Hepatitis C virus NS3 protein interacts with ELKS-{delta} and ELKS-{alpha}, members of a novel protein family involved in intracellular transport and secretory pathways

Rachmat Hidajat1, Motoko Nagano-Fujii1, Lin Deng1, Motofumi Tanaka1,2, Yuki Takigawa1, Sohei Kitazawa3 and Hak Hotta1

1 Division of Microbiology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan
2 Division of Gastroenterological Surgery, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan
3 Division of Molecular Pathology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan

Correspondence
Hak Hotta
hotta{at}kobe-u.ac.jp


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The NS3 protein of hepatitis C virus (HCV) has a serine protease activity in its N-terminal region, which plays a crucial role in virus replication. This region has also been reported to interact not only with its viral cofactor NS4A, but also with a number of host-cell proteins, which suggests a multifunctional feature of NS3. By means of yeast two-hybrid screening using an N-terminal region of NS3 as bait, a human cDNA encoding a region of ELKS-{delta}, a member of a novel family of proteins involved in intracellular transport and secretory pathways, was molecularly cloned. Using co-immunoprecipitation, GST pull-down and confocal and immunoelectron microscopic analyses, it was shown that full-length NS3 interacted physically with full-length ELKS-{delta} and its splice variant, ELKS-{alpha}, both in the absence and presence of NS4A, in cultured human cells, including Huh-7 cells harbouring an HCV subgenomic RNA replicon. The degree of binding to ELKS-{delta} varied with different sequences of the N-terminal 180 residues of NS3. Interestingly, NS3, either full-length or N-terminal fragments, enhanced secretion of secreted alkaline phosphatase (SEAP) from the cells, and the increase in SEAP secretion correlated well with the degree of binding between NS3 and ELKS-{delta}. Taken together, these results suggest the possibility that NS3 plays a role in modulating host-cell functions such as intracellular transport and secretion through its binding to ELKS-{delta} and ELKS-{alpha}, which may facilitate the virus life cycle and/or mediate the pathogenesis of HCV.

The primer sequences used in this study are available as supplementary material in JGV Online.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Hepatitis C virus (HCV) is the causative agent of non-A, non-B hepatitis (Choo et al., 1989). It has been estimated that more than 170 million individuals are infected with HCV worldwide, representing nearly 3 % of the world's population (WHO, 1999). The majority of patients remain chronically infected, suffering from chronic liver disorders such as chronic hepatitis, liver cirrhosis and hepatocellular carcinoma (HCC).

HCV, an enveloped RNA virus, belongs to the genus Hepacivirus of the family Flaviviridae. The HCV genome is a single-stranded, positive-sense RNA of approximately 9·6 kb, which contains a large open reading frame (ORF) flanked by 5'- and 3'-untranslated regions (Reed & Rice, 2000). The ORF encodes a polyprotein of approximately 3000 aa, which is cleaved by the cellular signal peptidase and two virally encoded proteases into at least 10 mature proteins: core, envelope glycoprotein 1 (E1), E2, p7, non-structural protein 2 (NS2), NS3, NS4A, NS4B, NS5A and NS5B (Hijikata et al., 1991, 1993).

NS3 is comprised of two domains, one possessing a serine protease activity and the other an RNA helicase activity, both of which are essential for virus replication. The protease domain resides in the N-terminal part of NS3, while the helicase domain is in the C-terminal part. NS3 enzymic activities are modulated by NS4A, which forms a complex with NS3 to stabilize and localize it to the perinuclear endoplasmic reticulum (ER) membranes (Reed & Rice, 2000; Wölk et al., 2000). NS3 also interacts with the other NS proteins, either directly or indirectly through host-cell proteins, to form the virus replication complex (Aizaki et al., 2004).

The interactions between NS3 and cellular proteins have been studied to elucidate its role(s) in viral pathogenesis (Reed & Rice, 2000; Tellinghuisen & Rice, 2002). The helicase domain of NS3 was shown to interact with protein kinase A (Aoubala et al., 2001; Borowski et al., 1999a). It was also reported that NS3 served as a substrate for protein kinase C (PKC) and inhibited PKC signalling via competition with its physiological substrates (Borowski et al., 1999b). NS3 also binds to histones H2B and H4 (Borowski et al., 1999c). The NS3 helicase domain has also been shown to be essential for NS3 protein methylation by the protein arginine methyltransferase 1 (Rho et al., 2001).

Studies on the NS3 serine protease domain have demonstrated that it can transform NIH3T3 cells (Sakamuro et al., 1995), rat fibroblasts (Zemel et al., 2001) and the human liver cell line QSG7701 (He et al., 2003), although the underlying mechanism(s) remains to be elucidated. Previously, we reported that the oncogenic properties of NS3 might involve an interaction with the tumour suppressor p53 (Ishido & Hotta, 1998; Muramatsu et al., 1997). The protease domain of NS3 has also been reported to bind to Sm-D1, a component of small nuclear ribonucleoprotein associated with autoimmune disease (Iwai et al., 2003). A more recent study demonstrated that the protease domain of NS3 interacts with LMP7, a component of the immunoproteasome, and downregulated the proteasome peptidase activity (Khu et al., 2004).

