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A cell-based bicistronic lentiviral reporter system for identification of inhibitors of the hepatitis C virus internal ribosome entry site
Journal of Virological Methods 158 (2009) 152–159
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Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
A cell-based bicistronic lentiviral reporter system for identification of inhibitors of the hepatitis C virus internal ribosome entry site Sofia Lourenc¸o a,1 , Sébastien Boni a,1 , Denis Furling b , Franc¸ois-Loïc Cosset c , Annie Cahour a,∗ a
Laboratoire de Virologie, CERVI, Unité Propre de Recherche et d’Enseignement Supérieur de l’UPMC, Université Paris 6 EA 2387, IFR 113, Groupe Hospitalier Pitié-Salpêtrière, 83 Boulevard de l’Hôpital, 75651 Paris Cedex 13, France INSERM & UPMC, Université Paris 6, UMRS 787, Institut de Myologie, 105 Boulevard de l’Hôpital, 75634 Paris Cedex 13, France c IFR 128, Inserm U758, Ecole Normale supérieure de Lyon, 46 Allée d’Italie, 69007 Lyon, France b
a b s t r a c t Article history: Received 10 December 2008 Received in revised form 27 January 2009 Accepted 5 February 2009 Available online 14 February 2009 Keywords: SIN lentiviral vector SIV HCV IRES inhibitors Reporters Gene therapy
This report describes the development, optimization and implementation of a persistent cell-based system to test inhibitors of hepatitis C (HCV) translation. The assay is based on a heterologous human immunodeficiency virus-1/simian immunodeficiency virus (HIV-1/SIV) lentiviral vector expressing the bicistronic cassette containing the firefly and renilla luciferase genes, respectively, as reporters, and the HCV internal ribosome entry site (IRES) inserted in between, under the control of the cytomegalovirus (CMV) promoter. The drug target in this assay is the HCV IRES, the activity of which leads to modulation of the renilla luciferase gene expression under its control, which is monitored by luminometry. The system has been validated using interferon (IFN), which is still the only consensual antiviral agent against HCV infection, associated with ribavirin. This bicistronic vector, extended to other viral IRESs and assayed in different cell lines, exhibited weak cell tropism, allowing its broad use in gene therapy, which frequently needs a multicistronic transfer vector to follow the expression of a gene of interest inside the target cells with the aid of a reporter, a drug selection marker, or a suicide gene, expressed from the same transcript. © 2009 Elsevier B.V. All rights reserved.
1. Introduction There is a major health need to identify and develop novel efficient antiviral compounds against hepatitis C virus (HCV) infection (WHO, 2000). Three percent of the human population is infected and more than 75% of seropositive individuals develop a chronic infection, which can lead to severe liver disease, culminating in cirrhosis and occasionally hepatocellular carcinoma (Alter, 2007). Currently there is no HCV vaccine and approximately 40% of all infected patients do not respond to interferon/ribavirin combination therapy. At present, there are several antiviral strategies targeting the essential viral enzymes as NS3-NS4A serine protease and the NS5B RNA-dependent RNA polymerase (De Francesco and Migliaccio, 2005; Pawlotsky and Gish, 2006). More recently, since the development of a cell culture system for HCV (Moradpour et al., 2007; Wakita et al., 2005), the first steps of the viral life cycle (attachment and entry) have become of prime importance for the design of antiviral agents (Pawlotsky et al., 2007). However, the HCV 5 noncoding region (5 NCR), which is the most conserved
∗ Corresponding author. Tel.: +33 145 82 6298; fax: +33 145 82 6314. E-mail address: [email protected] (A. Cahour). 1 These authors contributed equally. 0166-0934/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2009.02.007
among the different genotypes, with the exception of the genotype 3 (Masante et al., 2008), and directs HCV RNA translation in a cap-independent manner via an internal ribosome entry site (IRES), still remains an attractive therapeutic target (Dasgupta et al., 2004; Jubin, 2001). Indeed, potent inhibitors would not affect translational machinery of the host cell, due to predominant cap-dependent translation mechanisms used by most of cellular mRNAs. In previous reports a bicistronic pIRF vector was designed to assess HCV IRES efficiency in different contexts (Laporte et al., 2000, 2003) and under the influence of trans-acting viral factors (Boni et al., 2005; Lourenc¸o et al., 2008). Briefly, this vector contains a bicistronic cassette composed of the luciferase firefly (FLuc) and renilla (RLuc) genes linked by the HCV IRES and driven either by the T7 or the CMV promoter. However, this system is restricted to transient studies. The goal of this work was to develop a persistent reporter system to test inhibitors of HCV IRES translation and to determine the duration of inhibition. To that end, the use of a simian immunodeficiency virus (SIV)-based lentiviral vector (Schnell et al., 2000) expressing the bicistronic cassette described above was preferred to the establishment of a system based on selection of a drug that might modify the cell environment. Lentiviral vectors appear relatively easy to produce in adequate quantities, unlike adenovirus-based vectors, and can achieve gene transfer and
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expression at satisfactory levels in both growth-arrested and differentiated target cells for a prolonged period. There is now a third generation of lentiviral vectors that are rendered nonpathogenic by self-inactivation (SIN) (Zufferey et al., 1998). The safety of the system used in that study is reinforced due to the fact that production of lentiviral particles was achieved by cotransfecting 293T cells with the SIV-transfer vector based on SIVmac251 (Mangeot et al., 2000) and heterologous helper constructs such as HIV-1 8.71 plasmid coding for the HIV-1 viral genes, and the vesicular stomatitis virus G envelope (VSV-G) for pseudotyping (Naldini et al., 1996a), thus avoiding any homologous recombinations, so the particles are incapable of independent propagation. The present study reports the optimization of this heterologous HIV-1/SIV lentiviral system in different types of cells, and its validation as a bicistronic reporter vector with IFN known to target the HCV 5 NCR (Hazari et al., 2005). Thereafter, this vector appeared as a promising tool for gene therapy which requires multigene expression of the same transcript to follow the expression of the transgene of interest.
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2.3. Production and titration of vector particles Reporter HIV-1/SIV-derived particles were generated upon cotransfection of 293T cells with the transfer vector, and 8.71 and HCMV-G constructs, using the transient calcium phosphate method as described previously (Naldini et al., 1996a). To that end, 6 × 106 293T cells per 100 cm2 tissue culture dish (Costar) were seeded 24 h prior to transfection. Viral supernatants were harvested 48 h post-transfection, cleared by low-speed centrifugation at 800 × g for 5 min, then treated with 1 M DNAse-1 (Roche) in the presence of 1 M magnesium chloride (Sigma) for 15 min at 37 ◦ C in order to eliminate any contaminating plasmid DNA. In a last step, the vector was concentrated by ultracentrifugation at 50,000 × g for 2 h at 4 ◦ C and the viral pellet resuspended in 70 l of cold phosphatebuffered saline (PBS), was aliquoted (15 l) in Eppendorf tubes and stored at −80 ◦ C until use. The titer of vector particles was normalized by measuring the p24 HIV-1 capsid protein concentration of the viral stock, using the Alliance HIV-1 p24 (capsid antigen) ELISA kit (PerkinElmer) according to the supplier. Titers obtained ranged from 80 to 100 ng p24 antigen (Ag)/l.
2. Materials and methods 2.4. Cell transduction with lentiviral vectors 2.1. Cells Human embryonic kidney 293T cells, human hepatoblastoma HepG2 and Huh7 cells, porcine kidney PK15 cells and murine embryonic NIH3T3 fibroblasts were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen), supplemented with 10% fetal calf serum (FCS) and 1% penicillin–streptomycin. Human primary myoblasts were originally isolated as previously described (Edom et al., 1994) from the quadriceps of a 5-day-old infant, in accordance with French legislation on ethics. Cells were grown in Ham’s F-10 medium (Invitrogen), supplemented with 20% FCS and 1% penicillin–streptomycin. For myoblast differentiation, growth medium was replaced by serum-free DMEM medium supplemented with 100 g/ml of transferrin and 10 g/ml insulin.
2.2. Plasmids and vectors All transfer vectors were produced with the SIV minimal lentiviral vector pGAE-0 previously described (Mangeot et al., 2000, 2002) and originally derived from SIVmac251. The strong ubiquitous internal CMV promoter is used to increase the expression level of the transgene. Different transfer vectors were designed, comprising the following reporter genes amplified with appropriate primers containing restriction sites used for cloning: the EGFP gene inserted at the AgeI/XhoI sites of the multicloning sequence, giving pGAE–EGFP, the LacZ gene inserted at the AgeI/XhoI sites, giving pGAE–LacZ, and the luciferase genes linked by the HCV IRES, constituting the bicistronic cassette, inserted in two steps; FLuc gene was first inserted at the AgeI/HpaI sites of the vector, followed by the sequence HCV IRES-RLuc at the HpaI/XhoI sites, giving pGAE–FHR. Plasmids pGAE–FCR and pGAE–FER were obtained in the same way, except that CSFV and EMCV IRESs, respectively, were inserted first in BamHI/PstI sites in pIRLuc construct in place of the HCV IRES, and second the IRES-RLuc fragment was inserted in pGAE–FLuc at HpaI/XhoI sites. The packaging HIV 8.71 vector (a gift from Dr. Naldini) expresses the complete sets of structural and accessory viral proteins with the exception of env and vpu, and is devoid of the packaging sequence (Naldini et al., 1996b). The plasmid HCMV-G (kindly given by Dr. Yee) expresses the vesicular stomatitis virus G (VSV-G) envelope protein allowing efficient pseudotyping of lentiviral particles and broad cell tropism (Yee et al., 1994).
