Claire Gondeau

Lydiane Pichard-Garcia

Patrick Maurel
email: patrick.maurel@inserm.fr

£ To the best of their knowledge, authors have no real or perceived

Keywords: small interfering RNA, miR, microRNA, 5 hepatoma cell line, replicon, hepatocyte, virion, antiviral drugs, RNA interference, pharmaceutical screening £

Investigations on the biology of hepatitis C virus (HCV) have been hampered by the lack of small animal models. Efforts have therefore been directed to designing practical and robust cellular models of human origin able to support HCV replication and production in a reproducible, reliable and consistent manner. Many different models based on different forms of virions and hepatoma or other cell types have been described including virus-like particles, pseudotyped particles, subgenomic and full length replicons, virion productive replicons, immortalised hepatocytes, fetal and adult primary human hepatocytes. This review focuses on these different cellular models, their advantages and disadvantages at the biological and experimental levels, and their respective use for evaluating the effect of antiviral molecules on different steps of HCV biology including virus entry, replication, particles generation and excretion, as well as on the modulation by the virus of the host cell response to infection.

Introduction

Hepatitis C virus (HCV) is an enveloped RNA virus which belongs to the Flaviviridae family and is the unique member of the Hepacivirus genus [START_REF] Choo | Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome[END_REF][START_REF] Houghton | Molecular biology of the C viruses: implications for diagnosis, development and control of viral disease[END_REF]. Within the last twenty years, HCV has emerged as a common cause of liver disease with an estimate of 170 million people infected annually on a worldwide basis. HCV infection is characterized by viral persistence and chronic liver disease in approximately 80% of cases. The complications of chronic hepatitis C include cirrhosis in 20% of cases and hepatocellular carcinoma, which has an incidence of up to 4 to 5% per year in patients with HCV-related cirrhosis. HCV-related end stage liver disease is now the principal indication for liver transplantation in industrialized countries [START_REF] Pawlotsky | Pathophysiology of hepatitis C virus infection and related liver[END_REF]. Efficient vaccine against HCV does not exist and the only available treatment which associates pegylated interferon α (IFN-Peg) and ribavirine is efficient in only 50 % of patients chronically infected with a virus of genotype 1 (the most frequent in Europe, Japan and North America) or 4, and in approximately 80 % of the patients infected with a virus of genotype 2 and 3 (Chevaliez & Pawlotsky, 2007a). It is therefore of major importance to develop new and efficient therapeutic strategies [START_REF] Wakita | HCV research and anti-HCV drug discovery: toward the next generation[END_REF].

HCV biology

HCV virions

HCV genome consists of an approximately 9.6 kb-long single strand RNA coding for a polyprotein of approximately 3000 amino acids [START_REF] Choo | Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome[END_REF]. The polyprotein is translated from an IRES (internal ribosomal entry site), a highly conserved region of the genome, and is cleaved by host and viral proteases. Host proteases generate the capside, envelope viral glycoproteins E1 and E2 and protein p7, while the viral proteases NS2/NS3 (cysteine protease) and NS3/NS4A (serine protease) generate the non-structural proteins (NS2 to NS5B). Non-structural proteins are involved in the replication of HCV genome and the building up of virions [START_REF] Suzuki | Hepatitis C viral life cycle[END_REF]. There are six major genotypes. The sequence similarity within two different genotypes is in the range of 70% at both nucleotide and amino acid levels [START_REF] Martell | Hepatitis C virus (HCV) circulates as a population of different but closely related genomes: quasispecies nature of HCV genome distribution[END_REF][START_REF] Pawlotsky | Hepatitis C virus population dynamics during infection[END_REF]. The HCV virion consists of a complex (nucleocapside) between genomic RNA and the core protein, embedded within a membrane derived from the host cell and containing E1/E2 protein heterodimers [START_REF] Lavie | Assembly of a functional HCV glycoprotein heterodimer[END_REF]. Both E1 and E2 ectodomains are multi-glycosylated and these proteins play a major part in the virus entry in host cells. In the plasma, HCV virions exhibit a wide range of density (from <1.06 to 1.30 g/ml). The low density virions appear to be the most infectious in chimpanzee. They have been shown to form complex particles with low and very low density lipoproteins (LDL, VLDL, apoB100, apoE, apoCII and apoCIII) that are enriched in triglycerides [START_REF] Andre | Characterization of low-and very-low-density hepatitis C virus RNAcontaining particles[END_REF][START_REF] Andre | Hepatitis C virus particles and lipoprotein metabolism[END_REF][START_REF] Diaz | association of Hepatitis C virus with apolipoprotein B48-containing lipoproteins[END_REF]. These complexes are named lipoviroparticles (LVP) although the actual organisation of virion and lipoprotein within the £ To the best of their knowledge, authors have no real or perceived conflicts of interests.

LVPs is not yet clearly established. In infected patients, on average, 40% of the plasmaderived HCV RNA appears to be associated with triglyceride-rich LVP.

