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1 FRE 2736 CNRS-bioMérieux, IFR 128 Biosciences Lyon-Gerland, Lyon, France
2 Laboratoire Bioinformatique et RMN Structurales, Institut de Biologie et Chimie des Protéines, UMR 5086 CNRS-UCBL Lyon-I, IFR 128 Biosciences Lyon-Gerland, Lyon, France
3 MRC Virology Unit, Glasgow G11 5JR, UK
4 CNRS-Inserm U544, Institut de Virologie, Strasbourg, France
5 UMR 2714 CNRS-bioMérieux, IFR 128 Biosciences Lyon-Gerland, Lyon, France
Correspondence
Christine Bain
bain{at}transgene.fr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) and hepatocytes, cells that are all capable of supporting HCV replication. THP-1 cells, monocyte-derived M and DCs, and Huh7 cells were infected by using adenoviruses (Ad) encoding Core, CE1E2 and a Core sequence modified so that the Core protein is wild type, but no ARFPs are expressed (C
ARFP). THP-1 cells and DCs infected with Ad encoding Core or CE1E2 produced significant levels of interleukin-6 (IL-6), IL-8, MCP-1 and MIP-1
, whereas production of these chemokines with AdCARFP was reduced or abolished. Similar effects on IL-8 production were observed in Huh7 cells and on IL-6 and MIP-1 in M. Wild-type Core sequence, but not CARFP, could trans-activate the IL-8 promoter and this activation was not associated with activation of p38/p42–44MAPK. This study illustrates, for the first time, the critical importance of ARFP expression in immunomodulatory functions attributed to Core expression and suggests a potential involvement of ARFP in mechanisms associated with HCV pathogenesis.
Present address: Transgene, AFSSA Lyon, 31 avenue Tony Garnier, 69364 Lyon Cedex 07, France. 
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), computer-based sequence analyses suggested that the viral genome contains an alternative reading frame (ARF) within the Core coding sequence (Walewski et al., 2001). Synthesis of Core-derived ARF proteins (ARFPs) was shown in cell-free extracts and/or Escherichia coli, but expression of an ARFP has yet to be demonstrated in mammalian cells. Furthermore, no consensus has yet been reached either on the mechanisms involved in initiation of ARFP synthesis or on the exact frameshifting position (Baril & Brakier-Gingras, 2005; Boulant et al., 2003; Vassilaki & Mavromara, 2003; Xu et al., 2001). The role of these proteins in the virus life cycle or in HCV-associated pathogenesis is unknown, but detection of ARFP-specific antibodies and T cell-mediated immune responses in HCV patients suggests that ARFPs are produced during natural infection (Bain et al., 2004).
HCV Core has been assigned a large array of functional, often contradictory, activities: pro- and anti-apoptotic effects, regulation of cell growth, transcriptional activities and immunosuppressive properties (Bergqvist et al., 2003; Ray et al., 1998). Earlier studies based on the use of transgenic mice expressing HCV proteins, including Core, have provided a set of evidence linking HCV protein expression and development of steatosis, oxidative stress, inflammation and, in some instances, hepatocellular carcinomas (Lerat et al., 2002; Okuda et al., 2002). More recently, expression of HCV Core has been linked, at least in vitro, to the induction of cytokine profiles associated with the development of fibrogenesis via a direct action on hepatic stellate cells (Bataller et al., 2004). The recent discovery of novel ARFPs certainly challenges various activities attributed to Core.
Experimental data strongly support infection by HCV during natural infection of different cell types. Beside the primary target cells, hepatocytes, HCV genomes (both positive- and negative-strand RNA) have been found within the population of peripheral blood mononuclear cells, in cells belonging to the monocyte/macrophage (M) lineage, as well as in T- and B-cell lineages (Laskus et al., 2000; Sansonno et al., 1996). Dendritic cells (DCs) isolated from HCV chronic patients have also been described as harbouring HCV replicative intermediate. In vitro, cells supporting HCV replication include M, monocyte-derived DCs, the hepatoma cell line Huh7 and the THP-1 monocytic cell line (Caussin-Schwemling et al., 2001; Lindenbach et al., 2005; Ponzetto et al., 2005; Radkowski et al., 2004; Wakita et al., 2005; Zhong et al., 2005).