A total of 2 × 105 cells were used for transduction and seeded onto 12-well plates (Costar) the day before. Transductions were carried out in 800 l total volume of serum-free medium to which viral stocks were added at various concentrations (0–400 ng p24 antigen per well) and 6 g/ml Polybrene (Sigma). Then, after gentle agitation for 2 h at 37 ◦ C, cells were incubated until 48–72 h with fresh and complete medium. Transduction efficiencies were calculated using the percentage of EGFP-positive cells analyzed by flow cytometry, or by luminometry for cells expressing -galactosidase, both of these methods being described below. Whole-cell population was used rather than selected clones in all of our experiments. 2.5. Semi-quantitative RT-PCR The total RNA was isolated from the Huh7 cells treated with different doses of IFN 16 h after transduction using Trizol (Invitrogen) and its concentration in 20 l-sample of nuclease-free water was measured spectrophotometrically. An equal quantity of each RNA was included as template in each of the semiquantitative one-step RT-PCRs using the Access kit (Promega) and the following primer pairs—FLuc: 5 -CCCTGGAAGATGGAAGCGTT3 and 5 -TTTGCAGCCTACCGTAGTGT-3 ; RLuc: 5 -GCCTCCTGGATCACTACAAGTA-3 and 5 -AAAAGAACCCAGGGTCGGACTC-3 ; and glyceraldehyde-phosphatase dehydrogenase (GAPDH) as an internal quantitative control: 5 -AGAACATCATCCCTGCCTCTACTG-3 and 5 -CATGTGGGCCATGAGGTCCA-3 . FLuc and RLuc fragments were amplified in the same reaction as follows: one cycle of reverse transcription at 50 ◦ C for 30 min and 20 cycles of amplification at 94 ◦ C for 15 s, 50 ◦ C for 30 s, and 72 ◦ C for 1 min. The PCR products were subjected to electrophoresis on 2% agarose gel. The intensity of FLuc and RLuc bands was quantified using Image J software, and converted into arbitrary units and set to 100% in absence of treatment. 2.6. FACS analysis 48 h post-transduction, the samples were washed twice with PBS, fixed with a PBS 4% paraformaldehyde solution for 15 min at 4 ◦ C, and subjected to flow cytometry using a FACScan analyzer (Becton-Dickinson, BD Biosciences). EGFP fluorescence was detected following excitation with an argon ion laser source at 488 nm. Percentage of EGFP-positive cells was determined in comparison with the negative control of non-infected cells using Cell
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Quest computer software. For each sample, 5000 events were collected. 2.7. Reporter microscopic analysis Natural green fluorescence of the EGFP was examined at the indicated time points under a fluorescence microscope (Olympus) at 484 nm. Adherent cells were washed twice with PBS and fixed with PBS 4% paraformaldehyde as described above. Hoechst nucleus blue staining was then performed by incubating fixed cells for 10 min in the presence of a 0.5 g/ml solution of Hoechst 33258 (Sigma) in PBS, and observed at 340 nm. 2.8. Luminometric analyses Luciferase activities were assayed on cell lysates using the dual luciferase reporter assay system (Promega). Briefly, infected cells plated onto 24-well plates were washed twice with PBS after discarding the supernatant, and lysed in a suitable volume of lysis buffer with gentle agitation for 15 min at room temperature. Then, FLuc and RLuc activities were measured sequentially according to the supplier’s protocol. Errors were calculated as the standard deviation of three calculated activities for each point per experiment repeated twice, and expressed as percentages of the average activity. -Galactosidase expression was also determined by luminometry using the -Glo assay system (Promega) according to the supplier’s protocol. The values were given as for luciferase activities in relative light units (RLU).
3. Results 3.1. Efficient transduction of various cell lines and primary myoblasts Reporter lentiviral vectors described in Fig. 1 were produced with satisfactory titers after transfection of 293T cells, and induced expression of the corresponding reporter. Usually, supernatants collected at 48 h post-transfection produced better transduction efficiencies than those collected at 72 h. The resulting supernatants, concentrated by ultracentrifugation, titrated at approximately 100 ng/l of p24 antigen. Transduction efficiency was assessed first with the pGAE–EGFP particles both in hepatoma cell lines (HepG2 and Huh7) and human primary myoblasts. Flow cytometry analysis showed a positive correlation between the percentage of EGFPpositive cells and the increasing quantity of viral particles (Fig. 2A). A plateau was reached for a vector particle dose of 200 ng of p24 or higher, with approximately 90% of EGFP-expressing cells (i.e. 90% for myoblasts, 92.6% for HepG2 and 88.5% for Huh7). Similar results were obtained also with the murine NIH3T3 and the porcine PK15 cell lines (94% and 90%, respectively; data not shown). Transduction efficiency is similar in the three cell types, except that the myoblasts appeared less transducible at low vector amounts (i.e. 0–50 ng p24 Ag). Thereafter, transduction efficiencies were defined also for the vector expressing the -galactosidase (pGAE–LacZ). As shown in Fig. 2B, a plateau of -galactosidase activity was reached between 200 and 400 ng p24 Ag of the corresponding vector, reflecting the maximal transduction efficiency observed with the EGFP vector.
Fig. 1. Schematic representation of SIV-derived SIN vector and helper constructs. (A) SIV transfer vector derived from pGAE-0 is represented in the upper panel. Cis genetic elements are symbolized with shadowed boxes, whereas promoters are depicted in white boxes. pCMV, early cytomegalovirus promoter; RU5, LTR; U3, partially deleted 3 LTR; E/DLS, 5 encapsidation and dimerization sequence; gag, 5 gag region; cPPT, central polypurine tract; CTS, central termination sequence; RRE, rev-responsive element; SA/ESE, splicing acceptor/splicing donor. Transgenes used in this study are depicted in the lower panel: EGFP and LacZ, the genes encoding the enhanced green fluorescent protein and the -galactosidase respectively, giving the following corresponding monocistronic lentiviral vectors pGAE–EGFP and pGAE–LacZ. FLuc (firefly luciferase) and RLuc (renilla luciferase) represent the two reporter genes of the following bicistronic vectors: pGAE–FHR, with the HCV IRES, pGAE–FER, with the EMCV IRES, pGAE–FCR, with the CSFV IRES. (B) 8.71 packaging construct expressing the accessory proteins gag, pol, tat and rev, with the exception of env and vpu, and lacking the packaging sequence . (C) pHCMV-G plasmid encoding the VSV-G glycoprotein for pseudotyping the lentiviral particles.
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Fig. 2. Transduction efficiency of different cell types. Dose–response transduction experiments with pGAE–EGFP (A) and pGAE–LacZ (B) vectors were performed, using heterologous HIV-1/SIV lentiviral system, in hepatoma cell lines Huh7 (open bars) and HepG2 (shaded bars), and in human primary myoblasts (solid bars) as described in Section 2, in 12-well plates with increasing amounts of vector particles (0–400 ng p24 Ag) as indicated. Percentage cells expressing EGFP was determined by flow cytometry (A) and luminometric activity of cells expressing -galactosidase was measured by the aid of -Glo kit after their lysis according to the supplier’s protocol. Each column represents the average transduction efficiency from three calculated activities for each point per experiment repeated twice, and error bars indicate the standard deviations.
3.2. Efficient transduction of cells using a bicistronic lentiviral cassette Taking into account the performances obtained with a single transgene, and faced with the frequent need to use multigene
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transfer vectors to follow the transgene of interest in the target cell, the system was extended to a bicistronic construct containing two reporter genes separated with the HCV IRES. The SIV-derived lentiviral pGAE–FHR vector containing the bicistronic cassette FLuc-HCV IRES-RLuc, described previously (Laporte et al., 2000), inserted at the AgeI/XhoI restriction sites of the pGAE-0 vector, was developed and produced as described above. As depicted in Fig. 3, luminometric activities were measured at different vector doses and relative HCV efficiency was expressed through the ratio RLuc/FLuc (R/F). Since FLuc is translated in a cap-dependent fashion, it therefore represents the control of transduction efficiency. For each vector amount in hepatoma and in PK15 cells, FLuc has a higher activity than RLuc, in contrast to NIH3T3 cells where the RLuc activity is the highest. For all cell lines, the ratio of R/F is relatively constant according to the vector amount, validating a stable expression of the transgenes. The R/F ratios are around 0.40 and 0.32 for HepG2 and Huh7, respectively, and slightly lower in the PK15 cell line (0.21), which is not susceptible to HCV infection. Unexpectedly, the murine NIH3T3 cells that were supposedly not permissive to HCV infection showed a very high ratio (2.78). To investigate a putative cellular tropism determined by the IRES studied, two new constructs were designed. They were both derived from the pGAE-0 transfer vector, with insertion of the bicistronic cassette; however, the HCV IRES sequence was replaced by either the CSFV IRES, giving pGAE–FCR, or the EMCV IRES, giving pGAE–FER. The four cell lines and the primary myoblasts were transduced with the three different bicistronic lentiviral vectors and the luminometric activities of FLuc and RLuc were determined. As observed with HCV IRES, FLuc activity was higher in human or porcine cells than in murine cells (data not shown). The R/F ratios reported in Table 1 indicate that the activity of CSFV IRES is comparable to that of HCV IRES, except for the porcine cell line PK15 (supposed to be the preferred host), where it is more efficient. The EMCV IRES exhibited an increased ratio in human hepatoma cell lines when compared with HCV or CSFV IRES, and its highest efficiency was found in the human myoblasts and not in the murine cells. Surprisingly, the HCV IRES activity is low in hepatoma cells and
Fig. 3. Transduction efficiency of different cell lines, hepatoma cells Huh7 and HepG2, and NIH3T3 and PK15 cells in 24-well plates as described in Section 2, using bicistronic lentiviral transfer vector containing the HCV IRES element, pGAE–FHR. Luciferase activities at increasing vector doses (from 0 to 200 ng p24 Ag) as indicated, were measured by luminometry and expressed in relative light units (RLU). FLuc activity (cap-dependent) (solid bars); RLuc activity (HCV IRES-dependent) (open bars). Relative HCV IRES efficiency, estimated from the RLuc/FLuc ratio, was represented by the black line and appeared constant, independently of the vector amount. Each column or point represents the average transduction efficiency from three calculated activities for each point per experiment repeated twice, and error bars indicate the standard deviations.