To identify another possible cellular target(s) that interacts with the NS3 protease domain, we screened a human cDNA library using a yeast two-hybrid system. We have shown here that NS3, through its protease domain, binds to ELKS-{delta} and its splice variant, ELKS-{alpha} (Nakata et al., 1999, 2002). Since a mouse homologue of ELKS-{delta}, namely Rab6-interacting protein 2B (Rab6IP2B), was reported to affect intracellular transport by binding to Rab6 (Monier et al., 2002), we tested the possible effects of NS3–ELKS-{delta} interaction on intracellular transport and secretory pathways using a secreted alkaline phosphatase (SEAP) assay. We observed that NS3 augmented the cellular secretion of SEAP and that the increase in SEAP secretion was proportional to the degree of binding between NS3 and ELKS-{delta}. These results collectively suggest the possibility that NS3 affects intracellular transport and/or secretion pathways by interacting with ELKS-{delta} and ELKS-{alpha}.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Plasmid construction.
cDNA fragments encoding an N-terminal portion of NS3 (aa 1027–1208) were amplified from sera of HCV-1b-infected patients by RT-PCR as described previously (Ogata et al., 2002, 2003), with minor modifications, using the primers NS3-F and NS3-181R (see Supplementary Table in JGV Online). The amplified fragments were subcloned in frame to the LexA DNA-binding domain of pHybLex/Zeo (Invitrogen) to generate pLex-NS3-H-31 and pLex-NS3-H-45 for expression in yeast. Frame-shift mutants of bait (pLex-NS3-H-31-FS) and prey [pB42-ELKS-{delta}(787–1063)-FS] were generated by digesting the parental plasmids with EcoRI and BamHI, respectively, followed by blunt-end formation and self-ligation. They served as negative controls. pB42-ATF6-{alpha} (Tong et al., 2002) was also used as a negative control in this study.

A mammalian expression plasmid encoding Myc-tagged full-length NS3 [pcDNA3.1/NS3F(MKC1a)] was constructed by amplifying a cDNA fragment from pBSNS3/1027–1657 (Muramatsu et al., 1997) using primers NS3/M/B and NS3FHindIII (Supplementary Table), followed by subcloning into pcDNA3.1/myc-His(–)C (Invitrogen). Expression plasmids for chimeric forms of full-length NS3 were constructed, in which the N-terminal 180 residues were derived from clinical isolates, nos 42, 45, H05-5 and H17-2 (Ogata et al., 2002, 2003; GenBank accession nos AB072084, AB072086, AB072048 and AB072055, respectively), while the C-terminal 451 residues were derived from MKC1a (GenBank accession no. D45172). These plasmids were designated pcDNA3.1/NS3(M-42), pcDNA3.1/NS3(M-45) pcDNA3.1/NS3(M-H05-5) and pcDNA3.1/NS3(M-H17-2), respectively.

A GST fusion protein-expressing plasmid was also constructed. A vector plasmid, pcDNA3.1/GST, was constructed by introducing a 767 bp HincII fragment of pGEX-4T1 (Pharmacia) into EcoRI-digested, blunt-ended pcDNA3.1/myc-His(–)C. The full-length NS3-coding region of MKC1a was subcloned in frame to pcDNA3.1/GST to generate pcDNA3.1/GST-NS3. pBS-GST-NS4B-F (Tong et al., 2002) and pBS-GST-NS5A-F (Taguchi et al., 2004) served as negative controls in this study.

Expression plasmids for FLAG-tagged, full-length ELKS-{delta} and ELKS-{alpha} were constructed. cDNA fragments of 3889 and 3834 bp encoding N-terminally deleted ELKS-{delta} and ELKS-{alpha}, respectively, were obtained by digesting pDR2-ELKS-{delta} and pDR2-ELKS-{alpha} (kind gifts from Dr M. Emi, Institute of Gerontology, Nippon Medical School, Kawasaki, Kanagawa, Japan) with BamHI and XbaI and subcloned into the pcDNA3.1/N-FLAG vector (Tong et al., 2002). Subsequently, a 718 bp sequence of ELKS-{delta} or ELKS-{alpha} (from the ATG initiation codon to the BamHI site) was amplified by PCR from pDR2-ELKS-{delta} or pDR2-ELKS-{alpha} using the primers ELKS-F and ELKS-303R (Supplementary Table) and subcloned into the above plasmids. The resultant plasmids were designated pcDNA3.1/N-FLAG-ELKS-{delta} and pcDNA3.1/N-FLAG-ELKS-{alpha}. In addition, various deletion mutants of ELKS-{delta} were constructed by PCR using appropriate sets of primers (Supplementary Table), followed by subcloning into the expression vector.

Yeast two-hybrid screening.
Yeast two-hybrid screening was performed using a Hybrid Hunter kit (Invitrogen) and a human cDNA library, as reported previously with some modifications (Tong et al., 2002). In brief, the L40 yeast strain carrying pLex-NS3-H31 was transformed with the pYESTrp cDNA library prepared from HeLa cells (Invitrogen). Transformants were screened for growth on YC-WHUKZ300 plates lacking tryptophan, histidine, uracil and lysine but containing Zeocin. The resultant colonies were tested for {beta}-galactosidase ({beta}-Gal) activity according to the manufacturer's protocol.