HCV cellular entry

HCV virion enter host cells through a multistep process involving sequencial attachment to several entry factors [START_REF] Dubuisson | Early steps of the hepatitis C virus life cycle[END_REF] (Fig 1). It is likely that each step is necessary for the next one and for full coordination of the entry. The participation of glycosaminoglycans [START_REF] Barth | Viral and cellular determinants of the hepatitis C virus envelope-heparan sulfate interaction[END_REF][START_REF] Morikawa | The roles of CD81 and glycosaminoglycans in the adsorption and uptake of infectious HCV particles[END_REF] and LDL receptor (LDLR) [START_REF] Agnello | Hepatitis C virus and other flaviviridae viruses enter cells via low density lipoprotein receptor[END_REF][START_REF] Molina | The low-density lipoprotein receptor plays role in the infection of primary human hepatocytes by hepatitis C virus[END_REF] as attachment factors to concentrate HCV at the host cell membrane has been documented and is consistent with the fact that both E1 and E2 are highly glycosylated and with the nature of the HCV LVPs [START_REF] Andre | Characterization of low-and very-low-density hepatitis C virus RNAcontaining particles[END_REF] (Fig 1, step 1). After initial recruitment at the membrane, HCV particles appear to bind to high affinity entry factors in a sequential manner. These factors involve the scavenger receptor BI (SR-BI), tetraspanin CD81 and proteins of tight junctions (TJ). SR-BI belongs to the CD36 family of lipid transporters and regulators of lipid homeostasis and is considered as the major high density lipoprotein (HDL) receptor (M. [START_REF] Krieger | Scavenger receptor class B type I is a multiligand HDL receptor that influences diverse physiologic systems[END_REF]. However, it is a promiscuous receptor of other lipoproteins including LDL, VLDL, acetylated and oxidized LDL [START_REF] Acton | Expression cloning of SR-BI, a CD36-related class B scavenger receptor[END_REF][START_REF] Connelly | Scavenger receptor BI: a scavenger receptor with a mission to transport high density lipoprotein lipids[END_REF]. SR-BI has been shown to interact with the soluble E2 protein with high affinity and its implication as an HCV entry factor is well documented (Bartosch et al., 2003;[START_REF] Dreux | Receptor complementation and mutagenesis reveal SR-BI as an essential HCV entry and functionally imply its intra-and extra-cellular domains[END_REF][START_REF] Maillard | The interaction of natural hepatitis C virus with human scavenger receptor SR-BI/Cla1 is mediated by ApoB-containing lipoproteins[END_REF][START_REF] Scarselli | The human scavenger receptor class B type I is a novel candidate receptor for the hepatitis C virus[END_REF][START_REF] Voisset | High density lipoproteins facilitate hepatitis C virus entry through the receptor class B type I[END_REF][START_REF] Von Hahn | Oxidized low-density lipoprotein inhibits hepatitis C virus cell entry in human hepatoma cells[END_REF]. Interestingly however, HDLs do not appear to compete with HCV entry but rather favor it [START_REF] Voisset | High density lipoproteins facilitate hepatitis C virus entry through the receptor class B type I[END_REF][START_REF] Zeisel | Scavenger receptor class B type I is a key host factor for hepatitis C virus infection required for an entry step closely linked to CD81[END_REF]. Recently SR-BII, a form generated by alternative splicing of SR-BI gene, has been shown to bind E2 and to be involved in HCV cell entry as well. Apparently, SR-BI and SR-BII may trigger the entry of different HCV variants (Grove et al., 2007). Finally, Dreux et al. [START_REF] Dreux | Receptor complementation and mutagenesis reveal SR-BI as an essential HCV entry and functionally imply its intra-and extra-cellular domains[END_REF] recently demonstrated that SR-BI is an essential HCV entry factor by investigating the impact of ectopic expression of human SR-BI in the entry of HCVpp and HCVcc in rat hepatoma cells that do not express SR-BI (Fig 1 ,step 2).

Tetraspanin CD81 has been well characterized as a major factor for HCV cellular entry (Bartosch et al., 2003;[START_REF] Buck | Direct infection and replication of naturally occurring hepatitis C virus genotypes 1, 2, 3 and 4 in normal human hepatocyte cultures[END_REF][START_REF] Cormier | CD81 is an entry coreceptor for hepatitis C virus[END_REF][START_REF] Hsu | Hepatitis C virus glycoproteins mediate pH-dependent cell entry of pseudotyped particles[END_REF][START_REF] Kapadia | Initiation of hepatitis C virus infection is dependent on cholesterol and cooperativity between CD81 and scavenger receptor B type I[END_REF][START_REF] Koutsoudakis | The level of CD81 cell surface expression is a key determinant for productive entry of C virus into host cells[END_REF][START_REF] Molina | Serum-derived hepatitis C virus infection of primary human hepatocytes is CD81 dependent[END_REF][START_REF] Petracca | analysis of hepatitis C virus envelope-CD81 binding[END_REF][START_REF] Pileri | Binding of hepatitis C virus to CD81[END_REF][START_REF] Wunschmann | Characterization of hepatitis C virus (HCV) and HCV E2 interactions with CD81 and the low-density lipoprotein receptor[END_REF]Zhang et al., 2004) (Fig 1, step 3). E2 protein has been shown to exhibit a very high affinity (K d ~10 -9 M) for the large extracellular loop of CD81 [START_REF] Petracca | analysis of hepatitis C virus envelope-CD81 binding[END_REF][START_REF] Pileri | Binding of hepatitis C virus to CD81[END_REF]. CD81 expression and HCV entry are dependent on the plasma membrane sphingomyelin/ceramide ratio and cholesterol content, suggesting that CD81 must be located in tetraspanin-enriched microdomains to function as a viral entry mediator [START_REF] Voisset | Ceramide enrichment of the plasma membrane induces CD81 internalization and inhibits hepatitis C virus entry[END_REF]. Recent investigations suggest that both CD81 and SR-BI may function cooperatively to mediate HCV entry in target cells [START_REF] Kapadia | Initiation of hepatitis C virus infection is dependent on cholesterol and cooperativity between CD81 and scavenger receptor B type I[END_REF][START_REF] Zeisel | Scavenger receptor class B type I is a key host factor for hepatitis C virus infection required for an entry step closely linked to CD81[END_REF], while CD81 expression is absolutely required to confer Huh-7 cell permissiveness to HCV infection [START_REF] Akazawa | CD81 expression is important for the permissiveness of Huh7 cell clones for heterogeneous hepatitis C virus infection[END_REF]. Interestingly, a soluble form of E2 has been suggested to link both CD81 and SR-BI [START_REF] Heo | Hepatitis C virus E2 links soluble human CD81 and SR-B1 protein[END_REF]. A recent work confirms these finding at the in vivo level [START_REF] Meuleman | Anti-CD81 antibodies can prevent a hepatitis C virus infection in vivo[END_REF].

Recent investigations suggest that two proteins entering the constitution of tight junctions (TJ) claudin (CLDN) [START_REF] Evans | Claudin-1 is a hepatitis C virus co-receptor required for a late step in entry[END_REF] and occludin (OCLN) [START_REF] Ploss | Human occludin is a hepatitis C virus entry factor required for infection of mouse cells[END_REF] play a part in HCV entry. CLDNs constitute a family of four transmembrane domain proteins with two extracellular loops that are essential for the formation of TJs by forming homo or heterodimers on opposing cells, and for cell polarisation [START_REF] Furuse | Claudin-1 and -2: novel integral membrane proteins localizing at tight junctions with no sequence similarity to occludin[END_REF]. OCLN is a four transmembrane domain protein as well, present in the TJ complex of polarized epithelial cells. It appears to be associated with the CLDN-based strands and seems to play a role by £ To the best of their knowledge, authors have no real or perceived conflicts of interests.

