Analysis of the subcellular localization of hepatitis C virus E2 glycoprotein in live cells using EGFP fusion proteins

François Kien, Jean-Daniel Abraham, Catherine Schuster and Marie Paule Kieny

INSERM U544, Institut de Virologie, 3 rue Koeberlé, 67000 Strasbourg, France

Correspondence
Catherine Schuster
Catherine.schuster{at}viro-ulp.u-strasbg.fr


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Hepatitis C virus (HCV) E1 and E2 glycoproteins assemble intracellularly to form a non-covalently linked heterodimer, which is retained in the endoplasmic reticulum (ER). To study the subcellular localization of E2 in live cells, the enhanced green fluorescent protein (EGFP) was fused to the N terminus of E2. Using fluorescence and confocal microscopy, we have confirmed that E2 is located in the ER, where budding of HCV virions is thought to occur. Immunoprecipitation experiments using a conformation-sensitive antibody and a GST pull-down assay showed that fusion of EGFP to E2 interferes neither with its heterodimeric assembly with E1, nor with proper folding of the ectodomain, nor with the capacity of E2 to interact with human CD81, indicating that the EGFP–E2 fusion protein is functional. As a tool to study binding of E2 to target cells, we also described the expression of an EGFP–E2 fusion protein at the cell surface.


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Hepatitis C virus (HCV) is the main aetiological agent of non-A, non-B hepatitis. It is a positive-strand RNA virus belonging to the Flaviviridae family (Francki et al., 1991). Its genome encodes a polyprotein of 3010 amino acids, which is cleaved co- and post-translationally by cellular and viral proteases to generate mature viral proteins (Reed & Rice, 2000). The two glycoproteins, E1 and E2, are type I integral transmembrane proteins (Dubuisson, 2000; Op De Beeck et al., 2001). E2 has been reported to interact with E1 to form a stable non-covalently linked heterodimer and heterogeneous disulfide-linked aggregates (Choukhi et al., 1998; Deleersnyder et al., 1997; Dubuisson et al., 1994), which are believed to result from a non-productive folding pathway. Immunolocalization studies and glycan analysis have shown that HCV glycoproteins are located in the endoplasmic reticulum (ER) (Deleersnyder et al., 1997; Dubuisson et al., 1994; Duvet et al., 1998). Deletion of the transmembrane (TM) domains of E1 and E2 and expression of chimeric proteins in which the TM domains were exchanged for corresponding domains of proteins transported to the plasma membrane have shown that the TM domains play a major role in the localization of the glycoprotein complex (Cocquerel et al., 1998, 1999; Flint & McKeating, 1999; Flint et al., 1999). In addition, these multifunctional TM domains are reponsible for retention of the proteins in the ER without recycling through the Golgi apparatus (Cocquerel et al., 1999; Duvet et al., 1998) and are involved in the assembly of non-covalent E1–E2 heterodimers (Cocquerel et al., 1998, 2000; Michalak et al., 1997; Op De Beeck et al., 2000; Patel et al., 2001).

The specific receptor allowing penetration of HCV into target cells has not been unambiguously identified to date. Low-density lipoprotein receptor (Agnello et al., 1999), human CD81 (hCD81) tetraspanin (Pileri et al., 1998) and glycosaminoglycans (Chen et al., 1997) may all act as receptors for HCV, either sequentially or for different viral quasispecies. However, several reports suggest that additional, as yet unidentified cellular proteins are involved in virus binding and entry (Meola et al., 2000; Petracca et al., 2000). Recently, Scarselli et al. (2002) have proposed the human scavenger receptor class B type I as a novel candidate receptor for HCV.

Previous studies of the subcellular localization of HCV glycoproteins have used indirect immunofluorescence and immunoelectron microscopic techniques (Deleersnyder et al., 1997; Dubuisson et al., 1994). Utilization of the Aequorea victoria green fluorescent protein (GFP) for visualizing gene expression and protein localization has provided a powerful tool to investigate subcellular localization of various recombinant proteins in live cells (Chalfie et al., 1994). To examine the subcellular localization of HCV E2 glycoprotein in live mammalian cells, we have constructed a recombinant vaccinia virus (VV) expressing the enhanced green fluorescent protein (EGFP) fused to E2. Since the TM domain of E2 is known to be multifunctional, EGFP was fused to the N terminus rather than to the C terminus of E2 (Fig. 1a). The corresponding coding sequences were assembled using overlap extension PCR amplification on infectious p90/HCV-FL-long pU HCV clone cDNA (Kolykhalov et al., 1997) and pEGFP-C1 plasmid vector (Clontech). The resulting sequence was cloned within the thymidine kinase gene of plasmid pTG9148 (Transgene). The recombinant VV expressing the EGFP–E2 fusion protein (vvIV215) or the core, E1 and E2 proteins (vvIV205) were generated by homologous recombination (Kieny et al., 1984). A VV recombinant (vvIV218) expressing the EGFP protein was used as a control. Expression and size of the recombinant proteins in human hepatic cell lines were confirmed using Western blot analysis with a GFP-specific monoclonal antibody (mAb) (Clontech) and a conformation-insensitive E2-specific mAb (H47) (not shown).