Cell culture and protein expression.
Vaccinia virus T7 hybrid expression was performed as reported previously (Muramatsu et al., 1997). In brief, HeLa cells were inoculated with recombinant vaccinia virus expressing T7 RNA polymerase (vTF7-3). After virus adsorption for 1 h, cells were transfected with expression plasmids using Lipofectin (Invitrogen) and incubated overnight. Cells were then analysed for possible protein–protein interactions (see below). For immunofluorescence and the SEAP secretion assay, a plasmid-based expression method was employed using Fugene 6 transfection reagent (Roche). Cells were then incubated for 48 h before analysis.

A Huh-7 cell line stably harbouring an HCV subgenomic RNA replicon was prepared as described previously (Lohmann et al., 2001; Taguchi et al., 2004; Takigawa et al., 2004), using pFK2884Gly (a kind gift from Dr R. Bartenschlager, University of Heidelberg, Heidelberg, Germany). A cured Huh-7 cell line was prepared by maintaining the stable HCV replicon-harbouring cell line in the presence of {alpha}-interferon (1000 IU ml–1) for 1 month.

Double-staining immunofluorescence.
Cells expressing Myc-tagged NS3 and FLAG-tagged ELKS-{delta} were fixed with 4 % paraformaldehyde at room temperature for 15 min and permeabilized with 100 % methanol at –20 °C for 3 min. Cells were then blocked with 5 % normal donkey serum for 1 h at room temperature. Primary antibodies used were anti-Myc mouse monoclonal antibody (Santa Cruz Biotech) and anti-FLAG rabbit polyclonal antibody (Sigma). FITC-conjugated goat anti-mouse IgG (MBL) and Cy3-conjugated donkey anti-rabbit IgG (Chemicon) were used as secondary antibodies, respectively. Stained cells were observed with a laser-scanning confocal microscope (LSM510 version 3.0; Carl Zeiss) or a fluorescence microscope (BX51; Olympus) attached to a DP70 CCD camera.

Immunoelectron microscopy.
Cells expressing Myc-tagged NS3 and FLAG-tagged ELKS-{delta} were fixed with 4 % paraformaldehyde and 0·2 % glutaraldehyde for 30 min at room temperature. After washing with PBS, cells were centrifuged at 1500 r.p.m. for 5 min. The cell pellet was dehydrated in a series of 70, 80 and 90 % ethanol, embedded in LR White resin (London Resin) and kept at –20 °C for 2 days to facilitate resin polymerization. After ultrathin sectioning, samples were etched in 3 % H2O2 for 5 min at room temperature and washed with PBS. For labelling, sections were incubated with anti-Myc mouse monoclonal antibody and anti-FLAG rabbit antiserum for 1 h. After rinsing with PBS, sections were incubated with goat anti-mouse IgG and goat anti-rabbit IgG conjugated to 5 and 10 nm gold particles (Sigma), respectively, for 1 h. Sections were post-stained with uranyl acetate and lead citrate and observed under a transmission electron microscope (JEM 1200EX; JEOL).

Co-immunoprecipitation.
Cells expressing Myc-tagged NS3 and FLAG-tagged ELKS-{delta} or ELKS-{alpha} were lysed in ice-cold RIPA buffer (1x PBS, 1 % NP-40, 0·5 % sodium deoxycholate, 0·1 % SDS) with freshly added protease inhibitors (PMSF and aprotinin). Lysates were centrifuged at 10 000 g for 10 min at 4 °C and the supernatants were mixed with 0·25 µg normal rabbit IgG and 20 µl protein G–agarose beads (Pharmacia) at 4 °C for 30 min to eliminate non-specific binding. The pre-cleared lysates were incubated with anti-Myc rabbit polyclonal antibody for 1 h at 4 °C, followed by incubation with 20 µl protein G–agarose beads for another 1 h. After five washes with ice-cold RIPA buffer, the immunoprecipitates were analysed by immunoblotting, as described below.

Immunoblotting.
Samples were subjected to SDS-PAGE and blotted electrophoretically on to a PVDF membrane (Immobilon-P; Millipore). After blocking in PBS containing 5 % skimmed milk, membranes were incubated with anti-FLAG or anti-Myc mouse monoclonal antibody for 1 h. Membranes were then incubated with peroxidase-labelled goat anti-mouse IgG (MBL) for 1 h. After three washes, protein bands were visualized by enhanced chemiluminescence (Amersham Biosciences). Densitometric analysis was performed using publicly available software (tnimage-3.3.14; available at http://brneurosci.org/tnimage.html).

GST pull-down assay.
Lysates were prepared from cells transiently expressing GST-tagged NS3 and FLAG-tagged ELKS-{delta} or ELKS-{alpha}. Lysates were mixed with 20 µl glutathione-conjugated Sepharose 4B beads at 4 °C for 90 min. The beads were washed five times with ice-cold RIPA buffer and subjected to immunoblot analysis using anti-FLAG antibody. To verify that there was a comparable amount of protein in each sample, lysates were directly (without pull-down) subjected to immunoblotting.

