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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ABSTRACT |
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MAIN TEXT |
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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. 1
a). 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 EGFPE2 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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As an indicator of intracellular trafficking of the protein, we examined the sensitivity of EGFPE2 to endoglycosidase treatment after pulsechase labelling with 100 µCi 35S-Protein Labelling Mix ml-1 and immunoprecipitation with mAb H53. Immunoprecipitates were digested with either endo--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 EGFPE2 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 EGFPE2 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 EGFPE2, 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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Expression of EGFPE2 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 EGFPE2 fusion protein-specific staining was localized at restricted areas in the cell (Fig. 3c
). Moreover, EGFPE2 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 EGFPE2 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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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 EGFPE2 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 EGFPE2TMrabies 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 EGFPE2TMrabies fusion protein was further analysed with mAb H53 in pulsechase experiments as described above. EGFPE2TMrabies 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 EGFPE2TMrabies 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 EGFPE2TMrabies 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 EGFPE2TMrabies by Western blot following incubation with GSTCD81 (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 EGFPE2TMrabies and hCD81 was detected using this technique. This result is consistent with previous reports demonstrating a modulation of the E2hCD81 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 EGFPE2TMrabies 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 EGFPE2TMrabies 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 EGFPE2TMrabies 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 EGFPE2 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 EGFPE2 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.
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ACKNOWLEDGEMENTS |
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Received 28 October 2002;
accepted 21 November 2002.