By using adenoviral (Ad) constructs, we evaluated comparatively the effect of either wild-type (wt) Core coding sequence (AdCore or AdCE1E2) or a Core sequence modified so that the Core protein is wt, but no ARFP is expressed (AdCARFP), on the pattern of soluble factors released by different cell types known to be sites of replication of HCV during natural infection. A first screening on the THP-1 monocytic cell line, as a surrogate for primary monocytes, was further expanded on monocyte-derived DCs and M, and Huh7 hepatoma cells.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) and are specific, respectively, to the Core25–45 and ARFP67–93 domains.
Cell cultures.
Cells were cultured in complete Dulbecco's modified Eagle's medium (DMEM) (human Huh7 hepatoma cells) or complete RPMI medium (human THP-1 monocytic leukaemia cells, DCs or M), consisting of DMEM or RPMI medium supplemented with 10 % fetal calf serum, 2 mM L-glutamine and 100 IU penicillin/streptomycin ml–1 (Sigma).
DCs and M were differentiated from monocytes obtained from healthy donors by elutriation or CD14-positive selection (Etablissement Français du Sang). Immature DCs (iDCs) were obtained after incubation of monocytes for 6 days in complete RPMI medium supplemented with 200 U interleukin-4 (IL-4) ml–1 (Peprotech) and 200 ng granulocyte–macrophage colony-stimulating factor (GM-CSF) ml–1 (Peprotech). M were obtained after incubation of monocytes for 8 days in complete RPMI medium supplemented with 100 ng M colony-stimulating factor (M-CSF) ml–1 (Peprotech) and 10 µg GM-CSF ml–1.
Design of the CARFP gene.
A synthetic gene, named CARFP, encoding the Core protein derived from the HCV-J sequence (Kato et al., 1990) was synthesized by GeneArt (http://www.geneart.com/) according to the following constraints: (i) the amino acid sequence of the Core protein is totally conserved; (ii) no ARFP, resulting from either a frameshift (Boulant et al., 2003; Xu et al., 2001) or internal initiation in the +1 or +2 open reading frames, can be expressed (Baril & Brakier-Gingras, 2005; Vassilaki & Mavromara, 2003); (iii) RNA secondary structures that could be involved in frameshifting events, such as ribosomal entry sites, AT- or GC-rich sequences or repeat sequences, are abolished. In order to fulfil these conditions, 13 and 26 stop codons were introduced, respectively, in the +1 and +2 reading frames (Fig. 3a). The absence of any ARF product was confirmed by using three plasmids containing the green fluorescent protein (GFP) coding sequence in the 0, +1 or +2 frame of the CARFP coding sequence: pQB1-CARFP-GFP(0) (GFP cloned after nt 420), pQB1-CARFP-GFP(+1) (GFP cloned after nt 421) or pQB1-CARFP-GFP(+2) (GFP cloned after nt 422). Seventy-two hours after transfection, GFP expression was analysed in Huh7 cells by flow cytometry. Transfection efficiency was normalized by using the pCMV DsRed-Express system (Clontech), using analysis of DsRed fluorescent protein expression by flow cytometry.
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Plasmids pQBCore-GFP(0) and pQBCore-GFP(+1), expressing Core in fusion with the GFP reporter gene, were constructed as described elsewhere (Boulant et al., 2003).
pGWiz plasmids encoding Core and CE1E2 were described previously (Himoudi et al., 2002). A pGWiz plasmid encoding CARFP was constructed as follows: the CARFP sequence was amplified by PCR and inserted into the commercial pGWiz plasmid between SalI and NotI restriction sites. pGWiz-GFP, a control plasmid expressing GFP under the cytomegalovirus (CMV) promoter, was purchased from Gene Therapy Systems, Inc.
Recombinant Ad constructs.
Ad constructs encoding CE1E2, Core, GFP and -galactosidase (-Gal) were described previously (Himoudi et al., 2002). After amplification of the corresponding sequences by PCR, AdCARFP was constructed as described previously (Siavoshian et al., 2004). Ad preparations did not present endotoxin contamination, as assessed by Limulus–amoebocyte assay (Marcel Mérieux Laboratory).