SEAP secretion assay.
A SEAP secretion assay was performed to measure the possible effect of NS3 on the secretory pathway. SEAP is a genetically engineered secreted form of alkaline phosphatase (Cullen & Malim, 1992) and the SEAP secretion assay has been widely used to monitor cellular secretory function including Rab6-mediated intracellular transport (Echard et al., 2000; Martinez et al., 1994). HeLa cells were transiently transfected with 200 ng pcDNA3.1-derived NS3 expression plasmid, 50 ng pSEAP2-Control (BD Biosciences) and 1 ng pRL-SV40 (Promega), in the presence or absence of 200 ng pcDNA3.1/N-Flag-ELKS-{delta}. pRL-SV40 was used to express Renilla luciferase as an internal control. After 48 h, culture medium was collected for the SEAP secretion assay, while the cells were processed for the Renilla luciferase assay. SEAP activity was measured using the SEAP Reporter Gene Assay (Roche) and Renilla luciferase activity measured using the Renilla Luciferase Assay System (Promega), according to the manufacturers' instructions. SEAP activity in each sample was normalized against Renilla luciferase activity. Since pSEAP2-Control and pRL-SV40 use the same SV40 early promoter, the marginal effect of NS3 on promoter activity (an increase of ~20 %, as determined by Renilla luciferase activity) was nullified and the effect on SEAP secretion could be determined by this assay.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Identification of ELKS-{delta} as an NS3-interacting protein using a yeast two-hybrid assay
To identify a human protein(s) that physically interacts with the NS3 protease domain (aa 1027–1208) (Fig. 1a), the L40 yeast strain harbouring pLex-NS3-H-31 was transformed with the pYESTrp-based HeLa cDNA library and screened for growth on YC-WHUKZ300 selection plates. Out of 5·8x107 primary transformants screened, 260 colonies grew on the selection plates. Further screening for {beta}-Gal activity identified a clone that showed strong reactivity. DNA sequence analysis revealed that the yeast clone carried a pYESTrp-driven sequence that completely matched the sequence for a C-terminal portion of ELKS-{delta} (GenBank accession no. AB053470; Nakata et al., 2002). The cloned cDNA fragment was designated ELKS-{delta}(787–1063).



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Fig. 1. Identification of ELKS-{delta} as an NS3-binding protein by yeast two-hybrid assay. (a) Schematic diagram of the HCV genome. The N-terminal serine protease domain of NS3 (aa 1027–1208) used as bait is also depicted. (b) A representative result showing specific interaction between the NS3 protease domain and ELKS-{delta}(787–1063). The L40 yeast strain co-transfected with the indicated plasmids was grown on tryptophan-deficient media containing the antibiotic Zeocin and subsequently assayed for {beta}-Gal activity. Development of a blue colour within 30 min indicated strong interaction between the two proteins of interest. ++, Strong interaction; +, moderate interaction; –, no interaction.

 
The specificity of the interaction between NS3 and ELKS-{delta}(787–1063) was tested by transfecting the parental (naïve) L40 strain with various combinations of expression plasmids, including two different NS3 sequences (H-31 and H-45) and frame-shift mutants of NS3-H-31 and ELKS-{delta}(787–1063). The results demonstrated specific interaction between the NS3 protease domain and ELKS-{delta}(787–1063) in yeast (Fig. 1b).

NS3 interacts with ELKS-{delta} and its splice variant, ELKS-{alpha}, in cultured human cells
The interaction between NS3 and ELKS-{delta} was further investigated in mammalian cells and a specific interaction between the protease domain of NS3 and ELKS-{delta}(787–1063) was observed (data not shown). Moreover, full-length NS3 (aa 1027–1657) was shown to interact with ELKS-{delta}(787–1063) in HeLa cells (Fig. 2a, lane 2). We then narrowed down the region of ELKS-{delta} responsible for the interaction with full-length NS3 (Fig. 2b). C-terminal truncation of ELKS-{delta} up to aa 1008 did not affect the interaction with NS3. However, further deletion up to aa 995 or 979 abolished the ability to interact with NS3. N-terminal truncation of the initial fragment up to aa 846 did not affect the interaction with NS3, while further deletion up to aa 876 or 886 completely abolished it. These results mapped a minimum NS3-interacting region somewhere between aa 846 and 1008 of ELKS-{delta}. ELKS-{delta}(846–1008) consistently interacted with NS3. Similar results were obtained using a yeast two-hybrid system (data not shown).