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Fig. 1. (a) Schematic representation drawn to scale of the E2, EGFP and chimeric proteins EGFP–E2 and EGFP–E2–TMrabies used in this study. EGFP–E2, signal sequence of E2 fused to EGFP and E2; EGFP–E2–TMrabies, signal sequence of E2 fused to EGFP, to the ectodomain of E2 lacking its C-terminal 56 amino acids and to the TM and cytoplasmic domain of rabies G protein. The signal sequence of E2 is from aa 371 to 383 (positions on the polyprotein), the ectodomain of E2 is from aa 384 to 717 and the TM domain of E2 from aa 718 to 746. The TM and cytoplasmic domain of the rabies G protein is from aa 458 to 524. (b) Sensitivity of the chimeric proteins to glycosidase digestion. HepG2 cells infected with the appropriate recombinant viruses were pulse-labelled for 15 min and chased for 3 h. Cell lysates were used for immunoprecipitation with mAb H53 (conformation-sensitive anti-E2 mAb). Immunoprecipitates were left untreated (C), or treated with endo H (H) or PNGase F (F) before analysis by 10 % SDS-PAGE and autoradiography. HCV-specific products are indicated on the right of each panel. Deglycosylated proteins are indicated by asterisks. Sizes (kDa) of molecular mass markers are indicated on the left.

 
In order to examine the proper folding and conformation of the protein, EGFP–E2 was expressed and labelled with 100 µCi 35S-Protein Labelling Mix (NEN) ml-1 in HepG2 cells, immunoprecipitated with a conformation-sensitive E2-specific mAb (H53) and analysed by SDS-PAGE (Dubuisson & Rice, 1996). The EGFP–E2 protein, like native E2 (Fig. 1b), was recognized by mAb H53, indicating that the conformation of the EGFP–E2 fusion protein was similar to that of native E2.

As an indicator of intracellular trafficking of the protein, we examined the sensitivity of EGFP–E2 to endoglycosidase treatment after pulse–chase labelling with 100 µCi 35S-Protein Labelling Mix ml-1 and immunoprecipitation with mAb H53. Immunoprecipitates were digested with either endo-{beta}-N-acetylglucosaminidase H (endo H; Roche Boehringer Mannheim) or peptide N-glycosidase F (PNGase F; New England Biolabs), or left untreated. Both native E2 and EGFP–E2 were shown to be sensitive to endo H and PNGase F endoglycosidases, indicative of their retention in the ER (Fig. 1b). This result indicates that fusion of EGFP to the N terminus of the E2 protein does not modify the localization of this glycoprotein.

Having demonstrated that addition of EGFP to its N terminus interferes neither with the correct folding nor with the sensitivity of E2 to endoglycosidase treatment, we investigated whether the fusion could affect known functional properties of E2. Indeed, E2 has been reported to interact with E1 to form a stable, non-covalently linked heterodimer and E2 has been shown to interact with hCD81.

Association of the EGFP–E2 fusion protein with E1 was investigated by co-immunoprecipitation of E2 and E1 using mAb H53. The proteins were expressed in trans (Cocquerel et al., 2001) using vvIV215 and AdIV243 (a recombinant adenovirus expressing HCV E1 protein) in HepG2 cells, labelled and immunoprecipitated as described above. vvIV205 was used as a control. As shown in Fig. 2(a), E1 was co-immunoprecipitated with EGFP–E2, demonstrating that this protein interacts with E1. Similar results were obtained using a GFP-specific polyclonal antibody (data not shown). This result demonstrated that addition of EGFP to the N terminus of the E2 protein does not alter its association with E1.



