021 Lille cedex, France
2 Laboratoire de Vectorologie Rétrovirale et Thérapie Génique, INSERM U412, IFR74, Ecole Normale Supérieure de Lyon, Lyon, France
Correspondence
Jean Dubuisson
jean.dubuisson{at}ibl.fr
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ABSTRACT |
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These authors contributed equally to this work.
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MAIN TEXT |
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The TM domains of HCV glycoproteins have been shown to play a major role in the biogenesis of the E1E2 heterodimer (reviewed by Op De Beeck et al., 2001). These domains are less than 30 amino acid residues long and are composed of two hydrophobic stretches separated by a short segment containing one or two fully conserved charged residues (Cocquerel et al., 2002
). The charged residues present in the middle of the TM domains of HCV glycoproteins have been shown to play a major role in ER retention and heterodimerization (Cocquerel et al., 2000
). Here, we used site-directed mutagenesis to investigate the specific role of each of the charged residues (Asp and Arg) present in the TM domain of E2.
Alanine scanning insertion mutagenesis has demonstrated that the TM domains of HCV glycoproteins play a direct role in E1E2 heterodimerization (Op De Beeck et al., 2000). In addition, replacement of the charged residues present in the TM domain of E2 (Asp and Arg) by Ala residues has also been shown to alter heterodimerization (Cocquerel et al., 2000
). To better understand the individual role of Asp and Arg in this function, individual mutants were produced, and we analysed the effect of these mutations on E1E2 heterodimerization. Site-directed mutagenesis was performed by enzymic inverse PCR (Stemmer & Morris, 1992
). Several mutants were produced and expressed with the phCMV plasmid (Negre et al., 2000
) containing the sequence of E1E2 polyprotein (Bartosch et al., 2003
). The effect of the mutations was analysed in our heterodimerization assay as previously described (Op De Beeck et al., 2000
) (Fig. 1
). The ability of the mutants to form a noncovalent E1E2 complex was analysed by immunoprecipitation with a conformation-sensitive E2-specific monoclonal antibody (mAb) (H53) that has been shown to specifically precipitate native E1E2 complexes (Cocquerel et al., 1998
; Duvet et al., 1998
). Cells expressing mutated HCV proteins were pulse-labelled with [35S]methionine/[35S]cysteine (Promix, Amersham) for 30 min and chased for 4 h. These conditions are appropriate to detect the peak of heterodimer formation (data not shown). As shown by Western blotting analysis, E1 expression was constant, whatever the E2 mutant tested (data not shown). Since mAb H53 is E2-specific, and because the E2 domain containing the H53 epitope can fold independently of E1 (Michalak et al., 1997
), the amount of E1 coprecipitated by mAb H53 is a good indicator of the assembly of the noncovalent heterodimer. To evaluate the percentage of heterodimerization, E1/E2 ratios were measured for each mutant and compared with the ratio obtained with wild-type proteins.
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Interestingly, despite their rare presence in TM helices, strongly polar residues are highly conserved, suggesting molecular interactions that either functionally or structurally favour these residues (Arkin & Brunger, 1998; Jones et al., 1994
). Interhelical polar interactions have been observed in some integral membrane protein structures available at high resolution (Kuhlbrandt et al., 1994
). The TM domain of E1 contains a positive charge (Lys) and one of the two charged residues in E2 is negative (Asp). In addition, our site-directed mutagenesis data indicate that the Asp residue in the TM domain of E2 plays a major role in E1E2 heterodimerization, whereas the Arg residue makes only a marginal contribution to this function. This would suggest that an ion pair might be formed between the Lys residue of E1 and the Asp residue of E2. However, replacement of the acidic residue of E2 (Asp) by a basic residue (Lys) did not alter E1E2 heterodimerization (data not shown).
These data are therefore not in favour of E1E2 heterodimerization being mediated by an ion pair. However, this does not exclude side-chain/side-chain interhelical hydrogen bonds between the Lys residue in E1 and the Arg residue in E2, as observed for some other residues in artificial -helices (Gratkowski et al., 2001
; Zhou et al., 2001
). Alternatively, by potentially changing the localization of E2 from an ER microdomain, mutation of the Asp residue might reduce the chances of E1 encountering E2 in the same microdomain for heterodimerization. If this is the case, the positively charged residues present in the TM domains of HCV glycoproteins would play only an indirect role in E1E2 heterodimerization, and other residues would be responsible for heterodimerization. Further investigations will be needed to identify the potential existence of such residues.