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Fig. 2. Determination of the minimum region of ELKS-{delta} required for interaction with full-length NS3 in cultured human cells. (a) A series of deletion mutants of FLAG-tagged ELKS-{delta} was expressed by the vaccinia–T7 hybrid expression method in HeLa cells without (odd-numbered lanes) or with (even-numbered lanes) Myc-tagged full-length NS3. Cell lysates were immunoprecipitated using anti-Myc antibody and probed by immunoblotting using anti-FLAG antibody (upper panel). Lysates were directly (without being immunoprecipitated with anti-Myc antibody) probed with anti-FLAG antibody to verify a comparable degree of expression of the ELKS-{delta} mutants (middle panel). Efficient immunoprecipitation of Myc-tagged NS3 with anti-Myc antibody was also verified (lower panel). (b) Schematic diagram of the various deletion mutants of ELKS-{delta}. ++, Strong interaction; –, no interaction.

 
Next, we tested the interaction of full-length NS3 with full-length ELKS-{delta} and its splice variant, ELKS-{alpha}. GST pull-down analysis demonstrated that full-length NS3 interacted with full-length ELKS-{delta} and ELKS-{alpha} (Fig. 3a). The specificity of the interaction between NS3 and ELKS-{delta} was confirmed by demonstrating that neither NS4B nor NS5A of HCV bound to full-length ELKS-{delta} under the same experimental conditions (Fig. 3b). Specific interaction between the two molecules was also confirmed by co-immunoprecipitation analysis, in which anti-FLAG antibody (directed against FLAG-tagged full-length ELKS-{delta} and ELKS-{alpha}) co-immunoprecipitated full-length NS3 (data not shown).



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Fig. 3. Full-length ELKS-{delta} and ELKS-{alpha} interact with full-length NS3 in cultured human cells. (a) FLAG-tagged full-length ELKS-{delta} (lanes 1 and 4), full-length ELKS-{alpha} (lanes 2 and 5) and a deletion mutant, ELKS-{delta}(787–1063) (lanes 3 and 6), were co-expressed in HeLa cells with GST (lanes 1–3) or full-length NS3 fused to GST (lanes 4–6). Cell lysates were pulled down with glutathione beads and probed with anti-FLAG antibody (upper panel). Lysates were directly (without being pulled down) probed with anti-FLAG antibody to confirm expression of ELKS-{delta} or ELKS-{alpha} (middle panel). Efficient pull-down was also verified (lower panel). (b) FLAG-tagged full-length ELKS-{delta} was co-expressed in HeLa cells with empty vector (lane 2), GST (lane 3), GST-tagged full-length NS3 (lane 4), full-length NS4B (lane 5) and full-length NS5A (lane 6). Cells lysates were pulled down by glutathione beads and probed with anti-FLAG antibody (upper panel). Lysates were directly probed with anti-FLAG antibody to confirm ELKS-{delta} expression (middle panel). Efficient pull down was also verified (lower panel). (c) FLAG-tagged full-length ELKS-{delta} (lanes 2 and 6), full-length ELKS-{alpha} (lanes 3 and 7) and a deletion mutant, ELKS-{delta}(787–1063) (lanes 4 and 8), were expressed in Huh-7 cells harbouring an HCV RNA replicon (lanes 5–8) or parental Huh-7 cells as a control (lanes 1–4). Cell lysates were immunoprecipitated with anti-NS3 polyclonal antibody and probed with anti-FLAG antibody (upper panel). Expression of ELKS-{delta} and ELKS-{alpha} (middle panel) and efficient immunoprecipitation of NS3 (lower panel) were also verified.

 
In order to determine whether NS3 interacted with ELKS proteins in the presence of the other HCV non-structural proteins, we expressed ELKS-{delta} and ELKS-{alpha} in Huh-7 cells harbouring an HCV subgenomic RNA replicon and subsequently immunoprecipitated NS3 using anti-NS3 polyclonal antibody. The result demonstrated that NS3 expressed in the context of HCV replication interacted with full-length ELKS-{delta} and ELKS-{alpha} in HCV RNA replicon-harbouring cells (Fig. 3c).

Sequence comparison of ELKS-{delta} and ELKS-{alpha}
The amino acid sequences of ELKS-{delta} and ELKS-{alpha} differ from each other in their C-termini due to alternative splicing (Fig. 4a). As described above, a minimum region responsible for the interaction with NS3 was mapped between aa 846 and 1008 of ELKS-{delta} (Fig. 2). Since full-length ELKS-{alpha} interacted with NS3 (Fig. 3), we then determined a minimum region of ELKS-{alpha} responsible for the interaction. Deletion mutational analysis revealed that ELKS-{alpha}(747–948), but not ELKS-{alpha}(763–948), ELKS-{alpha}(791–948) or ELKS-{alpha}(806–948), interacted with NS3 (Fig. 4b and c), suggesting that ELKS-{alpha}(747–948) is the minimum region responsible for the interaction with NS3.



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Fig. 4. Sequence comparison of C-terminal portions of ELKS-{delta} and ELKS-{alpha}. (a) Sequence alignment of a C-terminal portion of ELKS-{delta} and ELKS-{alpha}. Asterisks indicate identical residues. Hyphens indicate missing residues when compared with the other isoform. Underlined (aa 846–1008) is the minimum region of ELKS-{delta} required for interaction with NS3 (see Fig. 2). (b) A series of deletion mutants of FLAG-tagged ELKS-{alpha} was expressed without (odd-numbered lanes) or with (even-numbered lanes) Myc-tagged full-length NS3. ELKS-{delta}(787–1063) (lanes 3 and 4) served as a positive control. Cell lysates were immunoprecipitated using anti-Myc antibody and probed with anti-FLAG antibody (upper panel). Lysates were directly probed with anti-FLAG antibody to confirm expression of the ELKS-{alpha} mutants (middle panel). Efficient immunoprecipitation of NS3 was also verified (lower panel). (c) Schematic diagram of the ELKS-{alpha} deletion mutants. +, Moderate interaction [weaker than ELKS-{delta}(787–1063)]; –, no interaction.