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Fig. 2. (a) Heterodimerization of E1 and EGFP–E2 expressed in trans. HepG2 cells co-infected with vvIV215 and AdIV243, or infected with vvIV205, were pulse-labelled for 15 min and chased for 3 h. Cell lysates were used for immunoprecipitation with mAb H53. Immunoprecipitates were analysed by 10 % SDS-PAGE. HCV-specific products are indicated on the right. The size (kDa) of molecular mass markers are indicated on the left. (b) Interaction with hCD81. Lysates of BHK-21 cells infected with vvIV205 (Core–E1–E2), vvIV215 (EGFP–E2) or vvIV279 (EGFP–E2–TMrabies) were first incubated with purified recombinant GST–CD81 or GST for 2 h at 4 °C, then incubated with 50 µl glutathione–Sepharose 4B beads (50 % slurry) at 4 °C for 2 h. The beads were washed four times in 500 µl EBC buffer (140 mM NaCl, 0·5 % NP-40, 50 mM Tris/HCl, pH 8·0), pelleted at 500 g for 30 s and boiled in SDS-PAGE sample buffer. Proteins bound to the GST fusion proteins were resolved by SDS-PAGE and detected by Western blot using mAb H47. Sizes (kDa) of molecular mass markers are indicated on the left.

 
We then investigated whether fusion of E2 with EGFP would affect its binding to hCD81. Indeed, among the possible disadvantages of GFP as a protein tag is its large size (29 kDa), which could mask regions of E2 involved in interactions with hCD81. Lysates of BHK-21 cells infected with vvIV205 or vvIV215 were incubated with a soluble recombinant glutathione S-transferase (GST) fusion protein containing the large extracellular loop of hCD81 (GST–CD81) (Higginbottom et al., 2000) or with GST protein for 2 h at 4 °C. CD81–E2 and CD81–EGFP–E2 complexes were recovered by incubation (pull-down) with glutathione–Sepharose 4B beads for 2 h at 4 °C. Interaction with CD81 was then analysed by Western blot detection of native E2 and EGFP–E2 using mAb H47. As shown in Fig. 2(b), both E2 and EGFP–E2 were detected when incubated with GST–CD81. No binding of E2 or EGFP–E2 to GST was observed. This result showed that the EGFP–E2 fusion protein conserves its capacity to bind hCD81.

Expression of EGFP–E2 in HepG2 cells was detected in live cells using conventional fluorescence microscopy (not shown). In addition, infected cells were examined by laser scanning confocal microscopy (LSCM) at 24 h post-infection (p.i.). As shown in Fig. 3(g), EGFP staining was localized uniformly throughout the cytoplasm and nucleus (Ogawa et al., 1995). In contrast, the EGFP–E2 fusion protein-specific staining was localized at restricted areas in the cell (Fig. 3c). Moreover, EGFP–E2 fluorescence showed a diffuse granular pattern indicative of a vesicular localization and was concentrated mainly in the perinuclear space, reminiscent of the subcellular localization of native E2 (Deleersnyder et al., 1997; Dubuisson et al., 1994; Duvet et al., 1998). Immunoelectron microscopy using mAb H53 confirmed this ER localization (data not shown). This observation of the retention and accumulation of EGFP–E2 in the ER of live cells supports the hypothesis that budding of HCV particles, like that of flaviviruses (Mackenzie & Westaway, 2001), occurs into this compartment.



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Fig. 3. Expression and subcellular localization of EGFP-tagged HCV proteins. HepG2 cells were infected with vvIV215 (EGFP–E2), vvIV279 (EGFP–E2–TMrabies), or vvIV218 (EGFP). EGFP or its fusion derivatives were observed at 8, 18 and 24 h p.i.

 
In order to study the trafficking of the fluorescent protein in live cells, HepG2 cells were infected with vvIV215 and examined at various times after infection. Staining was first observed in the majority of cells at 4 h p.i. (not shown). At 8 h p.i. (Fig. 3a), EGFP–E2 displayed a vesicular pattern compatible with an ER-like distribution. At 18 and 24 h p.i. (Fig. 3b, c), EGFP–E2 accumulated in the perinuclear space and in the ER as expected. Fusion of EGFP thus provides a powerful tool for the analysis of the temporal and spatial trafficking of the HCV E2 envelope protein in live mammalian cells.