DNA transfection and Ad infection.
Huh7 cells were transfected with the different plasmids in the presence of Lipofectamine/Plus reagent (Invitrogen). Huh7 and THP-1 cells were infected with the appropriate recombinant Ad at an m.o.i. of respectively 100 and 200 infectious units (IU) per cell for 72 h before Western blot (WB) analysis or staining with appropriate mAb for immunofluorescence/flow-cytometry studies (Gerszten et al., 2001). For cytokine analysis, supernatants were collected either directly after 72 h infection or after washing and an additional 48 h incubation in medium alone.
iDCs and M were infected with Ad at an m.o.i. of 500 for 2 h in complete medium. Under these conditions, up to 70 % of iDCs and M were found to express GFP following infection with Ad encoding GFP (data not shown). Volume was then increased with complete RPMI medium and cells were incubated for 48 h before WB analysis or phenotype determination by flow cytometry.
His-tagged protein purification.
Seventy-two hours post-transfection, in the presence or absence of proteasome inhibitor (MG-132; Calbiochem) for the last 12 h, Huh7 cells were lysed and His-tagged proteins were purified as described elsewhere (Boulant et al., 2003). Briefly, His-tagged proteins were eluted with 100 µl Tris buffer containing 500 mM imidazole (eluate 1). After a second elution (eluate 2), both eluates were analysed by WB.
WB analysis.
Infected or transfected cells were incubated in lysis buffer containing 1 % Triton X-100 and WB was performed as described previously (Pinna et al., 2004). Membranes were incubated sequentially with anti-ARFP or anti-Core mAbs and horseradish peroxidase-conjugated goat anti-mouse IgG antibodies (Dako). Phosphoproteins were revealed with anti-phospho- and -total p38 and p42MAPK antibodies (Cell Signaling Technology).
Immunofluorescence detection of HCV proteins.
THP-1 cells were infected with different Ad for 72 h before fixation/permeabilization. Staining was carried out by incubation with anti-Core mAb, then tetramethylrhodamine isothiocyanate (TRITC)–rabbit anti-mouse antibody (Dako).
Flow-cytometry analyses.
Infectivity of the different Ad constructs on THP-1 cells was assessed 48 h post-infection by staining with an anti-hexon antibody (AdenoX-rapid titre; BD Biosciences) for 1 h at 37 °C. After three washes, a phycoerythrin (PE)-conjugated anti-mouse antibody was added at room temperature for 1 h and analysed by flow cytometry.
Control IgG1–fluorescein isothiocyanate (FITC), control IgG2a–PE, CD1a–FITC, HLA-DR–FITC (BD Biosciences), CD40–PE, CD83–PE (Immunotech), CD86–PE, CD80–PE, CD11c–PE (BD Biosciences) and CD14–PE (Dako) were used to assess cell-surface phenotype of DCs and M.
Semiquantitative RT-PCR.
Total RNA was extracted from THP-1 cells infected or not with the different recombinant Ad constructs by using an RNeasy extraction kit (Qiagen). Quality of the RNA was determined by using an RNA 6000 Nano Assay chip (Agilent Technologies) on an Agilent 2100 Bioanalyzer according to the manufacturer's instructions. cDNA was produced from 1 µg total RNA by reverse transcription (5 min at 65 °C followed by 1 h at 55 °C) using the oligo(dT)20 ThermoScript RT-PCR system plus Platinum Taq DNA polymerase (Invitrogen) in a volume of 20 µl. To exclude contamination of samples by genomic DNA, controls in which the reverse transcriptase was replaced by water during the cDNA synthesis step were included. PCR amplification was performed by using primers specific for Core: sense, 5'-GTGGAAGGCGACAACTATC-3', and antisense, 5'-GGCAGATTCCCTGTTGCATA-3'; and CARFP: sense, 5'-AGCCAGCCTAGAGGAAGGAG-3', and antisense, 5'-ATTTCCGGTTGCGTAATTCA-3'. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) amplification (primers: sense, 5'-CCACATCGCTCAGACACC-3', and antisense, 5'-GAGGTCCACCACCCTGTT-3') was used to demonstrate equal RNA load. PCR amplification was carried out over 10, 20, 30 or 40 cycles of denaturation (92 °C, 1 min), annealing (55 °C, 1 min) and extension (72 °C, 1 min) using a thermocycler (ABI PRISM; Applied Biosystems). PCR products were analysed by electrophoresis in a 3 % agarose gel. The expected sizes of amplified products are 334 bp for Core, 339 bp for CARFP and 992 bp for GAPDH.