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Table 1 Relative efficiencies of different IRESs in various cell types. Cells
IRESa HCV
Huh7 HepG2 NIH3T3 PK15 Myoblasts
0.32 0.40 2.78 0.21 0.52
CSFV ± ± ± ± ±
0.02b 0.03 0.04 0.01 0.04
0.15 0.41 1.84 2.02 0.60
EMCV ± ± ± ± ±
0.01 0.03 0.05 0.04 0.03
0.87 1 1.30 1.66 2.10
± ± ± ± ±
0.01 0.1 0.05 0.02 0.05
a Different IRESs tested correspond to bicistronic lentiviral vector as follows: HCV, pGAE–FHR; CSFV, pGAE–FCR; EMCV, pGAE–FER. b Relative IRES efficiencies estimated by the ratio RLuc/FLuc. Each value represents the average IRES activity from tirlicate translation samples, followed by the standard deviation (±).
high in murine cells when compared with the other tested IRESs, even if it is a known human liver host, indicating the important role played by the cell factors on the activity of high-structured IRES elements (Rijnbrand and Lemon, 2000) (see Section 4). 3.3. Transgene expression is stable over a long period To test the stability and the duration of transgene expression, infected cells were grown and maintained in culture for a long period. As shown in Fig. 4, a persistent and stable expression of FLuc and RLuc was measured in HepG2 (shaded bars) and Huh7 (open bars) cells up to 12 weeks after transduction, as represented by the R/F ratio. Similar results were observed by flow cytome-
Fig. 4. Lentivirally transduced cells maintain transgene expression over time. Myoblasts (solid bars) transduced with pGAE–EGFP expressing EGFP were counted by flow cytometry at different time points as described in Section 2, and indicated a constant percentage of positive cells up to 4 weeks. Hepatoma cells (HepG2: shaded bars; Huh7, open bars) transduced with pGAE–FHR, containing the HCV IRES, revealed that the relative HCV IRES activity, estimated through the RLuc/FLuc ratio, did not vary over time until 12 weeks. Each column represents the average transduction efficiency from three calculated activities for each point per experiment repeated twice, and error bars indicate the standard deviations.
try using the EGFP construct as well as in the NIH3T3 cells (data not shown). Human primary myoblasts (solid bars) also exhibited stable expression of EGFP during their proliferative life span, at least for 4 weeks after transduction, until they reached the replicative senescence stage that limits the proliferative capacity of human somatic cells. In addition, less than 1% of transduced cells treated with the non-nucleoside HIV reverse-transcriptase inhibitor nevirapine (2 М) (Roche, Meylan) expressed EGFP 48 h post-transduction (data not shown). This result, together with stable expression described above, rules out the possibility of passive transduction of EGFP as described previously (Liu et al., 1996; Nash and Lever, 2004). In addition, no toxicity induced by the vector in either infected cells was observed, even at the highest doses tested (2000 ng of p24 Ag/well in a 24-well plate) (not shown). 3.4. Assay of HCV IRES inhibition The stability of luciferase expression gave us the possibility to follow the HCV IRES activity for a long period and investigate whether this bicistronic lentiviral vector was suitable for setting up a cell-based assay to test HCV IRES inhibitors for a long term follow-up, compared to transient transfection. IFN ␣-2a (Roferon, Roche) and IFN -1a (Avonex, Biogen. Idec) were selected since they inhibit HCV replication by targeting the HCV IRES (Dash et al., 2005), this being valid for the six genotypes (Hazari et al., 2005). Experiments were carried out in Huh7, HepG2 and NIH3T3 cells 12 weeks post-transduction with the pGAE–FHR vector. Cells were seeded in 24-well plates and incubated with increasing concentrations of either IFN ␣-2a or -1a. Concentrations ranging from 1 to 1000 IU/ml were lower than the physiological concentrations. After 16 h, cells were lysed in reporter lysis buffer and the luciferase activities were measured and plotted against IFN concentrations (Fig. 5). Both IFNs ␣ and  induced a dramatic decrease of RLuc activity (open bars) in hepatoma cells Huh7 and HepG2 in a dosedependent manner. Thus, the HCV IRES efficiency was decreased by 50% at 100 IU/ml of IFN and reached almost 85% inhibition at 1000 IU/ml. However, this activity was not inhibited at any dose in NIH3T3 cells, indicating the importance of the cell line used to test HCV IRES inhibitors, probably due to host factors. However, each IFN dramatically inhibited FLuc activity expressed in a capdependent way, in all cell lines tested and more efficiently than RLuc IRES-dependent. We then examined whether IFN treatment could have degraded FLuc or RLuc mRNAs which could explain differences observed between their activities and regarding the cell line used. To that aim, semi-quantitative RT-PCR amplifications were performed on total RNA extracted from transduced Huh7 cells under increasing doses of IFN ␣-2a, as described in Section 2. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was similarly examined for comparison. PCR products were analyzed by agarose gel electrophoresis and quantitated using IMAGE J software (Fig. 6). All RNAs reporters did not decay under IFN pressure, regardless of their cap or IRES status. Same results were obtained in another experiment using IFN -1a and NIH3T3 cells (data not shown), indicating that IFN ␣ or  preferentially blocks protein translation without altering the stability of corresponding mRNA. Taken together, these data validate the bicistronic lentiviral vector as a tool available for assessing a potent inhibitory effect of a compound on the HCV IRES. 3.5. Potential for gene therapy of the bicistronic vector Since the expression of the EGFP and the FLuc and RLuc transgenes is stable and the tropism exhibited by the HCV IRES is weak, our bicistronic system might be envisaged as a useful tool for multigene vector strategies in gene therapy. It is feasible to replace one of the two reporters by a gene of interest and to follow its expression
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Fig. 5. Effects of interferons ␣-2a et -1a on the expression of luciferases from the dicistronic lentiviral vector. Huh7 and HepG2 hepatoma cells and NIH3T3 cells transduced with pGAE–FHR for 12 weeks, were treated with increasing concentrations of each IFN (0–1000 UI/ml). Luciferase activities, expressed in RLU, were plotted as mean percentage of the untreated controls. FLuc (solid bars), cap-dependent translation; RLuc (open bars), IRES-dependent translation. Each column represents the average HCV IRES efficiency from three treated samples for each point per experiment repeated twice, and error bars indicate the standard deviations.
over time through expression of luciferase or another reporter such as EGFP. The gene of interest should be inserted upstream of the IRES, the second gene being less expressed in a bicistronic lentiviral vector (Dupuy et al., 2005; Mizuguchi et al., 2000). However, the results obtained with the murine NIH3T3 cell line indicate that, in some cells or species, insertion of the gene of interest downstream of the IRES could be more efficient. Taking advantage of the in vitro differentiation capacity of the myoblasts, we investigated the capacity of the construct to sustain expression of the transgene in post-mitotic cells. Prolif-
erating human myoblasts were transduced with pGAE–EGFP at a vector particle dose corresponding to 200 ng p24 Ag, and mononucleated transduced myoblasts expressing EGFP were amplified (Fig. 7A). Then, subconfluent cultures were switched to a differentiation medium for 6 days. Under this permissive condition, the myoblasts will differentiate, fuse and form multinucleated myotubes. As shown in Fig. 7B, the post-mitotic myotubes containing several nuclei still expressed EGFP, indicating that expression of the transgene is maintained during myogenic differentiation and in post-mitotic cells. 4. Discussion
Fig. 6. Graphical representation of the translational inhibition of both capdependent (FLuc) and IRES-dependent (RLuc) luciferases, and the stability of corresponding mRNAs after treatment of Huh7 cells with different concentrations of IFN ␣-2a (100–1000 UI/ml). The values in the graph are normalized according to the untreated control and expressed as a percentage of it. The mRNA quantitation was made by measuring the band intensity on the gel using IMAGE J software, and the luciferases expression was monitored by luminometry as described in Fig. 5.
Because of the cytotoxic nature of vaccinia virus, and possible modifications in a cellular environment under continuous antibiotic selection, our aim was to develop a persistent cell-based reporter system for characterization and long-term evaluation of HCV IRES inhibitors. For this, the initial bicistronic reporter vector pIRF limited to transient expression was adapted to a lentiviral transfer vector, resulting in stable expression of the bicistronic cassette due to its integration into the host cell genome. A heterologous lentiviral system was used, based on crosspackaging between elements from different but related viruses present on distinct plasmids (i) human HIV-1 packaging construct and (ii) SIV-gene transfer vector. This vector (pGAE-0) represents a minimal genome, devoid of viral open reading frames but bearing the cis-elements required for viral infectivity, and deleted for a large part of the 3 long terminal repeat (LTR) U3 domain containing sites essential for SIV transcription, rendering it self-inactivated (SIN). Such a secure vector system could be envisioned to be useful as well
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Fig. 7. Sustained transgene expression during differentiation of transduced human primary myoblasts into myotubes. Subconfluent myoblasts transduced with pGAE–EGFP vector particles corresponding to 200 ng p24 Ag, seeded in a 12-well plate, were induced to differentiate into myotubes by changing culture medium to DMEM as described in Section 2. Six days later, infected cells were observed in fluorescence microscopy with appropriate filters. (A) Myoblasts without induction of differentiation. Nuclei are stained in blue with Hoechst and the cytoplasm appears fluorescent green, expressing the EGFP (B) myoblasts with differentiation. The arrow indicates a myotube with several blue nuclei, the cytoplasm still exhibiting green fluorescence (magnification 20×).