regulating TJ permeability [START_REF] Forster | Tight junctions and the modulation of barrier function in disease[END_REF]. CLDN1 was discovered as a positive candidate in a genetic screen aimed at identifying genes that render a nonpermissive cell line (293T) infectable by HCVpp (HCV pseudoparticles, see section 4) and HCVcc (HCV particles produced in cell culture, see section 4) [START_REF] Evans | Claudin-1 is a hepatitis C virus co-receptor required for a late step in entry[END_REF] (Fig 1 ,step 4). The residues involved in the CLDN-mediated entry of HCV particles have been mapped to the first Nterminal half extracellular loop of the protein although no direct interaction between HCV E1/E2 and CLDN1 has been demonstrated. Comparative analyses of the kinetics of the inhibitory effect of antibodies against CD81 and CLDN1 on HCV entry suggested that CLDN1 plays a role in a step of the process that occurs after interaction with CD81 [START_REF] Evans | Claudin-1 is a hepatitis C virus co-receptor required for a late step in entry[END_REF]. CLDN6 and CLDN9 which only differ by one amino acid, have been both reported to mediate HCVpp and HCVcc entry in Huh-7.5 cells [START_REF] Meertens | The tight junction proteins claudin-1, -6, and -9 are entry cofactors for hepatitis C virus[END_REF], while other members of the CLDN family do not [START_REF] Zheng | Claudin-6 and claudin-9 function as additional coreceptors for hepatitis C virus[END_REF]. There is apparently no obvious preference in CLDN1/6/9 usage by the various HCV genotypes. Using the intestinal Caco-2 cell line which displays increased polarisation in culture, Mee et al. observed that the role of CLDNs in HCV entry does not correlate with their function as TJ components, and that disruption of TJ increases viral entry suggesting that HCV uses nonjunctional forms of CLDN1 [START_REF] Mee | Effect of cell on hepatitis C virus entry[END_REF]. This would imply that HCV preferentially targets hepatocytes in which cell-cell contacts are damaged. Using fluorescence resonance energy transfer, Harris et al. observed that CD81 and CLDN1 colocalize and form coreceptor complexes in liver tissue [START_REF] Harris | CD81 and claudin 1 coreceptor association: role in hepatitis C virus entry[END_REF] (Fig 1,step 4). SRBI, CD81 and CLDN1 appear to be expressed in the basolateral-sinusoidal membrane of hepatocytes in man, consistent with the fact that HCV enters the liver via the sinusoidal blood [START_REF] Reynolds | Hepatitis C virus receptor expression in normal and diseased liver tissue[END_REF]. In addition, these authors demonstrated that HCV infection positively regulates CLDN1 expression in Huh-7.5 cells and in human liver. It is suspected that CLDNs favor cell to cell virus spread in the liver. However, there are several cell lines which although being resistant to HCV infection express SRBI, CD81 and CLDN1. Evidence has been recently provided that binding of the virions to CD81, triggers Rho GTPase-dependent actin rearrangement that allows lateral movement of the CD81-virion complexes to the TJ linking hepatocytes [START_REF] Brazzoli | CD81 is central regulator of cellular events required for hepatitis C virus infection of human hepatocytes[END_REF]. Besides these observations, it was demonstrated that human OCLN plays a critical part in the process of HCV entry into human and rodent cells [START_REF] Ploss | Human occludin is a hepatitis C virus entry factor required for infection of mouse cells[END_REF] (Fig 1 ,step 4). Silencing of OCLN, specifically inhibited both HCVpp and HCVcc infection of Huh-7.5 cells. The second extracellular loop of OCLN was identified as the specific site of this action. Using different cell lines as well as mouse and human proteins and combinations thereof, these authors demonstrated that CD81 and OCLN represent the minimal human-specific entry factors for mouse and hamster cell infection, while both SR-BI and CLDN1 exhibited minor speciesspecificity, if at all. While recent indications suggested that polarisation restricts HCV entry into hepatocytes (Jane McKeating, personnal communication) and [START_REF] Farquhar | Primary hepatocytes as targets for hepatitis C virus replication[END_REF][START_REF] Mee | Effect of cell on hepatitis C virus entry[END_REF], Benedicto et al. provided direct evidence that infection affects TJs [START_REF] Benedicto | Hepatitis C virus envelope components alter of hepatocyte tight junction-associated proteins and promote occludin retention in the endoplasmic reticulum[END_REF]. Using the full genomic replicon system (Huh-7/JFH1) these authors demonstrated that HCV infection alters the distribution of TJ proteins including CLDN1, OCLN and ZO-1 by downregulating their accumulation, in disagreement with data reported by others [START_REF] Reynolds | Hepatitis C virus receptor expression in normal and diseased liver tissue[END_REF]. Notably and in contrast, OCLN exhibited a marked accumulation in the endoplasmic reticulum (ER) of infected cells. E2 protein colocalized and coimmunoprecipitated with OCLN suggesting that both proteins establish a direct virus-host protein interaction. Both normal OCLN localization and TJ organization were rescued by antiviral treatments of cells. Most of these results were confirmed in a recent paper [START_REF] Liu | Tight junction proteins claudin-1 and occludin control hepatitis C virus entry and are downregulated during infection to prevent superinfection[END_REF]. In sum, these data provide new arguments allowing to establish a link between HCVmediated liver pathogenesis (notably cholestatic disorders) and the quality of hepatocytehepatocyte TJs.

£ To the best of their knowledge, authors have no real or perceived conflicts of interests.

Finally, it has been recently shown that EWI-2wint, a cleavage product of EWI-2 a partner of CD81, is a potent inhibitor of the E2-CD81 interaction [START_REF] Rocha-Perugini | The CD81 partner EWI-2wint inhibits hepatitis C virus entry[END_REF]. Although being expressed in different cell lines, EWI-2wint is not expressed in hepatocytes. It is therefore possible that the hepatotropism of HCV might result either from the presence of other specific entry factor(s) or from the absence of specific entry inhibitor(s).