Measurement of soluble factor production.
Soluble factors were screened on THP-1 cell supernatants by using both a Human Cytokine Array III (RayBiotech), allowing detection of 42 soluble factors (Fig. 2a), and a Bio-Plex Human Cytokine 17-Plex panel (Bio-Rad). Levels of IL-8, MCP-1 and MIP-1 produced by THP-1 cells were measured by ELISA (IL-8, MCP-1, BD Biosciences; MIP-1, R&D Systems). Levels of IL-6, IL-8, MCP-1 and MIP-1 produced by DCs and M were measured by using a custom 4-Plex panel (Bio-Rad).
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ARFP and 0.4 µg pGWiz-GFP. Firefly luciferase luminometric activity was measured by use of a Dual Luciferase kit (Promega) 48 h after transfection.
Statistics.
Statistical analysis was performed by using a t-test and significant P values (P < 0.05) compared with controls.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, this construct was designed such that ARFPs resulting from ribosomal frameshifting can be produced and purified (Boulant et al., 2003; Xu et al., 2001). However, this design does not allow purification of ARFPs that could be produced by internal initiation either at codon 26 or at codon 83 (Baril & Brakier-Gingras, 2005; Vassilaki & Mavromara, 2003). When His-tagged products from cell extracts were stained with an anti-ARFP mAb, a specific band was detected exclusively in eluate 1 at a molecular mass of 17 kDa (Fig. 1b). When this same membrane was stained, without prior stripping, with a highly diluted anti-Core mAb, a second band at 21 kDa was detected in both eluates (Fig. 1b, c), suggesting that, in cells transfected with plasmids expressing His-tagged Core or CE1E2, levels of Core are much higher than levels of ARFP.
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). These data illustrate that the genotype 1b sequence used in our study is capable of expressing an ARFP of approximately 17 kDa, albeit to undetectable levels unless His-tagged ARFP is enriched prior to WB detection.
AdCE1E2 and AdCore induce IL-8, MCP-1 and MIP-1 production by THP-1 cells
Immunomodulatory effects of Core were first screened in THP-1 cells. Supernatants of Ad-infected cells were first analysed by using Human Cytokine Array III membranes (RayBiotech), then by using Luminex Bead Array (LBA) technology, allowing qualitative or quantitative screening of 42 (Fig. 2a) and 17 soluble factors, respectively. As demonstrated by others (Fernandez et al., 2002), RANTES is produced at high levels by THP-1 cells infected with Ad vectors. This production, however, was not modulated by expression of HCV Core (Fig. 2a). Secretion of only three chemokines, namely IL-8 (CXCL-8), MCP-1 (CCL-2) and MIP-1 (CCL-4), was upregulated in cells infected with Ad constructs expressing Core (Fig. 2a, b). Surprisingly, neither IL-1 nor tumour necrosis factor alpha (TNF-
), known to be IL-8 inducers, was induced (Fig. 2a, b) (Chaly et al., 2000).
Construction of an Ad construct encoding Core and depleted of ARFP expression
As we have shown (Fig. 1) that an ARFP can be expressed naturally from Core-coding sequences, in order to clarify the relative contribution of Core and ARFPs to chemokine induction, the Core-coding sequence was modified so that wt Core protein is expressed, but expression of any protein resulting from frameshifting or internal initiation is precluded (referred to as CARFP; Fig. 3a).