for testing inhibitors of HCV IRES over time, with the possibility of evaluating their pharmacokinetics, as for gene therapy. First, the system was optimized in different cell types with a unique reporter to confirm (i) its capacity to produce lentiviral particles at satisfactory titers, due to pseudotyping with the VSV-G glycoprotein, conferring on them a broad tropism and additional stability, and (ii) its efficient potential for gene transfer, using limited amounts of lentiviral vector, even in primary human myoblasts. Second, the same experimental conditions were extended to a bicistronic transfer vector containing the bicistronic cassette composed of the HCV IRES between the two luciferase genes, FLuc and RLuc, respectively, in order to evaluate the effects of candidate drugs on HCV IRES activity. The cell-based assay performed on transduced cells measured an inhibitory effect through the expression of RLuc gene, directly under the control of the HCV IRES. In a previous work, a promoter activity has been assigned to HCV IRES sequences in their DNA form (Dumas et al., 2003). Such a residual activity was demonstrated with the bicistronic transfer vector pGAE–FHR deleted of the CMV promoter (data not shown). However, this activity did not interfere with the inhibition test performed because (i) it is already present in the control without any antiviral reagent, and (ii) the expected response in such a test is an all or nothing. Otherwise, when subtle modulations of the HCV IRES are to be detected, IRES in RNA form is prefered to transfect cells (Boni et al., 2005; Lourenc¸o et al., 2008). The system was therefore validated with IFNs ␣-2a and -1a, IFN being the most efficient antiviral agent in HCV infection until now, at doses below those used during treatment (Chevaliez and Pawlotsky, 2007). Due to its sensitivity, rapidity and reproducibility, the assay can be formatted for high-throughput screening to identify HCV IRES inhibitors in libraries (in process). Furthermore, two other bicistronic transfer vectors were designed, differing by their IRES (pGAE–FCR with CSFV IRES or pGAE–FER, with EMCV IRES), to investigate a potent cellular tropism determined by the origin of the IRES considered. Surprisingly, no cell specificity was observed for any IRES tested, excepted for CSFV, which exhibited a preference for the porcine PK15 cell line. However, differences in IRES efficiency were observed with a given IRES in different cell types, as mentioned above and in previous studies (Laporte et al., 2000, 2003). In that work, the HCV IRES activity appeared higher in murine cells NIH3T3 than in its preferred host human hepatoma cells, and the CSFV IRES was the only one exhibiting an expected tropism in PK15 cells. These observations indicate the impact of cell trans-acting factors in the modulation of the IRES considered. For HCV IRES, some non-canonical factors also active on other viral IRESs, such as polypyrimidine tract-binding protein (PTB), La autoantigen and some ribonucleoproteins, have
been identified (Rijnbrand and Lemon, 2000), and more recently, the microRNA mi122 (Henke et al., 2008) and new ribonucleoproteins (Pacheto et al., 2008). However, concerning the PTB, its modulation of HCV IRES is still controversial, which may be due to its limited concentration in certain cell types (Robinson et al., 2008; Wang et al., 2008). In addition, the NIH3T3 cell line does not appear convenient for testing inhibitors of HCV IRES as its does not respond to IFN treatment. Thus, the choice of the cell line to be transduced depends on the final goal. In another clinical aspect, the bicistronic lentiviral vector pGAE–FHR developed here exhibited all potentialities for gene therapy: stability and duration of transgene expression, availability in various cell types due to the presence of the ubiquitous CMV promoter, and as a lentiviral vector, in both dividing and non-dividing target cells (Naldini et al., 1996b). The presence of the HCV IRES allows any combination between the transgene of interest and another reporter or suicide gene to follow its expression within the target cell, as it is required frequently in many potential gene transfer applications (Chinnasamy et al., 2006; De Felipe, 2002; Ngoi et al., 2004; Reiser et al., 2000). One condition needed to design a multicistronic transfer vector, is the IRES(s) considered to be strong enough to achieve expression of the transgene(s) at high levels in target cells. Observation of (i) good transduction efficiency of human primary myoblasts, the myogenic precursor cells responsible for muscle regeneration, and (ii) sustained expression of the transgene in post-mitotic and multinucleated myotubes, encouraged us in preliminary investigations in the important field of autologous cellular therapy based on genetically modified stem cells (Davies and Grounds, 2007). Further work is under way, using EGFP as a surrogate for therapeutic protein, to improve efficient gene delivery and sustained expression of the transgene in recipients, taking into account numerous reports in that field (Cockrell and Kafri, 2007). Importantly, since compartmentalization has been described for the HCV IRES within the infected organism, aside from other regions of the genome (Di Liberto et al., 2006; Laporte et al., 2003; Roque-Afonso et al., 2005), this characteristics should be considered as beneficial for both applications mentioned above with our bicistronic system. Although highly conserved among most of genotypes, the HCV IRES sequence has been reported to contain tissue-specific point mutations, some of which might prove useful. First, they might allow identification of inhibitors specific to IRES variants resistant to inhibitors of the wild-type IRES sequence. Second, they might improve transfer of a transgene in gene therapy, thus helping target cells in connection with a tissue-specific promoter.
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In conclusion, a heterologous HIV-1/SIV bicistronic lentiviral system has been developed for evaluation of inhibitors of HCV IRES activity and for multigene therapy due to its capacity (i) to produce high titer viral particles and (ii) to stably express the transgene in target cells for a long period. Preliminary results are encouraging and offer an opportunity to improve the system described for rapid screening of potent HCV IRES antiviral compounds and for efficient gene delivery in myoblasts for gene therapy of muscular diseases. Acknowledgements The authors are grateful to Philippe Emmanuel Mangeot for providing pGAE-0 SIV-derived lentiviral vector and for his expertise in lentiviral production. We are indebted to Dr. Naldini for providing gag-pol and env 8.71 plasmid, and to Dr Yee for the gift of the pseudotyping pVSV-G plasmid. We also thank Catherine Blanc of the flow cytometry platform of the Pitié-Salpêtrière site for her contribution and advice in FACS analysis. This work was supported in part by the Agence Nationale pour la Recherche sur le Sida, and the Foundation for Science and Technology of Lisbon. Sofia Lourenc¸o is a recipient of doctoral grant from the Foundation for Science and Technology of Lisbon (SFRH/BD/16218/2004), and Sébastien Boni of doctoral grant from the Ministère de l’Education Nationale de la Recherche et de la Technologie. References Alter, M., 2007. Epidemiology of hepatitis C virus infection. World J. Gastroenterol. 13, 2436–2441. Boni, S., Lavergne, J.P., Boulant, S., Cahour, A., 2005. Hepatitis C virus core protein acts as a trans-modulating factor on internal translation initiation of the viral RNA. J. Biol. Chem. 280, 17737–17748. Chevaliez, S., Pawlotsky, J.M., 2007. Interferon-based therapy of hepatitis C. Adv. Drug Deliv. Rev. 59, 1222–1241. Chinnasamy, D., Milsom, M.D., Shaffer, J., Neuenfeldt, J., Shaaban, A.F., Margison, G.P., Fairbairn, L.J., Chinnasamy, N., 2006. Multicistronic lentiviral vectors containing the FMDV 2A cleavage factor demonstrate robust expression of encoded genes at limiting MOI. Virol. J. 3, 14. Cockrell, A.S., Kafri, T., 2007. Gene delivery by lentivirus vectors. Mol. Biotechnol. 36, 184–204. Dasgupta, A., Das, S., Izumi, R., Venkatesan, A., Barat, B., 2004. Targeting internal ribosome entry site (IRES)-mediated translation to block hepatitis C and other RNA viruses. FEMS Microbiol. Lett. 234, 189–199. Dash, S., Prabhu, R., Hazari, S., Bastian, F., Garry, R., Zou, W., Haque, S., Joshi, V., Regenstein, F.G., Thung, S.N., 2005. Interferons alpha, beta, gamma each inhibit hepatitis C virus replication at the level of internal ribosome entry site-mediated translation. Liver Int. 25, 580–594. Davies, K., Grounds, M., 2007. Modified patient stem cells as preclude to autologous treatment of muscular dystrophy. Cell Stem Cell. 1, 595–596. De Felipe, P., 2002. Polycistronic viral vectors. Curr. Gene Ther. 2, 355–378. De Francesco, R., Migliaccio, G., 2005. Challenges and successes in developing new therapies for hepatitis C. Nature 436, 953–960. Di Liberto, G., Roque-Afonso, A.M., Kara, R., Ducoulombier, D., Fallot, G., Samuel, D., Feray, C., 2006. Clinical and therapeutic implications of hepatitis C virus compartmentalization. Gastroenterology 131, 76–84. Dumas, E., Staedel, C., Colombat, M., Reigadas, S., Chabas, S., Astier-Gin, T., Cahour, A., Litvak, S., Ventura, M., 2003. A promoter activity is present in the DNA sequence corresponding to the hepatitis C virus 5 UTR. Nucleic Acids Res. 31, 1275–1281. Dupuy, F.P., Mouly, E., Mesel-Lemoine, M., Morel, C., Abriol, J., Cherai, M., Baillou, C., Negre, D., Cosset, F.L., Klatzmann, D., Lemoine, F.M., 2005. Lentiviral transduction of human hematopoietic cells by HIV-1- and SIV-based vectors containing a bicistronic cassette driven by various internal promoters. J. Gene Med. 7, 1158–1171. Edom, F., Mouly, V., Barbet, J., Fiszman, M., Butler-Browne, G., 1994. Clones of human satellite cells can express in vitro both fast and slow myosin heavy chains. Dev. Biol. 164, 219–229. Hazari, S., Patil, A., Joshi, V., Sullivan, D.E., Fermin, C.D., Garry, R.F., Elliott, R.M., Dash, S., 2005. Alpha interferon inhibits translation mediated by the internal ribosome entry site of six different hepatitis C virus genotypes. J. Gen. Virol. 86, 3047–3053. Henke, J., Goergen, D., Zheng, J., Song, Y., Scüttler, C., Fehr, C., Jünemann, C., Niepmann, M., 2008. microRNA-122 stimulates translation of hepatitis C virus RNA. EMBO J. 27, 3300–3310.