HCV endocytosis and genome replication

After binding to entry factors, HCV appears to enter the cells by clathrin-mediated endocytosis [START_REF] Rouille | Subcellular localization of hepatitis C virus structural proteins in a cell culture system efficiently replicates the virus[END_REF][START_REF] Meertens | Hepatitis C virus entry requires a critical postinternalization step and delivery to early endosomes via clathrin-coated vesicles[END_REF] (Fig 1,step 5). Indeed, siRNA (small interfering RNA) directed to clathrin heavy chain and molecules that neutralise the acidic pH of endosomes produce a potent inhibition of the entry of HCVpp and HCVcc in various cell lines. ApoC-I, an exchangeable apolipoprotein that predominantly resides in HDL, was shown to increase HCV infectivity and the rate of fusion between viral and target membranes via a direct interaction with the virion surface [START_REF] Dreux | The exchangeable apolipoprotein ApoC-I promotes membrane fusion of hepatitis C[END_REF]. Consistent with this, recent studies demonstrated that the C-terminal region of ApoC-1 associates with surface components of virions during morphogenesis [START_REF] Meunier | Apolipoprotein c1 association with hepatitis C virus[END_REF]. The combination between interactions with receptors, low pH encountered in endosomes (Fig 1, step 6), and mutual chaperoning or cooperation activity of E1 and E2 is likely to trigger a conformational change on the envelope glycoproteins that leads to the fusion of the virus membrane with the endosome membrane (Bartosch et al., 2003a;[START_REF] Hsu | Hepatitis C virus glycoproteins mediate pH-dependent cell entry of pseudotyped particles[END_REF][START_REF] Tscherne | Time-and temperature-dependent activation of hepatitis C virus for low-pH-triggered entry[END_REF] The 5'-nontranslated region (5'-NTR) of HCV RNA harbours the IRES [START_REF] Suzuki | Hepatitis C viral life cycle[END_REF][START_REF] Wang | Translation of human hepatitis C virus RNA in cultured cells is mediated by an internal ribosome-binding mechanism[END_REF], a structurally complex region consisting of four domains which are critical for the regulation of the polyprotein translation and viral replication (Fig 2). The IRES binds sequentially the 40S ribosomal subunit, the eukaryotic initiation factors 3 and 2, and the 60S subunit via a GTP-dependent reaction. This eventually leads to the formation of the first peptide bond. The IRES activity appears to be further modulated by viral (core) [START_REF] Boni | Hepatitis C virus core protein acts as a trans-modulating factor on internal translation initiation of the viral RNA[END_REF] and host (La, heterologous ribonucleoprotein L, poly-C and pyrimidine trackbinding) proteins. It was recently reported that the heterogeneous nuclear ribonucleoprotein D increases HCV IRES activity through binding to the stem loop region II element [START_REF] Paek | RNA-binding protein hnRNP modulates internal ribosome entry site-dependent translation of hepatitis virus RNA[END_REF]. Moreover, the domain I of IRES is capable of binding a liver specific microRNA (miR-122) that appears to enhance HCV RNA replication rate in a cell specific manner [START_REF] Jopling | Positive and negative modulation of viral and cellular mRNAs by liver-specific microRNA miR-122[END_REF][START_REF] Randall | Cellular cofactors affecting hepatitis C virus infection and replication[END_REF]. The polyprotein precursor generated by translation is cleaved by host and viral proteases [START_REF] Suzuki | Hepatitis C viral life cycle[END_REF]. Host proteases generate the structural proteins core, E1, E2 and p7, while the viral proteases NS2/NS3 and NS3/4 generate respectively the non-structural proteins NS2 and NS3, and NS4A, NS4B, NS5A and NS5B [START_REF] Bartenschlager | Kinetic and structural analyses of hepatitis C virus polyprotein processing[END_REF][START_REF] Failla | An amino-terminal domain of the hepatitis C virus NS3 protease is essential for interaction with NS4A[END_REF][START_REF] Lin | Hepatitis C virus NS3 serine proteinase: trans-cleavage requirements and processing kinetics[END_REF][START_REF] Tanji | Hepatitis C virus processing: kinetics and mutagenic analysis of serine proteinasedependent cleavage[END_REF]. HCV genome replication is assumed to proceed within complexes associated with ER membranes and begins with the RNA-dependent RNA polymerase (NS5B)-mediated synthesis of a complementary replicative (negative) strand. The latter is then used as a template to synthesize the genomic (positive) strand by the same enzyme (Fig 2). Replication is thought to yield double stranded RNA (dsRNA) structures. In primary human hepatocytes, the rate of synthesis of the genomic strand appears to be 5 to 10 times greater than that of the replicative strand [START_REF] Castet | Alpha interferon inhibits hepatitis C virus replication in primary human hepatocytes infected in vitro[END_REF]. Like other viral RNA polymerases, NS5B has a high error rate (averaging 10 -4 to 10 -5 per base site) and lacks proofreading activity, so that mutations accumulate in newly generated HCV genomes (quasispecies) [START_REF] Farci | Evolution of hepatitis C viral quasispecies and hepatic injury in perinatally infected children followed prospectively[END_REF][START_REF] Pawlotsky | Hepatitis C virus population dynamics during infection[END_REF]. The building-up of the replication complexes is assumed to be initiated at ER membranes by NS4B, an integral membrane protein [START_REF] Egger | Expression of hepatitis C virus proteins induces distinct membrane alterations including a candidate viral replication complex[END_REF][START_REF] Gao | Interactions between viral nonstructural proteins and host protein hVAP-33 mediate the formation of hepatitis C virus RNA replication complex on lipid raft[END_REF].

£ To the best of their knowledge, authors have no real or perceived conflicts of interests.

Analysis of replication complexes showed that the replicating HCV RNA and the NS proteins colocalize on cytoplasmic membrane structures. These structures appear to be resistant to detergent treatment and cofractionate with a lipid raft-associated protein, caveolin-2, suggesting that they are lipid rafts derived from intracellular membranes [START_REF] Shi | Hepatitis C virus RNA replication occurs on a detergent-resistant membrane that cofractionates with caveolin-2[END_REF]. It is now admitted that NS proteins form complexes in both ER and Golgi membranes, but that HCV RNA replication takes part in the Golgi membranes [START_REF] Aizaki | Characterization of the hepatitis C virus RNA replication complex associated with lipid rafts[END_REF]. Many viral and host proteins have been shown to interact with NS5B to regulate the rate of RNA replication [START_REF] Suzuki | Hepatitis C viral life cycle[END_REF]. In the Huh-7.5/JFH1 HCVcc model, the E1/E2 heterodimer was retained in the ER and does not accumulate in other intracellular compartments or at the plasma membrane [START_REF] Rouille | Subcellular localization of hepatitis C virus structural proteins in a cell culture system efficiently replicates the virus[END_REF]. Core was found to be associated to lipid droplets but was not found in the nucleus or in association with mitochondria or other well-defined intracellular compartments. Moreover, core did not colocalize with E1/E2. Although subcellular structures similar to the previously reported "membranous web" were observed, they did not harbor virus-like bodies.