To verify that the CARFP sequence functions as expected, plasmids containing the GFP sequence inserted downstream of wt Core or CARFP gene in either the 0 or +1 frame [Core-GFP(0), Core-GFP(+1), CARFP-GFP(0) or CARFP-GFP(+1)] were constructed and co-transfected with pCMV DsRed plasmid into Huh7 cells. Transfection efficiency, as assessed by flow cytometry (Fig. 3b,) was equivalent between cells transfected with the different constructs, with >95 % of cells expressing DsRed. As expected, GFP was expressed when cloned in frame 0 of wt Core (32 % GFP+ cells) and CARFP (29 % GFP+ cells) (Fig. 3b). When cells were transfected with Core-GFP(+1) plasmid, 1.84 % of cells stained positive for GFP, confirming that frame +1 of the Core coding sequence is an ARF. In contrast, when the GFP sequence was inserted in frame +1 of the CARFP sequence, no GFP+ cells were detected, thus demonstrating that this CARFP sequence cannot lead to the expression of an ARFP.
This sequence was inserted in a recombinant Ad construct and its infectivity was investigated in the THP-1 cell line. As expected, infectivity of AdCARFP, as assessed by quantifying cells expressing the Ad hexon protein, is equivalent to that of AdCE1E2 or AdCore on the THP-1 cell line (Fig. 4a). Patterns of Core expression following infection with AdCore, AdCE1E2 or AdCARFP were analysed by WB (Fig. 4b) and immunofluorescence (Fig. 4c). A specific band at 20 kDa, revealed with an anti-Core antibody, is present in cells infected with AdCARFP, similarly to cells infected with AdCore or AdCE1E2 (Fig. 4b). Levels of Core expressed in cells infected with AdCARFP, as estimated by WB, were equivalent to those expressed by cells infected with AdCore, but lower than those expressed by cells infected with AdCE1E2. This difference is not due to variations in stability of Core transcripts, as demonstrated by semiquantitative RT-PCR (Fig. 4d), but may rather involve stabilization of Core by E1 and E2 proteins, as proposed previously (Himoudi et al., 2002). Furthermore, Core expressed from the CARFP sequence shows ring-like and dot structures similar to those of Core expressed from AdCE1E2 or AdCore (Fig. 4c).
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ARFP sequence does allow expression of a Core protein displaying characteristics equivalent to those of Core expressed from unmodified, wt Core sequences, whilst ARFP expression is abolished.
Triggering of chemokine production decreases when Core is expressed in the absence of ARFP in THP-1 cells
As previously observed by using LBA (Fig. 2b), THP-1 cells infected with AdCore and AdCE1E2 release higher levels of IL-8, MCP-1 and MIP-1 compared with uninfected cells or cells infected with AdGal as measured by ELISA (Fig. 5). IL-8 production by THP-1 cells infected with AdCARFP did not differ significantly from levels detected in cells infected with AdCore or AdCE1E2. Furthermore, levels of IL-8 were significantly higher in cells infected with AdCARFP than in cells infected with AdGal (Fig. 5). Conversely, in cells infected with AdCARFP, secretion of MCP-1 and MIP-1 was not triggered, reaching levels equivalent to those produced by either uninfected or AdGal-infected cells.
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by cells infected by AdCore or AdCE1E2 seems to require expression of ARFPs, Core expression may be sufficient to induce IL-8 secretion by THP-1 cells.
Effect of Core-encoding Ad constructs on monocyte-derived DCs and M
In order to confirm these results on primary cells, human blood monocytes were differentiated into either DCs or M by incubation in the presence of GM-CSF and respectively IL-4 or M-CSF (Fig. 6a). Effect of the different Ad constructs was evaluated based on the phenotype as well as the pattern of soluble factors secreted by iDCs and M.