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Contents lists available at ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
A cell-based bicistronic lentiviral reporter system for identification of inhibitors of the hepatitis C virus internal ribosome entry site Sofia Lourenc¸o a,1 , Sébastien Boni a,1 , Denis Furling b , Franc¸ois-Loïc Cosset c , Annie Cahour a,∗ a
Laboratoire de Virologie, CERVI, Unité Propre de Recherche et d’Enseignement Supérieur de l’UPMC, Université Paris 6 EA 2387, IFR 113, Groupe Hospitalier Pitié-Salpêtrière, 83 Boulevard de l’Hôpital, 75651 Paris Cedex 13, France INSERM & UPMC, Université Paris 6, UMRS 787, Institut de Myologie, 105 Boulevard de l’Hôpital, 75634 Paris Cedex 13, France c IFR 128, Inserm U758, Ecole Normale supérieure de Lyon, 46 Allée d’Italie, 69007 Lyon, France b
a b s t r a c t Article history: Received 10 December 2008 Received in revised form 27 January 2009 Accepted 5 February 2009 Available online 14 February 2009 Keywords: SIN lentiviral vector SIV HCV IRES inhibitors Reporters Gene therapy
This report describes the development, optimization and implementation of a persistent cell-based system to test inhibitors of hepatitis C (HCV) translation. The assay is based on a heterologous human immunodeficiency virus-1/simian immunodeficiency virus (HIV-1/SIV) lentiviral vector expressing the bicistronic cassette containing the firefly and renilla luciferase genes, respectively, as reporters, and the HCV internal ribosome entry site (IRES) inserted in between, under the control of the cytomegalovirus (CMV) promoter. The drug target in this assay is the HCV IRES, the activity of which leads to modulation of the renilla luciferase gene expression under its control, which is monitored by luminometry. The system has been validated using interferon (IFN), which is still the only consensual antiviral agent against HCV infection, associated with ribavirin. This bicistronic vector, extended to other viral IRESs and assayed in different cell lines, exhibited weak cell tropism, allowing its broad use in gene therapy, which frequently needs a multicistronic transfer vector to follow the expression of a gene of interest inside the target cells with the aid of a reporter, a drug selection marker, or a suicide gene, expressed from the same transcript. © 2009 Elsevier B.V. All rights reserved.
1. Introduction There is a major health need to identify and develop novel efficient antiviral compounds against hepatitis C virus (HCV) infection (WHO, 2000). Three percent of the human population is infected and more than 75% of seropositive individuals develop a chronic infection, which can lead to severe liver disease, culminating in cirrhosis and occasionally hepatocellular carcinoma (Alter, 2007). Currently there is no HCV vaccine and approximately 40% of all infected patients do not respond to interferon/ribavirin combination therapy. At present, there are several antiviral strategies targeting the essential viral enzymes as NS3-NS4A serine protease and the NS5B RNA-dependent RNA polymerase (De Francesco and Migliaccio, 2005; Pawlotsky and Gish, 2006). More recently, since the development of a cell culture system for HCV (Moradpour et al., 2007; Wakita et al., 2005), the first steps of the viral life cycle (attachment and entry) have become of prime importance for the design of antiviral agents (Pawlotsky et al., 2007). However, the HCV 5 noncoding region (5 NCR), which is the most conserved
∗ Corresponding author. Tel.: +33 145 82 6298; fax: +33 145 82 6314. E-mail address: [email protected] (A. Cahour). 1 These authors contributed equally. 0166-0934/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2009.02.007
among the different genotypes, with the exception of the genotype 3 (Masante et al., 2008), and directs HCV RNA translation in a cap-independent manner via an internal ribosome entry site (IRES), still remains an attractive therapeutic target (Dasgupta et al., 2004; Jubin, 2001). Indeed, potent inhibitors would not affect translational machinery of the host cell, due to predominant cap-dependent translation mechanisms used by most of cellular mRNAs. In previous reports a bicistronic pIRF vector was designed to assess HCV IRES efficiency in different contexts (Laporte et al., 2000, 2003) and under the influence of trans-acting viral factors (Boni et al., 2005; Lourenc¸o et al., 2008). Briefly, this vector contains a bicistronic cassette composed of the luciferase firefly (FLuc) and renilla (RLuc) genes linked by the HCV IRES and driven either by the T7 or the CMV promoter. However, this system is restricted to transient studies. The goal of this work was to develop a persistent reporter system to test inhibitors of HCV IRES translation and to determine the duration of inhibition. To that end, the use of a simian immunodeficiency virus (SIV)-based lentiviral vector (Schnell et al., 2000) expressing the bicistronic cassette described above was preferred to the establishment of a system based on selection of a drug that might modify the cell environment. Lentiviral vectors appear relatively easy to produce in adequate quantities, unlike adenovirus-based vectors, and can achieve gene transfer and
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expression at satisfactory levels in both growth-arrested and differentiated target cells for a prolonged period. There is now a third generation of lentiviral vectors that are rendered nonpathogenic by self-inactivation (SIN) (Zufferey et al., 1998). The safety of the system used in that study is reinforced due to the fact that production of lentiviral particles was achieved by cotransfecting 293T cells with the SIV-transfer vector based on SIVmac251 (Mangeot et al., 2000) and heterologous helper constructs such as HIV-1 8.71 plasmid coding for the HIV-1 viral genes, and the vesicular stomatitis virus G envelope (VSV-G) for pseudotyping (Naldini et al., 1996a), thus avoiding any homologous recombinations, so the particles are incapable of independent propagation. The present study reports the optimization of this heterologous HIV-1/SIV lentiviral system in different types of cells, and its validation as a bicistronic reporter vector with IFN known to target the HCV 5 NCR (Hazari et al., 2005). Thereafter, this vector appeared as a promising tool for gene therapy which requires multigene expression of the same transcript to follow the expression of the transgene of interest.
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2.3. Production and titration of vector particles Reporter HIV-1/SIV-derived particles were generated upon cotransfection of 293T cells with the transfer vector, and 8.71 and HCMV-G constructs, using the transient calcium phosphate method as described previously (Naldini et al., 1996a). To that end, 6 × 106 293T cells per 100 cm2 tissue culture dish (Costar) were seeded 24 h prior to transfection. Viral supernatants were harvested 48 h post-transfection, cleared by low-speed centrifugation at 800 × g for 5 min, then treated with 1 M DNAse-1 (Roche) in the presence of 1 M magnesium chloride (Sigma) for 15 min at 37 ◦ C in order to eliminate any contaminating plasmid DNA. In a last step, the vector was concentrated by ultracentrifugation at 50,000 × g for 2 h at 4 ◦ C and the viral pellet resuspended in 70 l of cold phosphatebuffered saline (PBS), was aliquoted (15 l) in Eppendorf tubes and stored at −80 ◦ C until use. The titer of vector particles was normalized by measuring the p24 HIV-1 capsid protein concentration of the viral stock, using the Alliance HIV-1 p24 (capsid antigen) ELISA kit (PerkinElmer) according to the supplier. Titers obtained ranged from 80 to 100 ng p24 antigen (Ag)/l.
2. Materials and methods 2.4. Cell transduction with lentiviral vectors 2.1. Cells Human embryonic kidney 293T cells, human hepatoblastoma HepG2 and Huh7 cells, porcine kidney PK15 cells and murine embryonic NIH3T3 fibroblasts were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen), supplemented with 10% fetal calf serum (FCS) and 1% penicillin–streptomycin. Human primary myoblasts were originally isolated as previously described (Edom et al., 1994) from the quadriceps of a 5-day-old infant, in accordance with French legislation on ethics. Cells were grown in Ham’s F-10 medium (Invitrogen), supplemented with 20% FCS and 1% penicillin–streptomycin. For myoblast differentiation, growth medium was replaced by serum-free DMEM medium supplemented with 100 g/ml of transferrin and 10 g/ml insulin.
2.2. Plasmids and vectors All transfer vectors were produced with the SIV minimal lentiviral vector pGAE-0 previously described (Mangeot et al., 2000, 2002) and originally derived from SIVmac251. The strong ubiquitous internal CMV promoter is used to increase the expression level of the transgene. Different transfer vectors were designed, comprising the following reporter genes amplified with appropriate primers containing restriction sites used for cloning: the EGFP gene inserted at the AgeI/XhoI sites of the multicloning sequence, giving pGAE–EGFP, the LacZ gene inserted at the AgeI/XhoI sites, giving pGAE–LacZ, and the luciferase genes linked by the HCV IRES, constituting the bicistronic cassette, inserted in two steps; FLuc gene was first inserted at the AgeI/HpaI sites of the vector, followed by the sequence HCV IRES-RLuc at the HpaI/XhoI sites, giving pGAE–FHR. Plasmids pGAE–FCR and pGAE–FER were obtained in the same way, except that CSFV and EMCV IRESs, respectively, were inserted first in BamHI/PstI sites in pIRLuc construct in place of the HCV IRES, and second the IRES-RLuc fragment was inserted in pGAE–FLuc at HpaI/XhoI sites. The packaging HIV 8.71 vector (a gift from Dr. Naldini) expresses the complete sets of structural and accessory viral proteins with the exception of env and vpu, and is devoid of the packaging sequence (Naldini et al., 1996b). The plasmid HCMV-G (kindly given by Dr. Yee) expresses the vesicular stomatitis virus G (VSV-G) envelope protein allowing efficient pseudotyping of lentiviral particles and broad cell tropism (Yee et al., 1994).