 
NS3 co-localizes with ELKS-{delta} in human cells
We visualized the co-localization of NS3 with ELKS-{delta} in Huh-7 cells by double-staining immunofluorescence analysis. When co-expressed in Huh-7 cells using plasmid-based expression methods, full-length NS3 co-localized with full-length ELKS-{delta} in the cytoplasm, both in the absence (Fig. 5a) and presence (Fig. 5b) of NS4A. Co-localization of NS3 with ELKS-{delta} was also observed in HCV RNA replicon-harbouring cells (Fig. 5c).



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Fig. 5. Full-length ELKS-{delta} co-localizes with full-length NS3 in cultured human cells. FLAG-tagged full-length ELKS-{delta} was co-expressed in Huh-7 cells with Myc-tagged full-length NS3 in the absence (a) or presence (b) of NS4A, or in HCV RNA replicon-harbouring Huh-7 cells (c) and examined by double-staining immunofluorescence analysis. The orange colour in the cytoplasm observed in the merged pictures indicates co-localization of the two proteins. (d) FLAG-tagged full-length ELKS-{delta} was co-expressed with Myc-tagged full-length NS3 in HeLa cells. Cells were simultaneously stained with antibodies to Myc (5 nm diameter gold particles, thin arrows) and FLAG (10 nm diameter gold particles, thick arrows). Close proximity between both particles indicates tight interaction between the two proteins.

 
Immunoelectron microscopic analysis also demonstrated co-localization of full-length NS3 with full-length ELKS-{delta} in close proximity to the ER membranes in the perinuclear region (Fig. 5d).

NS3 interacts differentially with ELKS-{delta} in an NS3 sequence-dependent manner
A considerable degree of sequence variation has been observed in the N-terminal 180 residues of NS3 (Ogata et al., 2002, 2003). We performed experiments to determine whether or not interaction with ELKS-{delta} varied with different NS3 sequences. We first used 198-residue fragments of NS3 obtained from four patients (nos 42, 45, H05-5 and H17-2) and found that the degree of interaction with ELKS-{delta} varied with the different sequences (data not shown). Next, we tested full-length NS3 sequences of five different isolates, a parental strain (MKC1a) and four chimeric forms (M-42, M-45, M-H05-5 and M-H17-2), which differed from each other by at most 10 residues within the N-terminal 180 residues, all having the remaining C-terminal 451 residues in common. These full-length forms of NS3 were each expressed with full-length ELKS-{delta} and the interactions examined. Consistent with the results obtained with the 198-residue fragments, the degree of interaction between full-length NS3 and ELKS-{delta} varied with the different NS3 sequences, with M-42 and M-45 showing the strongest interaction, and M-H05-5 the weakest (Fig. 6a and b). Sequence alignment of the N-terminal 180 residues of NS3 is shown in Fig. 6(c). Five residues (Val-1044, Leu-1106, Ala-1176, Val-1179 and Ile-1196) were unique to M-H05-5. Based on this sequence alignment alone, however, it was difficult to draw a conclusion as to which residue(s) most significantly affects the interaction with ELKS-{delta}.



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Fig. 6. NS3 interacts with ELKS-{delta} in a sequence-dependent manner. (a) FLAG-tagged full-length ELKS-{delta} was co-expressed with Myc-tagged full-length NS3 of different sequences by the plasmid-based expression method in HeLa cells. Cell lysates were immunoprecipitated using anti-Myc antibody and probed with anti-FLAG antibody (upper panel). Lysates were directly probed with anti-FLAG antibody to confirm ELKS-{delta} expression (middle panel). Efficient immunoprecipitation of NS3 was also verified (lower panel). (b) The intensities of the ELKS-{delta} bands that co-immunoprecipitated with NS3 were quantified using available software (tnimage-3.3.14) and normalized to ELKS-{delta} expression level. Results are shown as mean±SD obtained from two independent experiments. *, P<0·05, compared with M-H05-5; {dagger}, P<0·05, compared with M-H17-2. (c) Sequence alignment of the N-terminal 180 residues of NS3 of different isolates. Asterisks indicate residues identical to those of M-45. The remaining C-terminal 451 residues were identical among the five isolates (see Methods).

 
When NS4A was co-expressed, the interaction between NS3 and ELKS-{delta} became weaker, although still detectable, and the NS3 sequence-dependent difference in the interaction with ELKS-{delta} was not clearly observed (data not shown).