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. were infected with the different Ad constructs for 48 h and expression of Core was analysed by WB. As expected, a specific band at 21 kDa is revealed with an anti-Core antibody in lysate of both cell types infected with AdCE1E2, AdCore and AdCARFP (Fig. 6b). In order to investigate whether expression of HCV Core/ARFPs or HCV Core alone leads to a modulation of morphological or phenotypic characteristics of DCs and M, we analysed the expression of various cell-surface markers expressed by iDCs and M infected or not by the different Ad constructs. Monocytes, cultured in the presence of GM-CSF and IL-4 or M-CSF, differentiated efficiently into respectively iDCs, as characterized by the dendrite-forming cells, and M, displaying characteristic fried-egg morphology (Fig. 6a). Upregulation of CD1a and downregulation of CD14 expression confirmed differentiation of monocytes into iDCs, as illustrated by low levels of costimulatory molecules (CD80 and CD86) as well as the absence of CD83 expression. M, differentiated from monocytes in the presence of M-CSF, had conserved CD14 expression (Fig. 6c, d). Neither infection with Ad nor expression of HCV antigens induced morphological or phenotypic changes of iDCs or M (Fig. 6c, d). Overall, infection of M and DCs by HCV-expressing Ad vectors did not induce morphological or phenotypic maturation of these cells.
Core coding sequences upregulate secretion of pro-inflammatory cytokines/chemokines by both iDCs and M.
In parallel with phenotype, supernatants from iDCs and M infected with the different Ad constructs were first screened for soluble factor content by use of the Bio-Plex Human Cytokine 17-Plex panel. No production of IL-1, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12p70, IL-13, IL-17, GM-CSF, gamma interferon (IFN-
), TNF- or granulocyte colony-stimulating factor (G-CSF) was detected in supernatant of cells infected or not with the different Ad constructs. In addition to soluble factors retrieved from THP-1 cells, M, but not iDCs, released significant amounts of IL-6 at baseline. Although Ad infection by itself slightly increased production of IL-8, MCP-1 and MIP-1 and induced IL-6 secretion by iDCs, levels of these four cytokines/chemokines were enhanced significantly in iDCs infected with AdCore and AdCE1E2 (Fig. 7a, b). Such an effect was also shown on M for IL-6 and MIP-1 secretion, whilst amounts of MCP-1 and IL-8 did not change upon infection with AdCore and AdCE1E2 (Fig. 7c, d).
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infected with AdCARFP, levels of MCP-1 and MIP-1 were comparable to levels determined in cells infected with AdGal. Similar effects on both iDCs and M are shown for IL-6 secretion. However, in contrast with data from THP-1 cells, infection of iDCs with AdCARFP did not upregulate IL-8 secretion in comparison with infection with AdGal, and amounts of IL-8 were significantly lower than those produced after infection with AdCore or AdCE1E2 (Fig. 7a, b).
Altogether, these results indicate that modulation of the soluble factor profile by Core coding sequences in iDCs and M is dependent on ARFP and not Core protein expression.
Expression of Core in the absence of ARFP does not trans-activate the IL-8 promoter in the HCV-susceptible Huh7 hepatoma cell line
Huh7 cells, described to support HCV replication (Wakita et al., 2005), were next infected with the different Ad constructs and IL-8 secretion was quantified. Although basal levels of IL-8 were 1–3 logs higher in Huh7 cells than in M, iDCs and THP-1 cells, induction of IL-8 expression was nevertheless observed in Huh7 cells after infection with AdCore or AdCE1E2 (Fig. 8a). As previously observed in iDCs and, less dramatically, in THP-1 cells, expression of Core in the absence of ARFP in Huh7 cells did not trigger IL-8 secretion, in comparison with Huh7 cells infected with AdGal (Fig. 8a). Production of MCP-1 and MIP-1 is typically not observed with Huh7 cells (data not shown).