In addition to their effect on heterodimerization, replacement of the charged residues present in the TM domain of E2 by Ala residues has also been shown to alter their ER retention function (Cocquerel et al., 2000). To better understand the individual role of Asp and Arg in ER retention, the subcellular location of the mutants was analysed. To determine whether the mutated proteins reach the cell surface, non-permeabilized cells expressing these proteins were analysed by immunofluorescence. Control Triton X-100-permeabilized cells were analysed in parallel. As shown previously (Cocquerel et al., 2000
), the cell-surface expression of E2 was very low for the wild-type protein under our conditions of expression (Fig. 2
). Recently, it has been shown that when overexpressed in 293T cells, a fraction of HCV glycoproteins accumulates at the plasma membrane (Bartosch et al., 2003
; Drummer et al., 2003
; Hsu et al., 2003
). However, the proportion of E1E2 that leaves the ER in these conditions is low and is observed after longer periods of expression (Op De Beeck et al., 2004
). As shown in Fig. 2
, the double mutant (E2-LAL) was highly expressed at the cell surface. Interestingly, the E2-LAR and E2-DAL proteins had a behaviour similar to that of the E2-LAL double mutant in terms of cell-surface expression, indicating that both charged residues contribute equally to ER retention.
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Recently, HCV pseudotyped particles (HCVpp) that are assembled by displaying HCV glycoproteins on retroviral core particles have successfully been generated (Bartosch et al., 2003; Drummer & Poumbourios, 2004
; Hsu et al., 2003
). The data that have been accumulated on HCVpp strongly suggest that they mimic the early steps of HCV infection. To further characterize the potential role of the charged residues present in the TM domain of E2, we analysed whether the mutants would be incorporated into HCVpp and whether such particles would be infectious.
The level of E2 associated with HCVpp was slightly reduced for E2-LAL, E2-LAR and E2-DAL (Fig. 3), suggesting that these mutations slightly reduced the incorporation of E2 into HCVpp. Interestingly, when both charged residues were replaced by Leu, E2 associated with HCVpp had a slower migration profile (Fig. 3
), suggesting that when both charged residues are mutated, the processing of E2 glycans in the Golgi apparatus is different. This might be due to differences in the kinetics of E2 trafficking in the secretory pathway. Similarly, when only the Asp residue was replaced by Leu, the migration pattern of E2 was more diffuse. No E1 glycoprotein was incorporated into HCVpp when both charged residues were replaced by Leu, whereas E1 incorporation into HCVpp was similar to wild-type for the E2-DAL mutant and strongly reduced for the E2-LAR mutant (Fig. 3
). In addition, the Arg-to-Lys mutation did not affect E1 incorporation into HCVpp, and the Asp-to-Glu mutation slightly reduced E1 incorporation into HCVpp. These data indicate that mutations that affect E1E2 heterodimerization reduce the incorporation of E1 into HCVpp.
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There are many reports on the role of TM sequences in the entry function of viral fusion proteins. For instance, replacement of the TM domain of gp41 by a GPI anchor has been shown to result in a loss of syncytium formation (Salzwedel et al., 1993). Similarly, it has been demonstrated that replacement of the TM domain of the haemagglutinin of influenza virus by a GPI anchor leads to hemifusion but not to the formation of a stable fusion pore (Kemble et al., 1994
). In addition, site-directed mutagenesis in the TM domain of the G glycoprotein of vesicular stomatitis virus has revealed that Gly residues in the TM domain of a viral envelope protein may play a role in fusion activity (Cleverley & Lenard, 1998
). Interestingly, mutations of Gly residues present in the TM domain of E1 of Semliki Forest virus decreased the stability of the E1E2 heterodimer and reduced the fusion activity of the virus (Sjoberg & Garoff, 2003
). All these observations suggest that the TM domains of viral envelope glycoproteins might play a role in coordinating protein reorganization so that the fusion process can occur. We might therefore expect that mutation of the charged residues in HCV envelope glycoprotein E2 would alter such a coordinated reorganization.
HCV glycoprotein E2 contains two fully conserved charged residues in the middle of its TM domain, Asp and Arg. These residues play a major role in the biogenesis and/or entry functions of this protein. However, the Asp and Arg residues do not contribute equally to these functions. Altogether, these data suggest that the TM domain of E2 plays a major role in the coordinated assembly and reorganization of the E1E2 heterodimer and that charged residues are key elements in this process.
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ACKNOWLEDGEMENTS |
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Received 27 April 2005;
accepted 5 July 2005.