NS3 enhances SEAP secretion from the cell, possibly through its interaction with ELKS-{delta}
The binding domain of Rab6IP2, a murine homologue of ELKS-{delta}, has been reported to affect the endosome-to-Golgi retrograde transport pathway through its binding to Rab6 (Monier et al., 2002). CAST and ERC proteins, rat homologues of the ELKS protein family, have also been implicated in the modulation of neurotransmitter secretion (Deguchi-Tawarada et al., 2004; Ohtsuka et al., 2002; Wang et al., 2002). We thought that NS3 might affect the possible function of ELKS-{delta} and/or its splice variant(s), modulating intracellular transport and secretory function. Therefore, we performed a SEAP assay. The results demonstrated that SEAP secretion was significantly enhanced by NS3 of three isolates (MKC1a, M-42 and M-45), especially when ELKS-{delta} was expressed ectopically (Fig. 7a). In this context, we assumed that endogenous ELKS-{delta} was also expressed, although to a lesser extent. In contrast, NS3 of the other isolates (M-H-05-5 and M-H17-2) did not enhance SEAP secretion in the absence of ectopic ELKS-{delta}, while a mild enhancement by NS3 of M-H17-2 was observed when ELKS-{delta} was expressed ectopically. Interestingly, the degree of interaction between NS3 and ELKS-{delta} correlated well with the level of increase in SEAP secretion (Fig. 7b). Moreover, SEAP secretion was enhanced in HCV RNA replicon-harbouring Huh-7 cells compared with HCV RNA-negative Huh-7 cells that had been cured by interferon treatment (Fig. 7c). These results strongly suggested that NS3 affects the cellular secretory pathway by interacting with ELKS-{delta} and ELKS-{alpha}.



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Fig. 7. NS3 enhances SEAP secretion from cells in a sequence-dependent manner. (a) Myc-tagged full-length NS3 proteins of different sequences were co-expressed with SEAP (pSEAP2-Control) and Renilla luciferase (pRL-SV40) in HeLa cells, in the absence (open columns) or presence (filled columns) of ectopic, full-length ELKS-{delta}. An irrelevant protein (GST) served as a control for NS3. SEAP activity in the medium was measured and normalized to Renilla luciferase activities. The percentage increase in NS3-mediated SEAP secretion over the control is shown. Results are given as the mean±SD from three independent experiments. *, P<0·001; {dagger}, P<0·01, compared with the control; {ddagger}, P<0·05, compared with the value in the absence of ectopic ELKS-{delta} expression (open column). (b) Correlation between NS3-mediated enhancement of SEAP secretion (percentage increase) and the degree of interaction with ELKS-{delta}. Correlation coefficient (r)=0·97973485 (P<0·0001). (c) Parental Huh-7 cells, HCV RNA replicon-harbouring Huh-7 cells and HCV RNA-negative cured Huh-7 cells were co-transfected with pSEAP2-Control and pRL-SV40. SEAP activity in the medium was measured and normalized to Renilla luciferase activities. Results are given as the mean±SD of the relative SEAP secretion compared with the parental Huh-7. *, P<0·05, compared with parental and cured Huh-7 cells.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In the present study, we demonstrated that an N-terminal protease domain of HCV NS3 interacts physically with ELKS-{delta} and ELKS-{alpha} in yeast and cultured human cells (Figs 1–5). This interaction was also observed in the presence of NS4A (Fig. 5b) and in HCV RNA replicon-harbouring cells (Figs 3c and 5c), where NS3 is expressed in the context of HCV RNA replication to form a virus replication complex in a unique membranous structure (Aizaki et al., 2004). It was also demonstrated that the degree of interaction with ELKS-{delta} varied with different NS3 sequences (Fig. 6). The N-terminal 180 residues of M-42 and M-45, which bound to ELKS-{delta} more efficiently than M-H05-5, were derived from HCV isolates obtained from patients without HCC, whereas those of M-H05-5 and M-H17-2 were from patients with HCC (Ogata et al., 2002, 2003). It is intriguing to speculate that certain NS3 sequences with reduced ELKS-{delta} binding are more likely to interact with, and modulate the function of, another host factor(s), such as p53 (L. Deng and others, unpublished data; Ishido & Hotta, 1998; Muramatsu et al., 1997), thus facilitating the development of HCC. However, with the limited number of samples tested, it was difficult to draw a conclusion about the correlation between the NS3–ELKS-{delta} interaction and certain clinical symptoms, including HCC.