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; Moorman et al., 2005; M. Fiorucci, unpublished observations), we investigated the implication of these two kinases in the production of IL-8 by Huh7 cells infected with a Core-encoding Ad. As shown in Fig. 8(b), the pattern of phosphorylation of p38/p42–44MAPK was not modified in cells infected with the Core-encoding Ad, suggesting that these two kinases are not involved in the IL-8 production pathway in Huh7 cells expressing endogenous Core. We next evaluated the effect of endogenous expression of HCV proteins on the IL-8 gene promoter. Huh7 cells were co-transfected with plasmids encoding Core, CE1E2 or CARFP together with a plasmid encoding firefly luciferase under the control of the IL-8 promoter. A third plasmid encoding GFP was used to standardize transfection efficiency. As illustrated in Fig. 8(c), the IL-8 promoter was transactivated by Core and CE1E2 constructs, as described previously (Hoshida et al., 2005). In contrast, whilst the transfection efficiency of Huh7 cells and expression levels of Core were equivalent between Core, CE1E2 and CARFP constructs (data not shown), trans-activation of the IL-8 promoter by CARFP was significantly lower than trans-activation by Core and CE1E2 (P < 0.05) and did not differ from control (P > 0.05). Thus Core, when expressed in the absence of ARFP, is not able to trans-activate the IL-8 promoter (Fig. 8c) or, consequently, to trigger IL-8 production by Huh7 cells (Fig. 8a). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Ray et al., 1996a, b). The recent discovery of a novel HCV protein, issued from an ARF overlapping the Core coding sequence, certainly challenges various activities so far attributed to Core. We show, for the first time, that an ARFP is expressed in mammalian cells transfected with a prototype genotype 1b Core coding sequence. Although this protein is probably very unstable and/or expressed at very low levels, our data clearly indicate that: (i) various cell types that have been shown to support HCV infection and/or replication, including the human hepatoma Huh7 cell line and the THP-1 monocytic cell line, as well monocyte-derived primary DCs and M, secrete pro-inflammatory cytokines and chemokines when infected with Ad constructs encoding wt Core-coding sequences; (ii) this secretion probably involves ARFP expression, as infection with an Ad encoding a Core protein modified so that wt Core, but no ARFP, is expressed is not capable of triggering secretion of such factors; (iii) in Huh7 cells, IL-8 induction by wt Core coding sequences involves trans-activation of the IL-8 promoter and requires expression of ARFP.
Expression of ARFPs from the Core coding sequence has been demonstrated so far by use of reporter genes inserted in the +1 frame of truncated Core coding sequences. By using a mAb generated from a genotype 1a ARFP (Komurian-Pradel et al., 2004), we were able to specifically detect a protein of 17 kDa in cells transfected with a genotype 1b Core coding sequence. This protein probably corresponds to the chimeric protein potentially ending at position 144 proposed by Boulant et al. (2003). This protein could only be visualized if tagged at its N terminus and purified on nickel beads. Indeed, His–ARFP, in contrast to His–Core, could not be detected by WB without purification on nickel beads (data not shown). These results, together with the fact that the whole content of His–ARFP is recovered in the first elution step, indicate that levels of expression of His–ARFP remain quite low compared with those of His–Core.
Immunomodulatory effects of Core coding sequences were evaluated in different cell types known either to support HCV replication in vitro, such as the Huh7 human hepatoma cell line (Wakita et al., 2005), or to constitute extrahepatic targets of HCV infection in vivo, such as monocyte-derived DCs and M or the THP-1 monocytic cell line as a surrogate of primary monocytes (Bain et al., 2001; Ducoulombier et al., 2004; Goutagny et al., 2003; Radkowski et al., 2004). An overall very limited pattern of soluble factors was found to be upregulated by Core coding sequences. In THP-1 cells, infection with AdCore or AdCE1E2 led to the secretion of chemokines such as MCP-1, MIP-1 and IL-8. As RANTES is typically produced at a high level by THP-1 cells infected with Ad (Fernandez et al., 2002), such secretion of RANTES may affect the profile of soluble factors induced by expression of HCV proteins. However, as discussed later in this section, both in vitro and in vivo data corroborate involvement of HCV Core, and probably ARFPs, in the induction of cytokines and chemokines such as IL-6, IL-8 and MCP-1. A similar pattern, although slightly different, was retrieved in primary cells derived from human blood monocytes. iDCs expressed IL-6 in addition to these chemokines, whilst a more restricted pattern, including only IL-6 and MIP-1, was upregulated by expression of Core coding sequences in M. Others have previously observed triggering of pro-inflammatory cytokines/chemokines by primary monocytes from chronically HCV-infected individuals (Dolganiuc et al., 2003), by primary M infected in vitro with HCV (Radkowski et al., 2004) or by iDCs infected with AdCore (Li et al., 2006). As we have shown that not only the Core protein, but also an ARFP, are expressed from wt Core coding sequences, in order to discriminate effects due to Core from those due to ARFP expression, we constructed an Ad vector containing a Core coding sequence modified so that Core, but no ARFP, is expressed. Production of cytokines/chemokines detected in cells infected with AdCore or AdCE1E2 was not recovered in any cell type infected with AdCARFP. At the level of IL-8 production, we were able to show that, whilst the IL-8 promoter was trans-activated efficiently in Huh7 cells infected by AdCore or AdCE1E2, as reported by Hoshida et al. (2005), expression of Core in the absence of ARFP was not able to trans-activate the IL-8 promoter or, consequently, lead to IL-8 secretion. Whether expression of ARFP alone or co-expression of ARFP with Core is responsible for the observed cytokine profiles remains to be established, but clearly expression of Core alone is not sufficient.