A total of 2 × 105 cells were used for transduction and seeded onto 12-well plates (Costar) the day before. Transductions were carried out in 800 l total volume of serum-free medium to which viral stocks were added at various concentrations (0–400 ng p24 antigen per well) and 6 g/ml Polybrene (Sigma). Then, after gentle agitation for 2 h at 37 ◦ C, cells were incubated until 48–72 h with fresh and complete medium. Transduction efficiencies were calculated using the percentage of EGFP-positive cells analyzed by flow cytometry, or by luminometry for cells expressing -galactosidase, both of these methods being described below. Whole-cell population was used rather than selected clones in all of our experiments. 2.5. Semi-quantitative RT-PCR The total RNA was isolated from the Huh7 cells treated with different doses of IFN 16 h after transduction using Trizol (Invitrogen) and its concentration in 20 l-sample of nuclease-free water was measured spectrophotometrically. An equal quantity of each RNA was included as template in each of the semiquantitative one-step RT-PCRs using the Access kit (Promega) and the following primer pairs—FLuc: 5 -CCCTGGAAGATGGAAGCGTT3 and 5 -TTTGCAGCCTACCGTAGTGT-3 ; RLuc: 5 -GCCTCCTGGATCACTACAAGTA-3 and 5 -AAAAGAACCCAGGGTCGGACTC-3 ; and glyceraldehyde-phosphatase dehydrogenase (GAPDH) as an internal quantitative control: 5 -AGAACATCATCCCTGCCTCTACTG-3 and 5 -CATGTGGGCCATGAGGTCCA-3 . FLuc and RLuc fragments were amplified in the same reaction as follows: one cycle of reverse transcription at 50 ◦ C for 30 min and 20 cycles of amplification at 94 ◦ C for 15 s, 50 ◦ C for 30 s, and 72 ◦ C for 1 min. The PCR products were subjected to electrophoresis on 2% agarose gel. The intensity of FLuc and RLuc bands was quantified using Image J software, and converted into arbitrary units and set to 100% in absence of treatment. 2.6. FACS analysis 48 h post-transduction, the samples were washed twice with PBS, fixed with a PBS 4% paraformaldehyde solution for 15 min at 4 ◦ C, and subjected to flow cytometry using a FACScan analyzer (Becton-Dickinson, BD Biosciences). EGFP fluorescence was detected following excitation with an argon ion laser source at 488 nm. Percentage of EGFP-positive cells was determined in comparison with the negative control of non-infected cells using Cell
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Quest computer software. For each sample, 5000 events were collected. 2.7. Reporter microscopic analysis Natural green fluorescence of the EGFP was examined at the indicated time points under a fluorescence microscope (Olympus) at 484 nm. Adherent cells were washed twice with PBS and fixed with PBS 4% paraformaldehyde as described above. Hoechst nucleus blue staining was then performed by incubating fixed cells for 10 min in the presence of a 0.5 g/ml solution of Hoechst 33258 (Sigma) in PBS, and observed at 340 nm. 2.8. Luminometric analyses Luciferase activities were assayed on cell lysates using the dual luciferase reporter assay system (Promega). Briefly, infected cells plated onto 24-well plates were washed twice with PBS after discarding the supernatant, and lysed in a suitable volume of lysis buffer with gentle agitation for 15 min at room temperature. Then, FLuc and RLuc activities were measured sequentially according to the supplier’s protocol. Errors were calculated as the standard deviation of three calculated activities for each point per experiment repeated twice, and expressed as percentages of the average activity. -Galactosidase expression was also determined by luminometry using the -Glo assay system (Promega) according to the supplier’s protocol. The values were given as for luciferase activities in relative light units (RLU).
3. Results 3.1. Efficient transduction of various cell lines and primary myoblasts Reporter lentiviral vectors described in Fig. 1 were produced with satisfactory titers after transfection of 293T cells, and induced expression of the corresponding reporter. Usually, supernatants collected at 48 h post-transfection produced better transduction efficiencies than those collected at 72 h. The resulting supernatants, concentrated by ultracentrifugation, titrated at approximately 100 ng/l of p24 antigen. Transduction efficiency was assessed first with the pGAE–EGFP particles both in hepatoma cell lines (HepG2 and Huh7) and human primary myoblasts. Flow cytometry analysis showed a positive correlation between the percentage of EGFPpositive cells and the increasing quantity of viral particles (Fig. 2A). A plateau was reached for a vector particle dose of 200 ng of p24 or higher, with approximately 90% of EGFP-expressing cells (i.e. 90% for myoblasts, 92.6% for HepG2 and 88.5% for Huh7). Similar results were obtained also with the murine NIH3T3 and the porcine PK15 cell lines (94% and 90%, respectively; data not shown). Transduction efficiency is similar in the three cell types, except that the myoblasts appeared less transducible at low vector amounts (i.e. 0–50 ng p24 Ag). Thereafter, transduction efficiencies were defined also for the vector expressing the -galactosidase (pGAE–LacZ). As shown in Fig. 2B, a plateau of -galactosidase activity was reached between 200 and 400 ng p24 Ag of the corresponding vector, reflecting the maximal transduction efficiency observed with the EGFP vector.
Fig. 1. Schematic representation of SIV-derived SIN vector and helper constructs. (A) SIV transfer vector derived from pGAE-0 is represented in the upper panel. Cis genetic elements are symbolized with shadowed boxes, whereas promoters are depicted in white boxes. pCMV, early cytomegalovirus promoter; RU5, LTR; U3, partially deleted 3 LTR; E/DLS, 5 encapsidation and dimerization sequence; gag, 5 gag region; cPPT, central polypurine tract; CTS, central termination sequence; RRE, rev-responsive element; SA/ESE, splicing acceptor/splicing donor. Transgenes used in this study are depicted in the lower panel: EGFP and LacZ, the genes encoding the enhanced green fluorescent protein and the -galactosidase respectively, giving the following corresponding monocistronic lentiviral vectors pGAE–EGFP and pGAE–LacZ. FLuc (firefly luciferase) and RLuc (renilla luciferase) represent the two reporter genes of the following bicistronic vectors: pGAE–FHR, with the HCV IRES, pGAE–FER, with the EMCV IRES, pGAE–FCR, with the CSFV IRES. (B) 8.71 packaging construct expressing the accessory proteins gag, pol, tat and rev, with the exception of env and vpu, and lacking the packaging sequence . (C) pHCMV-G plasmid encoding the VSV-G glycoprotein for pseudotyping the lentiviral particles.
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Fig. 2. Transduction efficiency of different cell types. Dose–response transduction experiments with pGAE–EGFP (A) and pGAE–LacZ (B) vectors were performed, using heterologous HIV-1/SIV lentiviral system, in hepatoma cell lines Huh7 (open bars) and HepG2 (shaded bars), and in human primary myoblasts (solid bars) as described in Section 2, in 12-well plates with increasing amounts of vector particles (0–400 ng p24 Ag) as indicated. Percentage cells expressing EGFP was determined by flow cytometry (A) and luminometric activity of cells expressing -galactosidase was measured by the aid of -Glo kit after their lysis according to the supplier’s protocol. Each column represents the average transduction efficiency from three calculated activities for each point per experiment repeated twice, and error bars indicate the standard deviations.
3.2. Efficient transduction of cells using a bicistronic lentiviral cassette Taking into account the performances obtained with a single transgene, and faced with the frequent need to use multigene
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transfer vectors to follow the transgene of interest in the target cell, the system was extended to a bicistronic construct containing two reporter genes separated with the HCV IRES. The SIV-derived lentiviral pGAE–FHR vector containing the bicistronic cassette FLuc-HCV IRES-RLuc, described previously (Laporte et al., 2000), inserted at the AgeI/XhoI restriction sites of the pGAE-0 vector, was developed and produced as described above. As depicted in Fig. 3, luminometric activities were measured at different vector doses and relative HCV efficiency was expressed through the ratio RLuc/FLuc (R/F). Since FLuc is translated in a cap-dependent fashion, it therefore represents the control of transduction efficiency. For each vector amount in hepatoma and in PK15 cells, FLuc has a higher activity than RLuc, in contrast to NIH3T3 cells where the RLuc activity is the highest. For all cell lines, the ratio of R/F is relatively constant according to the vector amount, validating a stable expression of the transgenes. The R/F ratios are around 0.40 and 0.32 for HepG2 and Huh7, respectively, and slightly lower in the PK15 cell line (0.21), which is not susceptible to HCV infection. Unexpectedly, the murine NIH3T3 cells that were supposedly not permissive to HCV infection showed a very high ratio (2.78). To investigate a putative cellular tropism determined by the IRES studied, two new constructs were designed. They were both derived from the pGAE-0 transfer vector, with insertion of the bicistronic cassette; however, the HCV IRES sequence was replaced by either the CSFV IRES, giving pGAE–FCR, or the EMCV IRES, giving pGAE–FER. The four cell lines and the primary myoblasts were transduced with the three different bicistronic lentiviral vectors and the luminometric activities of FLuc and RLuc were determined. As observed with HCV IRES, FLuc activity was higher in human or porcine cells than in murine cells (data not shown). The R/F ratios reported in Table 1 indicate that the activity of CSFV IRES is comparable to that of HCV IRES, except for the porcine cell line PK15 (supposed to be the preferred host), where it is more efficient. The EMCV IRES exhibited an increased ratio in human hepatoma cell lines when compared with HCV or CSFV IRES, and its highest efficiency was found in the human myoblasts and not in the murine cells. Surprisingly, the HCV IRES activity is low in hepatoma cells and
Fig. 3. Transduction efficiency of different cell lines, hepatoma cells Huh7 and HepG2, and NIH3T3 and PK15 cells in 24-well plates as described in Section 2, using bicistronic lentiviral transfer vector containing the HCV IRES element, pGAE–FHR. Luciferase activities at increasing vector doses (from 0 to 200 ng p24 Ag) as indicated, were measured by luminometry and expressed in relative light units (RLU). FLuc activity (cap-dependent) (solid bars); RLuc activity (HCV IRES-dependent) (open bars). Relative HCV IRES efficiency, estimated from the RLuc/FLuc ratio, was represented by the black line and appeared constant, independently of the vector amount. Each column or point represents the average transduction efficiency from three calculated activities for each point per experiment repeated twice, and error bars indicate the standard deviations.