ELKS was initially identified as a protein fused to the RET proto-oncogene product in human papillary thyroid carcinoma (Nakata et al., 1999). Four other splice variants of the ELKS protein family were subsequently identified and designated ELKS-{beta}, -{gamma}, -{delta} and -{varepsilon}, with the prototype isoform being renamed ELKS-{alpha} (Nakata et al., 2002). A number of related proteins highly homologous to the ELKS proteins were identified in rat, named CAST (CAST1, CAST2{alpha} and CAST2{beta}; Deguchi-Tawarada et al., 2004; Ohtsuka et al., 2002) and ERC (ERC1a, ERC1b and ERC2; Wang et al., 2002). Another research group discovered two related mouse proteins that interacted specifically with a small GTPase protein, Rab6, and therefore the proteins were named Rab6IP2A and Rab6IP2B (Monier et al., 2002). Sequence alignment has revealed that ELKS, CAST, ERC and Rab6IP2 are orthologous members of the same protein family in human, rat and mouse. Their expression profiles in the body differ with different isoforms, with ELKS-{alpha}, CAST2{alpha}, Rab6IP2A, ERC1b and ERC2 expressed only in the brain, while ELKS-{delta}, Rab6IP2B and ERC1a are expressed ubiquitously outside of the brain (Nakata et al., 2002; Deguchi-Tawarada et al., 2004). In the present study, we mapped the minimum NS3-binding sequences to the regions spanning aa 846–1008 of ELKS-{delta} (Fig. 2) and aa 747–948 of ELKS-{alpha} (Fig. 4). Since the sequence of ELKS-{delta} from aa 846 to 1008 completely matches that of the corresponding region of ELKS-{varepsilon} (aa 874–1036), ELKS-{varepsilon} is most likely to interact with NS3. On the other hand, when compared with the minimum NS3-binding sequence of ELKS-{alpha} (aa 747–948), the corresponding region of ELKS-{beta} (aa 747–992) and ELKS-{gamma} (aa 475–720) have an identical insertion of 44 residues between positions 805 and 806 of ELKS-{alpha}. At present, we do not know whether or not this insertion affects the interaction with NS3.

Rab6IP2A and Rab6IP2B have been reported partially to inhibit the endosome-to-Golgi retrograde transport pathway through their binding to Rab6 (Monier et al., 2002). Similarly, CAST and ERC proteins have been postulated to modulate neurotransmitter release through interaction with Rab3A-interacting molecules that are localized in the pre-synaptic active zone and involved in exocytosis of neurotransmitters in a Rab3-dependent manner (Deguchi-Tawarada et al., 2004; Ohtsuka et al., 2002; Wang et al., 2002). It is thus possible that certain ELKS-binding proteins, including viral products, affect intracellular transport and secretory pathways, either positively or negatively, by counteracting or augmenting the function of ELKS proteins. Indeed, we observed that HCV NS3 enhanced SEAP secretion from cells. Interestingly, the degree of enhancement of secretion correlated well with the degree of binding between NS3 and ELKS-{delta} (Fig. 7b). It has been reported that Rab6 is expressed in hepatocytes, playing an important role in regulating intracellular transport at the level of the Golgi complex (Feldmann et al., 1995). In addition, ELKS-{delta} and its mouse counterpart Rab6IP2B are expressed ubiquitously in the body (Monier et al., 2002; Nakata et al., 2002). Therefore, it is reasonable to assume that, even in a natural setting, i.e. in HCV-infected hepatocytes in vivo, NS3 interacts with ELKS-{delta} to modulate Rab6-dependent intracellular transport and secretory pathways, thereby facilitating intracellular transport of viral components so that virion formation takes place efficiently. However, the precise role of this interaction in the virus life cycle and/or viral pathogenesis is still unknown and awaits further investigation.

We observed enhanced SEAP secretion from Huh-7 cells harbouring an HCV subgenomic RNA replicon, compared with secretion from parental Huh-7 cells and interferon-treated cured cells (Fig. 7c). In this context, it was recently reported that the rate of MHC class I traffic to the cell surface was inhibited in Huh-7 cells harbouring an HCV subgenomic RNA replicon and that the inhibition was probably mediated by the NS4A/B precursor protein (Konan et al., 2003). At present, we do not know the reason for the apparent discrepancy between their results (inhibited MHC class I traffic) and ours (enhanced SEAP secretion) in HCV RNA replicon-harbouring cells. It is possible that these events occur through different mechanisms, which selectively modulate intracellular transport and secretion of a particular protein(s).

Recently, Ducut Sigala et al. (2004) identified ELKS-{alpha} as an essential regulatory subunit of the I{kappa}B kinase complex, a modulator of nuclear factor-{kappa}B (NF-{kappa}B) transcription factor activation. They showed that ELKS-{alpha} was involved in induced expression of NF-{kappa}B target genes, including pro-inflammatory genes, and also in establishing a cellular anti-apoptotic status in response to tumour necrosis factor-{alpha}. These findings, together with our present observations, imply that NS3, through its interaction with ELKS-{delta} and ELKS-{alpha}, may modulate NF-{kappa}B activation, thereby facilitating HCV pathogenesis, such as inflammation and tumour formation in the liver. Further studies are needed to elucidate these issues.


   ACKNOWLEDGEMENTS
 
The authors are grateful to Dr M. Emi (Institute of Gerontology, Nippon Medical School, Kawasaki, Kanagawa, Japan) for providing the plasmids pDR2-ELKS-{delta} (p14) and pDR2-ELKS-{alpha} (p32). Thanks are also due to Dr R. Bartenschlager (University of Heidelberg, Heidelberg, Germany) for providing an HCV subgenomic RNA replicon (pFK5B2884Gly). This work was supported in part by grants-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and the Japan Society for the Promotion of Science.


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ABSTRACT
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DISCUSSION
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Received 27 December 2004; accepted 11 April 2005.



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