HCV virion release

The assembly of viral particules involves capside protein oligomerisation and interaction with domains I and III as well as nucleotides 24-41 of the viral genomic RNA (Y. [START_REF] Tanaka | Selective binding of hepatitis C virus core protein to synthetic oligonucleotides corresponding to the 5' untranslated region of the viral genome[END_REF]. Core oligomerisation has been shown to be mediated by the 72-91 primary sequence of the protein [START_REF] Matsumoto | Homotypic interaction and multimerization of hepatitis C virus core protein[END_REF][START_REF] Nakai | Oligomerization of hepatitis C virus core protein is crucial for interaction with the domain of E1 envelope protein[END_REF]. After nucleocapside has been formed, it interacts with envelope proteins E1 and E2 and is secreted from the cell through plasmatic membrane budding so that host membrane constituents are also present in the virus membrane [START_REF] Baumert | Hepatitis C virus structural proteins assemble into viruslike particles in insect cells[END_REF][START_REF] Ezelle | Generation of hepatitis C virus-like particles by use of a recombinant vesicular stomatitis virus vector[END_REF][START_REF] Murakami | Production of infectious hepatitis C virus particles in three-dimensional cultures of the cell line carrying the genome-length dicistronic viral RNA of genotype 1b[END_REF]. NS5A has been shown to play a key role in regulating the early phase of HCV particle formation by interacting with Core through and its C-terminal serine cluster [START_REF] Masaki | Interaction of hepatitis C virus nonstructural protein 5A with core protein is critical for the production of infectious virus particles[END_REF]. Huang et al. demonstrated that HCV assembles in ApoB and MTTP (microsomal triglyceride transfer protein) enriched vesicles and that the viral secretion is dependent on both ApoB expression and VLDL assembly from subdomains of the ER (H. Huang et al., 2007) (Fig 2). This suggests that HCV uses the lipoprotein/cholesterol export system at its own benefit to escape from hepatocytes, and this is consistent with the use of lipoprotein receptors such as LDLR and SR-BI as cellular entry sites. These results were confirmed recently [START_REF] Nahmias | Apolipoprotein B-dependent hepatitis C virus secretion is inhibited by the grapefruit naringenin[END_REF]. Brefeldin A, which blocks the communication between the ER and the Golgi, effectively inhibited ApoB protein secretion and HCVcc virion production in the Huh-7.5/JFH1 HCVcc model. Interestingly, HCV secretion was up-regulated by oleic acid and down-regulated by insulin, precisely mirroring ApoB secretion by the cells. Moreover, naringenin an inhibitor of MTTP strongly blocked virion release, while viral RNA replication was not affected. In addition, Chang et al. demonstrated that the level of apoE correlates with HCV infectivity in the Huh-7.5/JFH1 HCVcc model [START_REF] Chang | Human apolipoprotein e is required for infectivity and production of hepatitis C virus in cell culture[END_REF]. ApoE-specific monoclonal antibodies against ApoE as well as ApoE-specific siRNA strongly decreased the release of infectious virions. These findings demonstrate that apoE is required for HCV virion infectivity and production, suggesting that HCV virions are assembled as apoE-enriched lipoprotein particles (Fig 2).

Mechanisms of host cell response to infection and modulation by HCV

The host response of cells to virus and other microorganism infections (so-called innate immune response) involves several strategies aimed at controling and limiting the spread of infection. One of these is initiated by cellular sensors that recognise the presence of pathogen-associated molecular patterns and activate a cascade of signaling pathways leading £ To the best of their knowledge, authors have no real or perceived conflicts of interests.

to the expression of signaling proteins (type I interferons -IFNα/β-and inflammatory cytokines) and genes (Interferon stimulated genes, ISG) [START_REF] Gale | Evasion of intracellular host defence by hepatitis C virus[END_REF]. Another strategy by which the host may respond to viral infection is RNA interference (RNAi) [START_REF] Voinnet | Induction and suppression of RNA silencing: insights from viral infections[END_REF].

Interferon-stimulating gene pathways

Two proteins are likely to play a major part as sensors of intracellular viral HCV RNA, TRL3 and RIG-I. TLR3 (Toll-like receptor) is a member of the TLR familly that recognises dsRNAs [START_REF] Alexopoulou | Recognition of doublestranded RNA and activation of NF-kappaB by Toll-like receptor 3[END_REF]. It is expressed in many tissus including the liver [START_REF] Matsumoto | TLR3: interferon induction by double-stranded RNA including poly(I:C)[END_REF]. A recent report shows that TLR3 is localized not only on plasma membrane but also in the ER of unstimulated cells where it moves to dsRNAcontaining endosomes in response to dsRNA, and colocalizes with c-Src kinase in the lumen of endosomes containing dsRNA [START_REF] Johnsen | Toll-like receptor 3 associates with c-Src tyrosine kinase on endosomes to initiate antiviral signaling[END_REF]. The acidic pH of endosomes [START_REF] De Bouteiller | Recognition of double-stranded RNA by human toll-like receptor 3 and receptor signaling requires multimerization and an acidic pH[END_REF] and association to the c-Src kinase favor dsRNA binding to and dimerisation of TLR3, and initiation of signal transduction, respectively. As reviewed by others [START_REF] Bode | Subversion of innate host antiviral strategies by the hepatitis C virus[END_REF][START_REF] Matsumoto | TLR3: interferon induction by double-stranded RNA including poly(I:C)[END_REF], TLR3 mediates signal transduction through an adaptor protein TICAM-1/TRIF. Once activated, TRIF recruits several proteins including TRAF3, TRAF6, NAP1 and RIP1 to form a multifunctional complex. Within this complex, TRAF3 and NAP1 recruit two kinases, IKKε and TBK1, that phosphorylate IRF3 and IRF7, while RIP1 either mediates cell apoptosis or recruits IKKαβγ which in turns phosphoryales IkB and activates nuclear translocation of NFkB. Phosphorylated IRF3 and IFR7 translocate to the nucleus and cooperate with AP1 and NFkB to activate the transcription of IFNβ, while NFkB activates the expression of inflammarory cytokines. The TLR3-mediated pathway appears to be dependent on the virus genome and entry process, and on the type of host cell and other anti-viral properties of host cells. Interestingly, TLR3 signaling pathway seems to be defective in Huh7 cells [START_REF] Lanford | Antiviral effect and virus-host interactions in response to alpha interferon, gamma interferon, poly(i)-poly(c), tumor necrosis factor alpha, and ribavirin in hepatitis C virus subgenomic replicons[END_REF]Preiss et al., 2008).