Although slightly different patterns of soluble factors were induced by Core coding sequences depending on the hepatic or monocytic origin of the cells, all are cytokines or chemokines involved in inflammation and fibrosis processes. Indeed, MCP-1 and IL-8 are profibrogenic chemokines that have been associated with advanced liver diseases of various aetiologies, including HCV infection (Marra et al., 1993). Recent data describe an association in HCV-infected individuals between a polymorphism within the promoter region of the MCP-1 gene, elevated intrahepatic expression of MCP-1 and more severe hepatic inflammation and fibrosis (Muhlbauer et al., 2003). Similarly, elevated levels of IL-8 in the serum and liver of chronically HCV-infected patients correlated with hepatic inflammation, staging of fibrosis and non-response to antiviral treatment (al-Wabel et al., 1995; Mahmood et al., 2002). Serum levels of IL-6, also known as hepatic stimulating factor, as well as its soluble receptors, are correlated with liver function impairment, degree of liver fibrosis and evolution to hepatocellular carcinoma in patients with HCV-related chronic liver diseases (Migita et al., 2006; Soresi et al., 2006). Altogether, in spite of low levels of expression and/or stability of ARFP, our data indicate that production of these pro-inflammatory/profibrogenic factors by hepatic as well as extrahepatic cells that may become infected by HCV during natural infection is dependent on the expression of this protein. Whilst Ad-based expression systems lead to production of non-physiological levels of HCV antigens in a non-physiological site (nucleus), such systems have been, and are still, widely used to uncover potential functions of viral proteins, in particular when relevant infection assays are either lacking or tedious to implement (Joo et al., 2005; Li et al., 2006). Although an in vitro HCV replication assay has recently been developed (Wakita et al., 2005), its implementation is yet far from being widely adopted. It would nonetheless be important to confirm our observations in such a system.
Whilst fibrogenic effects of exogenous Core protein on hepatic stellate cells and monocytes have been clearly demonstrated in vitro (Bataller et al., 2004; Dolganiuc et al., 2004), our results show that production of IL-6, IL-8, MCP-1 and MIP-1 by cells bearing Core coding sequences may involve expression not only of Core protein, but also of the novel ARFPs. Our data, indicating that endogenous expression of ARFPs is essential for the production of fibrogenic chemokines, could provide a link between independent sets of clinical data: the observations that (i) IL-6, IL-8 and MCP-1 are produced at elevated levels in advanced liver disease during HCV infection (al-Wabel et al., 1995; Bataller et al., 2004; Mahmood et al., 2002; Migita et al., 2006; Muhlbauer et al., 2003; Polyak et al., 2001a; Soresi et al., 2006); (ii) IL-8 levels in the sera of chronic patients are correlated inversely with response to IFN- therapy (Polyak et al., 2001b); and (iii) prevalence of anti-ARFP antibodies may be higher in HCV patients with advanced liver disease and cancer (Branch et al., 2005). These observations would suggest that fine interactions between Core and ARFP proteins may participate in determining disease outcome during HCV pathogenesis.
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ACKNOWLEDGEMENTS |
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ARFP adenovirus, and bioMérieux SA, Transgene SA and the ANRS (Agence Nationale de Recherche sur SIDA) for financial support and grant fellowship for M. F. |
REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 18 September 2006;
accepted 14 December 2006.
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