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Table 1 Relative efficiencies of different IRESs in various cell types. Cells
IRESa HCV
Huh7 HepG2 NIH3T3 PK15 Myoblasts
0.32 0.40 2.78 0.21 0.52
CSFV ± ± ± ± ±
0.02b 0.03 0.04 0.01 0.04
0.15 0.41 1.84 2.02 0.60
EMCV ± ± ± ± ±
0.01 0.03 0.05 0.04 0.03
0.87 1 1.30 1.66 2.10
± ± ± ± ±
0.01 0.1 0.05 0.02 0.05
a Different IRESs tested correspond to bicistronic lentiviral vector as follows: HCV, pGAE–FHR; CSFV, pGAE–FCR; EMCV, pGAE–FER. b Relative IRES efficiencies estimated by the ratio RLuc/FLuc. Each value represents the average IRES activity from tirlicate translation samples, followed by the standard deviation (±).
high in murine cells when compared with the other tested IRESs, even if it is a known human liver host, indicating the important role played by the cell factors on the activity of high-structured IRES elements (Rijnbrand and Lemon, 2000) (see Section 4). 3.3. Transgene expression is stable over a long period To test the stability and the duration of transgene expression, infected cells were grown and maintained in culture for a long period. As shown in Fig. 4, a persistent and stable expression of FLuc and RLuc was measured in HepG2 (shaded bars) and Huh7 (open bars) cells up to 12 weeks after transduction, as represented by the R/F ratio. Similar results were observed by flow cytome-
Fig. 4. Lentivirally transduced cells maintain transgene expression over time. Myoblasts (solid bars) transduced with pGAE–EGFP expressing EGFP were counted by flow cytometry at different time points as described in Section 2, and indicated a constant percentage of positive cells up to 4 weeks. Hepatoma cells (HepG2: shaded bars; Huh7, open bars) transduced with pGAE–FHR, containing the HCV IRES, revealed that the relative HCV IRES activity, estimated through the RLuc/FLuc ratio, did not vary over time until 12 weeks. Each column represents the average transduction efficiency from three calculated activities for each point per experiment repeated twice, and error bars indicate the standard deviations.
try using the EGFP construct as well as in the NIH3T3 cells (data not shown). Human primary myoblasts (solid bars) also exhibited stable expression of EGFP during their proliferative life span, at least for 4 weeks after transduction, until they reached the replicative senescence stage that limits the proliferative capacity of human somatic cells. In addition, less than 1% of transduced cells treated with the non-nucleoside HIV reverse-transcriptase inhibitor nevirapine (2 М) (Roche, Meylan) expressed EGFP 48 h post-transduction (data not shown). This result, together with stable expression described above, rules out the possibility of passive transduction of EGFP as described previously (Liu et al., 1996; Nash and Lever, 2004). In addition, no toxicity induced by the vector in either infected cells was observed, even at the highest doses tested (2000 ng of p24 Ag/well in a 24-well plate) (not shown). 3.4. Assay of HCV IRES inhibition The stability of luciferase expression gave us the possibility to follow the HCV IRES activity for a long period and investigate whether this bicistronic lentiviral vector was suitable for setting up a cell-based assay to test HCV IRES inhibitors for a long term follow-up, compared to transient transfection. IFN ␣-2a (Roferon, Roche) and IFN -1a (Avonex, Biogen. Idec) were selected since they inhibit HCV replication by targeting the HCV IRES (Dash et al., 2005), this being valid for the six genotypes (Hazari et al., 2005). Experiments were carried out in Huh7, HepG2 and NIH3T3 cells 12 weeks post-transduction with the pGAE–FHR vector. Cells were seeded in 24-well plates and incubated with increasing concentrations of either IFN ␣-2a or -1a. Concentrations ranging from 1 to 1000 IU/ml were lower than the physiological concentrations. After 16 h, cells were lysed in reporter lysis buffer and the luciferase activities were measured and plotted against IFN concentrations (Fig. 5). Both IFNs ␣ and  induced a dramatic decrease of RLuc activity (open bars) in hepatoma cells Huh7 and HepG2 in a dosedependent manner. Thus, the HCV IRES efficiency was decreased by 50% at 100 IU/ml of IFN and reached almost 85% inhibition at 1000 IU/ml. However, this activity was not inhibited at any dose in NIH3T3 cells, indicating the importance of the cell line used to test HCV IRES inhibitors, probably due to host factors. However, each IFN dramatically inhibited FLuc activity expressed in a capdependent way, in all cell lines tested and more efficiently than RLuc IRES-dependent. We then examined whether IFN treatment could have degraded FLuc or RLuc mRNAs which could explain differences observed between their activities and regarding the cell line used. To that aim, semi-quantitative RT-PCR amplifications were performed on total RNA extracted from transduced Huh7 cells under increasing doses of IFN ␣-2a, as described in Section 2. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was similarly examined for comparison. PCR products were analyzed by agarose gel electrophoresis and quantitated using IMAGE J software (Fig. 6). All RNAs reporters did not decay under IFN pressure, regardless of their cap or IRES status. Same results were obtained in another experiment using IFN -1a and NIH3T3 cells (data not shown), indicating that IFN ␣ or  preferentially blocks protein translation without altering the stability of corresponding mRNA. Taken together, these data validate the bicistronic lentiviral vector as a tool available for assessing a potent inhibitory effect of a compound on the HCV IRES. 3.5. Potential for gene therapy of the bicistronic vector Since the expression of the EGFP and the FLuc and RLuc transgenes is stable and the tropism exhibited by the HCV IRES is weak, our bicistronic system might be envisaged as a useful tool for multigene vector strategies in gene therapy. It is feasible to replace one of the two reporters by a gene of interest and to follow its expression
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Fig. 5. Effects of interferons ␣-2a et -1a on the expression of luciferases from the dicistronic lentiviral vector. Huh7 and HepG2 hepatoma cells and NIH3T3 cells transduced with pGAE–FHR for 12 weeks, were treated with increasing concentrations of each IFN (0–1000 UI/ml). Luciferase activities, expressed in RLU, were plotted as mean percentage of the untreated controls. FLuc (solid bars), cap-dependent translation; RLuc (open bars), IRES-dependent translation. Each column represents the average HCV IRES efficiency from three treated samples for each point per experiment repeated twice, and error bars indicate the standard deviations.
over time through expression of luciferase or another reporter such as EGFP. The gene of interest should be inserted upstream of the IRES, the second gene being less expressed in a bicistronic lentiviral vector (Dupuy et al., 2005; Mizuguchi et al., 2000). However, the results obtained with the murine NIH3T3 cell line indicate that, in some cells or species, insertion of the gene of interest downstream of the IRES could be more efficient. Taking advantage of the in vitro differentiation capacity of the myoblasts, we investigated the capacity of the construct to sustain expression of the transgene in post-mitotic cells. Prolif-
erating human myoblasts were transduced with pGAE–EGFP at a vector particle dose corresponding to 200 ng p24 Ag, and mononucleated transduced myoblasts expressing EGFP were amplified (Fig. 7A). Then, subconfluent cultures were switched to a differentiation medium for 6 days. Under this permissive condition, the myoblasts will differentiate, fuse and form multinucleated myotubes. As shown in Fig. 7B, the post-mitotic myotubes containing several nuclei still expressed EGFP, indicating that expression of the transgene is maintained during myogenic differentiation and in post-mitotic cells. 4. Discussion
Fig. 6. Graphical representation of the translational inhibition of both capdependent (FLuc) and IRES-dependent (RLuc) luciferases, and the stability of corresponding mRNAs after treatment of Huh7 cells with different concentrations of IFN ␣-2a (100–1000 UI/ml). The values in the graph are normalized according to the untreated control and expressed as a percentage of it. The mRNA quantitation was made by measuring the band intensity on the gel using IMAGE J software, and the luciferases expression was monitored by luminometry as described in Fig. 5.
Because of the cytotoxic nature of vaccinia virus, and possible modifications in a cellular environment under continuous antibiotic selection, our aim was to develop a persistent cell-based reporter system for characterization and long-term evaluation of HCV IRES inhibitors. For this, the initial bicistronic reporter vector pIRF limited to transient expression was adapted to a lentiviral transfer vector, resulting in stable expression of the bicistronic cassette due to its integration into the host cell genome. A heterologous lentiviral system was used, based on crosspackaging between elements from different but related viruses present on distinct plasmids (i) human HIV-1 packaging construct and (ii) SIV-gene transfer vector. This vector (pGAE-0) represents a minimal genome, devoid of viral open reading frames but bearing the cis-elements required for viral infectivity, and deleted for a large part of the 3 long terminal repeat (LTR) U3 domain containing sites essential for SIV transcription, rendering it self-inactivated (SIN). Such a secure vector system could be envisioned to be useful as well
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Fig. 7. Sustained transgene expression during differentiation of transduced human primary myoblasts into myotubes. Subconfluent myoblasts transduced with pGAE–EGFP vector particles corresponding to 200 ng p24 Ag, seeded in a 12-well plate, were induced to differentiate into myotubes by changing culture medium to DMEM as described in Section 2. Six days later, infected cells were observed in fluorescence microscopy with appropriate filters. (A) Myoblasts without induction of differentiation. Nuclei are stained in blue with Hoechst and the cytoplasm appears fluorescent green, expressing the EGFP (B) myoblasts with differentiation. The arrow indicates a myotube with several blue nuclei, the cytoplasm still exhibiting green fluorescence (magnification 20×).