RIG-I (Retinoic acid Induced Gene-I), an interferon sensitive gene (ISG), is a ubiquitous cytosolic CARD-containing DexD/H RNA helicase [START_REF] Yoneyama | The RNA helicase RIG-I has an essential function in double-stranded RNAinduced innate antiviral responses[END_REF]. RIG-I senses dsRNAs with secondary structures or with uncapped 5' triphosphate ends generated by several viruses with negative strand RNA, including HCV. A recent report identified the PolyU/UC region of the 3'-NTR of HCV as the major pathogen-associated molecular patterns of this virus [START_REF] Saito | Innate immunity induced by composition-dependent RIG-I recognition of hepatitis C virus RNA[END_REF]. Recent work suggested that in the absence of RNA, RIG-I is inhibited by intra-molecular interactions between the CARD and C-terminus domains, while RNA binding strongly stimulates ATPase activity and signaling [START_REF] Gee | Essential role of the N-terminal domain in the regulation of RIG-I ATPase activity[END_REF]. RIG-I activates the mitochondria-bound protein, IPS-1/MAVS/VISA/Cardif protein [START_REF] Hiscott | MasterCARD: a priceless link to innate immunity[END_REF] (referred to here as MAVS) through the N-terminal tandem CARD of RIG-I and the Nterminal CARD of MAVS (T. [START_REF] Saito | of innate antiviral defenses through a shared repressor domain in RIG-I and LGP2[END_REF][START_REF] Vitour | Regulation of interferon production by RIG-I and LGP2: a in self-control[END_REF]. MAVS associates with several proteins including TANK, FADD and RIP1 [START_REF] Lin | Negative regulation of the retinoic acid-inducible gene I-induced antiviral state by the ubiquitinediting protein A20[END_REF][START_REF] Meylan | Cardif is an adaptor protein in the RIG-I antiviral pathway and is targeted by hepatitis C virus[END_REF][START_REF] Xu | VISA is an adapter protein required for virus-triggered IFN-beta signaling[END_REF] and with proteins of the TRAF family, such as TRAF3 or TRAF6 [START_REF] Saha | Regulation of antiviral responses by a direct and specific interaction between TRAF3 and Cardif[END_REF]. Within this multifunctional complex, MAVS activates the IKKαβγ complex and the TBK1/IKKε kinases, thus provoking activation of NF-κB and IRF3, respectively (Fitzgerald et al., 2003;[START_REF] Sharma | Triggering the interferon antiviral response through an IKK-related pathway[END_REF]. TBK1 and IKKε present strong homologies and they both phosphorylate IRF3 [START_REF] Matsui | Cutting edge: Role of TANK-binding kinase 1 and inducible IkappaB kinase in IFN responses viruses in innate immune cells[END_REF]). Yet they differ in many aspects. IKKε, but not TBK1, associates with MAVS at the mitochondria and induces a subset of IFN-induced genes through phosphorylation of STAT1 which then allows binding of the ISGF3 complex to their promoter elements [START_REF] Tenoever | Activation of TBK1 and IKKvarepsilon kinases by vesicular stomatitis virus infection and the role of viral ribonucleoprotein in the development of interferon antiviral immunity[END_REF]. In contrast to IRF3, IRF7 is an ISG the expression of which is induced by IFNα/β after IRF3-mediated IFN induction. It thus appears that IFNα/β induction has two phases, early and late. IRF3 plays a £ To the best of their knowledge, authors have no real or perceived conflicts of interests. major part in both phases while IFR7 plays its major part in the late process [START_REF] Sato | Distinct and essential roles of transcription factors IRF-3 and IRF-7 in response to viruses for IFN-alpha/beta gene induction[END_REF]. Expression of IKKε can be induced in response to several factors, such as inflammatory stimuli and viral infections through the conjugated action of NF-κB and C/EBP transcription factors [START_REF] Kravchenko | IKKi/IKKepsilon plays a key role in integrating signals induced by pro-inflammatory[END_REF]. Interestingly, Huh-7.5 cells which are used for production of HCVcc particles (see section 4) appear to be deficient in RIG-I activity due to a mutation in the first CARD domain of the protein [START_REF] Sumpter | Regulating intracellular antiviral defense and permissiveness to hepatitis C virus RNA replication through a cellular RNA helicase, RIG-I[END_REF].