In the absence of an efficient tissue culture system to replicate HCV, studying HCV interactions with host cell-surface proteins can prove difficult. With the intention of developing a reagent to study HCV E2 interactions with host cell-surface components, we constructed a recombinant VV expressing an EGFP–E2 fusion protein transported to the cell surface. This fusion protein consisted of EGFP fused to the N terminus of a modified E2 protein resulting from the fusion of the ectodomain of E2 (truncated E2 protein ending at aa 661) to the TM and cytoplasmic domains of the rabies virus G glycoprotein, which is naturally exported to the cell surface (Fig. 1a) (Dietzschold et al., 1978). Recombinant vvIV279 expressing the EGFP–E2–TMrabies protein was generated and expression of the fusion protein was demonstrated by Western blot using GFP-specific and E2-specific (H47) mAbs (data not shown). The EGFP–E2–TMrabies fusion protein was further analysed with mAb H53 in pulse–chase experiments as described above. EGFP–E2–TMrabies was shown to be recognized by mAb H53 (Fig. 1b) and two bands were detected after immunoprecipitation: a fast-migrating form of the expected size, which was sensitive to both endo H and PNGase F, and a slow-migrating form, which corresponds to the EGFP–E2–TMrabies protein harbouring additional glycan modifications, acquired during translocation of the recombinant protein to the plasma membrane. This slow-migrating species was indeed found to be resistant to endo H and sensitive to PNGase F, suggesting that EGFP–E2–TMrabies reaches at least the medial- or trans-Golgi apparatus. Interaction with hCD81 was further analysed using a GST pull-down assay, as described above. Detection of the fast-migrating form of EGFP–E2–TMrabies by Western blot following incubation with GST–CD81 (Fig. 2b) demonstrated that this protein is expressed in a functional configuration and can interact with hCD81. It is of note that no interaction between the slow-migrating form of EGFP–E2–TMrabies and hCD81 was detected using this technique. This result is consistent with previous reports demonstrating a modulation of the E2–hCD81 interaction following translocation of this HCV glycoprotein to the plasma membrane (Flint et al., 2000; Heile et al., 2001). Indeed, E2 isoforms harbouring complex glycans have been shown to bind hCD81 with poorer affinity.

Cell-surface expression of EGFP–E2–TMrabies was analysed by fluorescence and LSCM. At 8 h p.i. (Fig. 3d), the recombinant protein displayed a vesicular pattern of fluorescence compatible with an ER-like distribution. At 18 and 24 h p.i. (Fig. 3e, f), the EGFP–E2–TMrabies protein displayed some residual granular staining indicative of a vesicular localization, but was mainly localized on the plasma membrane. Immunoelectron microscopy using the conformation-sensitive E2-specific mAb H53 confirmed cell-surface expression (data not shown). These results suggested that EGFP–E2–TMrabies recombinant protein first concentrates in the ER, where it exhibits distinctive patterns of localization ranging from a diffuse vesicular pattern to accumulation in the perinuclear space. Cell-surface localization follows further maturation. This observation also demonstrates that replacement of the TM domain of HCV E2 by the anchoring domain of rabies G glycoprotein leads to the expression of EGFP–E2 at the cell surface, indirectly confirming that the TM domain of E2 plays a major role in the subcellular localization and ER retention of this viral glycoprotein in live cells (Cocquerel et al., 1998; Flint et al., 1999; Forns et al., 2000; Patel et al., 2001; Takikawa et al., 2000).

We believe that the biologically functional EGFP–E2 fusion proteins described in this report constitute powerful new tools to study directly the subcellular localization of HCV E2 glycoprotein in live cells, as well as interaction of this HCV glycoprotein with host cell-surface proteins.


   ACKNOWLEDGEMENTS
 
We are grateful to C. M. Rice (Washington University School of Medicine, Saint Louis, USA) for providing the p90/HCV-FL-long pU clone, ATM no. PH 13149611755 and to Transgene S.A. for generously supplying shuttle plasmids to generate recombinant vaccinia and adenoviruses. We thank J. Dubuisson (Institut de Biologie, Lille, France) for kindly providing H47 and H53 mAbs. Plasmid containing the hCD81 cDNA was a generous gift of S. Levy (Stanford University, Stanford, USA). Robert Drillien is thanked for many helpful discussions. We are grateful to M. Dimitrova for help with the GST pull-down assay. The contribution of Anne Steffan and Mathieu Erhardt to the fluorescence, electron and confocal microscopy experiments is highly appreciated. The Inter-Institute Confocal Microscopy Platform used in this study was co-financed by the Région Alsace, the CNRS, the Université Louis Pasteur and the Association de la Recherche pour le Cancer (ARC). This work was supported by a grant from ARC (no. 76035703). F. K. was successively supported by grants from ADRERUS and from ARC. J. D. A. was successively supported by grants from ARC and BioMérieux, France.


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Received 28 October 2002; accepted 21 November 2002.