for testing inhibitors of HCV IRES over time, with the possibility of evaluating their pharmacokinetics, as for gene therapy. First, the system was optimized in different cell types with a unique reporter to confirm (i) its capacity to produce lentiviral particles at satisfactory titers, due to pseudotyping with the VSV-G glycoprotein, conferring on them a broad tropism and additional stability, and (ii) its efficient potential for gene transfer, using limited amounts of lentiviral vector, even in primary human myoblasts. Second, the same experimental conditions were extended to a bicistronic transfer vector containing the bicistronic cassette composed of the HCV IRES between the two luciferase genes, FLuc and RLuc, respectively, in order to evaluate the effects of candidate drugs on HCV IRES activity. The cell-based assay performed on transduced cells measured an inhibitory effect through the expression of RLuc gene, directly under the control of the HCV IRES. In a previous work, a promoter activity has been assigned to HCV IRES sequences in their DNA form (Dumas et al., 2003). Such a residual activity was demonstrated with the bicistronic transfer vector pGAE–FHR deleted of the CMV promoter (data not shown). However, this activity did not interfere with the inhibition test performed because (i) it is already present in the control without any antiviral reagent, and (ii) the expected response in such a test is an all or nothing. Otherwise, when subtle modulations of the HCV IRES are to be detected, IRES in RNA form is prefered to transfect cells (Boni et al., 2005; Lourenc¸o et al., 2008). The system was therefore validated with IFNs ␣-2a and -1a, IFN being the most efficient antiviral agent in HCV infection until now, at doses below those used during treatment (Chevaliez and Pawlotsky, 2007). Due to its sensitivity, rapidity and reproducibility, the assay can be formatted for high-throughput screening to identify HCV IRES inhibitors in libraries (in process). Furthermore, two other bicistronic transfer vectors were designed, differing by their IRES (pGAE–FCR with CSFV IRES or pGAE–FER, with EMCV IRES), to investigate a potent cellular tropism determined by the origin of the IRES considered. Surprisingly, no cell specificity was observed for any IRES tested, excepted for CSFV, which exhibited a preference for the porcine PK15 cell line. However, differences in IRES efficiency were observed with a given IRES in different cell types, as mentioned above and in previous studies (Laporte et al., 2000, 2003). In that work, the HCV IRES activity appeared higher in murine cells NIH3T3 than in its preferred host human hepatoma cells, and the CSFV IRES was the only one exhibiting an expected tropism in PK15 cells. These observations indicate the impact of cell trans-acting factors in the modulation of the IRES considered. For HCV IRES, some non-canonical factors also active on other viral IRESs, such as polypyrimidine tract-binding protein (PTB), La autoantigen and some ribonucleoproteins, have
been identified (Rijnbrand and Lemon, 2000), and more recently, the microRNA mi122 (Henke et al., 2008) and new ribonucleoproteins (Pacheto et al., 2008). However, concerning the PTB, its modulation of HCV IRES is still controversial, which may be due to its limited concentration in certain cell types (Robinson et al., 2008; Wang et al., 2008). In addition, the NIH3T3 cell line does not appear convenient for testing inhibitors of HCV IRES as its does not respond to IFN treatment. Thus, the choice of the cell line to be transduced depends on the final goal. In another clinical aspect, the bicistronic lentiviral vector pGAE–FHR developed here exhibited all potentialities for gene therapy: stability and duration of transgene expression, availability in various cell types due to the presence of the ubiquitous CMV promoter, and as a lentiviral vector, in both dividing and non-dividing target cells (Naldini et al., 1996b). The presence of the HCV IRES allows any combination between the transgene of interest and another reporter or suicide gene to follow its expression within the target cell, as it is required frequently in many potential gene transfer applications (Chinnasamy et al., 2006; De Felipe, 2002; Ngoi et al., 2004; Reiser et al., 2000). One condition needed to design a multicistronic transfer vector, is the IRES(s) considered to be strong enough to achieve expression of the transgene(s) at high levels in target cells. Observation of (i) good transduction efficiency of human primary myoblasts, the myogenic precursor cells responsible for muscle regeneration, and (ii) sustained expression of the transgene in post-mitotic and multinucleated myotubes, encouraged us in preliminary investigations in the important field of autologous cellular therapy based on genetically modified stem cells (Davies and Grounds, 2007). Further work is under way, using EGFP as a surrogate for therapeutic protein, to improve efficient gene delivery and sustained expression of the transgene in recipients, taking into account numerous reports in that field (Cockrell and Kafri, 2007). Importantly, since compartmentalization has been described for the HCV IRES within the infected organism, aside from other regions of the genome (Di Liberto et al., 2006; Laporte et al., 2003; Roque-Afonso et al., 2005), this characteristics should be considered as beneficial for both applications mentioned above with our bicistronic system. Although highly conserved among most of genotypes, the HCV IRES sequence has been reported to contain tissue-specific point mutations, some of which might prove useful. First, they might allow identification of inhibitors specific to IRES variants resistant to inhibitors of the wild-type IRES sequence. Second, they might improve transfer of a transgene in gene therapy, thus helping target cells in connection with a tissue-specific promoter.
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In conclusion, a heterologous HIV-1/SIV bicistronic lentiviral system has been developed for evaluation of inhibitors of HCV IRES activity and for multigene therapy due to its capacity (i) to produce high titer viral particles and (ii) to stably express the transgene in target cells for a long period. Preliminary results are encouraging and offer an opportunity to improve the system described for rapid screening of potent HCV IRES antiviral compounds and for efficient gene delivery in myoblasts for gene therapy of muscular diseases. Acknowledgements The authors are grateful to Philippe Emmanuel Mangeot for providing pGAE-0 SIV-derived lentiviral vector and for his expertise in lentiviral production. We are indebted to Dr. Naldini for providing gag-pol and env 8.71 plasmid, and to Dr Yee for the gift of the pseudotyping pVSV-G plasmid. We also thank Catherine Blanc of the flow cytometry platform of the Pitié-Salpêtrière site for her contribution and advice in FACS analysis. This work was supported in part by the Agence Nationale pour la Recherche sur le Sida, and the Foundation for Science and Technology of Lisbon. Sofia Lourenc¸o is a recipient of doctoral grant from the Foundation for Science and Technology of Lisbon (SFRH/BD/16218/2004), and Sébastien Boni of doctoral grant from the Ministère de l’Education Nationale de la Recherche et de la Technologie. References Alter, M., 2007. Epidemiology of hepatitis C virus infection. World J. Gastroenterol. 13, 2436–2441. Boni, S., Lavergne, J.P., Boulant, S., Cahour, A., 2005. Hepatitis C virus core protein acts as a trans-modulating factor on internal translation initiation of the viral RNA. J. Biol. Chem. 280, 17737–17748. Chevaliez, S., Pawlotsky, J.M., 2007. Interferon-based therapy of hepatitis C. Adv. Drug Deliv. Rev. 59, 1222–1241. Chinnasamy, D., Milsom, M.D., Shaffer, J., Neuenfeldt, J., Shaaban, A.F., Margison, G.P., Fairbairn, L.J., Chinnasamy, N., 2006. Multicistronic lentiviral vectors containing the FMDV 2A cleavage factor demonstrate robust expression of encoded genes at limiting MOI. Virol. J. 3, 14. Cockrell, A.S., Kafri, T., 2007. Gene delivery by lentivirus vectors. Mol. Biotechnol. 36, 184–204. Dasgupta, A., Das, S., Izumi, R., Venkatesan, A., Barat, B., 2004. Targeting internal ribosome entry site (IRES)-mediated translation to block hepatitis C and other RNA viruses. FEMS Microbiol. Lett. 234, 189–199. Dash, S., Prabhu, R., Hazari, S., Bastian, F., Garry, R., Zou, W., Haque, S., Joshi, V., Regenstein, F.G., Thung, S.N., 2005. Interferons alpha, beta, gamma each inhibit hepatitis C virus replication at the level of internal ribosome entry site-mediated translation. Liver Int. 25, 580–594. Davies, K., Grounds, M., 2007. Modified patient stem cells as preclude to autologous treatment of muscular dystrophy. Cell Stem Cell. 1, 595–596. De Felipe, P., 2002. Polycistronic viral vectors. Curr. Gene Ther. 2, 355–378. De Francesco, R., Migliaccio, G., 2005. Challenges and successes in developing new therapies for hepatitis C. Nature 436, 953–960. Di Liberto, G., Roque-Afonso, A.M., Kara, R., Ducoulombier, D., Fallot, G., Samuel, D., Feray, C., 2006. Clinical and therapeutic implications of hepatitis C virus compartmentalization. Gastroenterology 131, 76–84. Dumas, E., Staedel, C., Colombat, M., Reigadas, S., Chabas, S., Astier-Gin, T., Cahour, A., Litvak, S., Ventura, M., 2003. A promoter activity is present in the DNA sequence corresponding to the hepatitis C virus 5 UTR. Nucleic Acids Res. 31, 1275–1281. Dupuy, F.P., Mouly, E., Mesel-Lemoine, M., Morel, C., Abriol, J., Cherai, M., Baillou, C., Negre, D., Cosset, F.L., Klatzmann, D., Lemoine, F.M., 2005. Lentiviral transduction of human hematopoietic cells by HIV-1- and SIV-based vectors containing a bicistronic cassette driven by various internal promoters. J. Gene Med. 7, 1158–1171. Edom, F., Mouly, V., Barbet, J., Fiszman, M., Butler-Browne, G., 1994. Clones of human satellite cells can express in vitro both fast and slow myosin heavy chains. Dev. Biol. 164, 219–229. Hazari, S., Patil, A., Joshi, V., Sullivan, D.E., Fermin, C.D., Garry, R.F., Elliott, R.M., Dash, S., 2005. Alpha interferon inhibits translation mediated by the internal ribosome entry site of six different hepatitis C virus genotypes. J. Gen. Virol. 86, 3047–3053. Henke, J., Goergen, D., Zheng, J., Song, Y., Scüttler, C., Fehr, C., Jünemann, C., Niepmann, M., 2008. microRNA-122 stimulates translation of hepatitis C virus RNA. EMBO J. 27, 3300–3310.
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