RNA interference

Micro RNAs (miR) constitute a class of non-coding RNA that are transcribed from miR-encoding genes by RNA polymerase II. miR transcripts are processed to mature 19-21 nucleotide length miR duplexes by two enzymes, Drosha in the nucleus and, after export to the cytosol by exportin 5, Dicer. The miR duplex then binds the RNA induced silencing complex (RISC) which targets the complementary mRNAs after hybrization to the antisense miR strand. Depending on the degree of homology between the antisense miR and the target mRNA, the latter is either degraded or its rate of translation is reduced, making RNAi a powerfull tool for gene silencing. Most of miR binding sites are located in the 3'-NTR of mRNAs. This process of RNAi has been first recognized in plants and invertebrates as a mechanism of antiviral protection. However, the contribution of RNAi as another means of anti-HCV defence has only been recently explored [START_REF] Pedersen | Interferon modulation of cellular microRNAs as an antiviral mechanism[END_REF]. During HCV genome replication, dsRNA as well as hairpin structures are known to be formed [START_REF] Pedersen | Interferon modulation of cellular microRNAs as an antiviral mechanism[END_REF][START_REF] Wang | Hepatitis C virus core protein is a potent inhibitor of RNA silencing-based antiviral response[END_REF]. It is likely that these species are processed by Dicer so that siRNAs are produced and may induce HCV RNA silencing. However, the possibility that RNAi has a potent impact on HCV replication has been questioned [START_REF] Randall | Cellular cofactors affecting hepatitis C virus infection and replication[END_REF]. Ironically, the first miR whose effect on HCV has been documented in detail, i.e. miR-122, was shown to increase and not decrease HCV RNA accumulation, suggesting that this virus is able to use the cellular miR machinery to its own benefit. Jopling et al. demonstrated that miR-122 which is expressed at a high level in the human liver (as well as in Huh-7 cells) is required for an efficient replication of the viral RNA of genotypes 1 and 2a, although neither the rate of translation nor the turnover of viral RNA were affected [START_REF] Jopling | Positive and negative modulation of viral and cellular mRNAs by liver-specific microRNA miR-122[END_REF][START_REF] Jopling | Modulation of hepatitis C virus RNA abundance by a liver-specific MicroRNA[END_REF]. They identified two sites of miR-122 binding separated by 14 nucleotides in the extreme 5'-NTR of HCV RNA and showed that occupation of both site is required for increased RNA accumulation [START_REF] Jopling | Position-dependent function for a tandem miR-122-binding site located in the hepatitis C virus RNA genome[END_REF]. Although the mechanism of this stimulation is not yet fully understood, it is possible that either the miR-122/RISC-HCV genome complex recruits factors that enhance viral RNA abundance, or that miR-122 stimulates HCV translation by enhancing the association of ribosomes with the viral RNA at an early initiation stage [START_REF] Henke | microRNA-122 stimulates translation of hepatitis C virus RNA[END_REF]. Interestingly, the finding that ectopic expression of miR-122 in human kidney-derived cells substantially enhances HCV replication suggests that miR-122 may be responsible, at least in part, for the hepatic tropism of the virus [START_REF] Chang | Liver-specific miR-122 enhances the replication of hepatitis C virus in nonhepatic cells[END_REF]. It has to be emphasized that miR-122 has been shown to plays a significant role in promoting plasma cholesterol, lipid synthesis and inhibiting fatty-acid oxidation in the adult liver [START_REF] Esau | miR-122 regulation of lipid metabolism revealed by in vivo antisense targeting[END_REF]. Although this suggests that miR-122 is a possible therapeutic target for metabolic and cardiovascular diseases, it provides a direct functional link between HCV replication and lipid homeostasis. Recently, miR-199a* has been reported to target domain II of the IRES region of HCV and to strongly inhibit viral replication in HCVcc replicon systems of genotype 1b (Con1) and 2a (JFH1) [START_REF] Murakami | Regulation of the hepatitis C virus genome replication by miR-199a(*)[END_REF]. This effect was shown to be IFN-independent since the expression of several genes known to be controlled by IFN was not affected by miR-199a*. Whether miR-199a* triggers the degradation of viral RNA or inhibits its translation is presently unknown. However, the fact that miR-199a* also targets the 5'-NTR of HCV RNA suggests that the unexpected effect of £ To the best of their knowledge, authors have no real or perceived conflicts of interests. miR-122 does not stem from the fact that it targets sequences in the 5'-NTR. miR-122 and miR-199a* appear therefore to produce opposite effects on HCV replication. The observation that miR-122 is expressed in the liver at a greater level than miR-199a* and the finding that inhibiting the RNAi machinery (notably Drosha and Dicer) reduces HCV replication and infection in the Huh-7.5/J6 (2a)/JFH1 HCVcc replicon system suggest that the proviral effect of miR-122 dominates on the anti-viral effect of miR-199a* [START_REF] Randall | Cellular cofactors affecting hepatitis C virus infection and replication[END_REF]. Interestingly, using the Huh-7 cells and the JFH1 replicon system Pedersen et al. demonstrated that IFNα/β upregulates several miRs including miR-196, -296, -351, -431, and -448 which inhibit HCV infection and RNA replication. Moreover and in the meantime, IFN downregulates miR122 [START_REF] Pedersen | Interferon modulation of cellular microRNAs as an antiviral mechanism[END_REF]. It appears therefore that IFN is capable of orchestrating a large arsenal of antiviral agents not only including ISGs, but miRs as well (Fig

2).

Interferon signaling pathway and viral resistance

At present the treatment associating pegylated IFNα and ribavirin represents the only available therapy against HCV infection (Chevaliez & Pawlotsky, 2007a;Meurs & Breiman, 2007). Several reports have shown that IFNα has a direct antiviral effect on HCV replication in different culture models including replicons (Huh-7), productive replicons (Huh-7.5), immortalized non-neoplastic hepatocytes (PH5CH8), fetal and adult primary human hepatocytes [START_REF] Buck | Direct infection and replication of naturally occurring hepatitis C virus genotypes 1, 2, 3 and 4 in normal human hepatocyte cultures[END_REF][START_REF] Castet | Alpha interferon inhibits hepatitis C virus replication in primary human hepatocytes infected in vitro[END_REF][START_REF] Ikeda | Human hepatocyte clonal cell lines that support persistent replication of hepatitis C virus[END_REF][START_REF] Lanford | Antiviral effect and virus-host interactions in response to alpha interferon, gamma interferon, poly(i)-poly(c), tumor necrosis factor alpha, and ribavirin in hepatitis C virus subgenomic replicons[END_REF][START_REF] Lazaro | Hepatitis C virus replication in transfected and serum-infected cultured human fetal hepatocytes[END_REF][START_REF] Lindenbach | Complete replication of hepatitis C virus in cell culture[END_REF]. On binding its receptor, IFN triggers the formation of a complex between a heterodimer of IFN receptors IFNAR1 and IFNRA2 and two members of the Janus kinase family, Jak1 and Tyk2. This initiates a cascade of phosphorylation reactions that leads to the activation of STAT1 and STAT2 which in addition to IRF9 form the ISGF3 complex. ISGF3 then behaves as a transcription factor. It translocates to the nucleus and transactivates a battery of ISGs that exhibit interferon sensitive responsive elements (ISRE) in their promoter. ISG proteins exhibit antiviral activity such as IRF7 (which participates to IFNα production amplification; see above), protein kinase R (PKR), 2'-5' oligoadenylate synthase (OAS), ISG56, ISG20, RIG-I, etc [START_REF] Gale | Evasion of intracellular host defence by hepatitis C virus[END_REF]. Recently, strong evidence was provided that among other ISGs, viperin, a putative radical S-adenosyl-L-methionine (SAM) enzyme, is a potent inhibitor of HCV replicon [START_REF] Jiang | Identification of three interferon-inducible cellular enzymes that inhibit the replication of hepatitis C virus[END_REF]. This finding suggests that viperin represents a novel antiviral pathway that works together with other antiviral proteins, such as ISG20 and PKR, to mediate the IFN response against HCV infection.

However, the success of IFN therapy is limited to approximately 50% of patients in a genotype-dependent manner (Le [START_REF] Guillou-Guillemette | Genetic diversity of the hepatitis C virus: impact and issues in the therapy[END_REF]. IFN signaling pathway as well as RNAi appear therefore to be subverted by HCV. Indeed, recent studies have demonstrated that the virus has developed strategies to bloc innate cell response and inhibit the antiviral effect of IFN [START_REF] Bode | Subversion of innate host antiviral strategies by the hepatitis C virus[END_REF][START_REF] Gale | Evasion of intracellular host defence by hepatitis C virus[END_REF][START_REF] Randall | Interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures[END_REF]. Several HCV proteins interfere with the antiviral response of host cells to eventually repress the release of IFNβ, and with the IFNα signaling. NS3/NS4A has been reported to block IRF3 activation by proteolytic cleavage of TRIF adaptor (which controls in part the TLR3-mediated signaling) and MAVS (in the RIG-I-mediated signaling), as well as the ISG56-mediated response [START_REF] Foy | Regulation of interferon regulatory factor-3 by the hepatitis C virus serine protease[END_REF][START_REF] Li | Immune evasion by hepatitis C virus NS3/4A protease-mediated cleavage of the Tolllike receptor 3 adaptor protein TRIF[END_REF][START_REF] Meylan | Cardif is an adaptor protein in the RIG-I antiviral pathway and is targeted by hepatitis C virus[END_REF], although some of these observations have been questionned recently [START_REF] Nozaki | Tandem of lactoferrin-derived anti-hepatitis C virus peptide enhance antiviral activity in cultured human hepatocytes[END_REF]. HCV core inhibits STAT1 and induces the expression of SOCS3 which blocks ISFG3 formation [START_REF] De Lucas | Hepatitis C virus core protein downregulates transcription of interferon-induced antiviral genes[END_REF][START_REF] Yoshida | Activation of STAT3 by the hepatitis C virus core protein leads to cellular transformation[END_REF], although SOCS3 increased expression has also been observed in IFN-resistant replicons that lack core protein [START_REF] Zhu | Defective Jak-Stat activation in cells is associated with hepatitis C viral IFN-alpha resistance[END_REF]. E2 and NS5A have been shown to inhibit the activity of PKR which blocks translation by £ To the best of their knowledge, authors have no real or perceived conflicts of interests. phosphorylating eIF2α, via protein-protein interaction (Gale et al., 1998;[START_REF] Taylor | Inhibition of the interferon-inducible protein kinase PKR by HCV E2 protein[END_REF]. NS5A has been reported as well to interact with and inhibit the HCV genome cleavage by the ISG OAS/RnaseL [START_REF] Taguchi | Hepatitis C virus NS5A protein interacts with 2',5'-oligoadenylate synthetase and inhibits antiviral activity of IFN in an IFN sensitivity-determining regionindependent manner[END_REF]. In addition, NS5A induces the expression of IL8 which in turn negatively affects the antiviral effect of IFN (S. [START_REF] Girard | An altered cellular response to interferon and up-regulation of interleukin-8 induced by the hepatitis C viral protein NS5A uncovered by microarray analysis[END_REF][START_REF] Khabar | The alpha chemokine, interleukin 8, inhibits the antiviral action of alpha[END_REF]. While inhibiting the host innate or the IFN-induced defences, HCV proteins also protect infected cells from apoptosis [START_REF] Bode | Subversion of innate host antiviral strategies by the hepatitis C virus[END_REF]. In this respect, HCV core protein has been shown to inhibit TNFα-or FAS-mediated activation of caspase 8, p21 and p53 expression, or to enhance Bcl-XL, Bcl2 and cyclin D1 expression or the Raf1/MAPK signaling pathways [START_REF] Hassan | Activation of RB/E2F signaling pathway is required for the modulation of hepatitis C virus core protein-induced cell growth in liver and non-liver cells[END_REF][START_REF] Saito | Hepatitis C virus core protein inhibits tumor necrosis factor alpha-mediated apoptosis by a protective effect involving cellular FLICE inhibitory protein[END_REF]. E2 was reported to inhibit apoptosis by negatively regulating the cytochrome c release from mitochondria after TRAIL activation [START_REF] Lee | E2 of hepatitis C virus inhibits apoptosis[END_REF]. NS3/NS4A protects liver damage in animals treated with lethal doses of TNFα combined with D-galactosamine, while NS3 interacts with and affects p53 activity [START_REF] Frelin | The hepatitis C virus and immune evasion: non-structural 3/4A transgenic are resistant to lethal tumour necrosis factor alpha mediated liver disease[END_REF][START_REF] Tanaka | Singlepoint mutations of hepatitis C virus NS3 that impair p53 interaction and anti-apoptotic activity of NS3[END_REF]. Finally, NS5A has been shown to attenuate liver damage in animals treated with TNFα, to decrease p53 DNA binding and to activate PI3K [START_REF] Gong | HCV NS5A abrogates p53 protein function by interfering with p53-DNA binding[END_REF][START_REF] Majumder | Hepatitis C virus NS5A protein impairs TNF-mediated hepatic apoptosis, but not by an anti-FAS antibody, in transgenic mice[END_REF][START_REF] Street | Hepatitis C virus NS5Amediated activation of phosphoinositide 3-kinase results in stabilization of cellular and stimulation of beta-catenin-responsive transcription[END_REF]. However, some of these observations appear to be controversial depending on the cellular model used and this is not unexpected (see below). In a recent work (Hazari et al., 2007), HCV replicons with high and low activation of the IFN-promoter were cultured for a prolonged period of time in the presence of IFNα. Stable replicon cell lines with resistant phenotype were isolated and characterized for their ability to sustain viral replication in the presence of IFN. Reduced signaling and IFN-resistant phenotype was found in all Huh-7 cell lines even after eliminating HCV, suggesting that cellular factors were involved. Resistant phenotype in the replicons was not due to lack of interferon receptor expression but instead to defect in the JAK-STAT signaling pathway due to strongly reduced expression of Tyk2 and Jak-1 protein and phosphorylation of STAT 1 and STAT 2 proteins. These results emphasize the fact that depending on the cellular clone investigated, different cellular behavior may be observed in the resistance to IFN.

In addition to interfere with IFN signaling pathway, viruses in general [START_REF] Hemmes | The NS3 protein of Rice hoja blanca tenuivirus suppresses RNA silencing in plant and insect hosts by binding both siRNAs and miRNAs[END_REF] and HCV in particular are able to interfere with RNAi-dependent antiviral effect. Using cell lines that express constitutively green fluorescent protein (GFP) and in which HCV protein expression is inducible, Ji et al. demonstrated that silencing of GFP gene by siRNA was inhibited in the presence of the full length HCV polyprotein ; HCV E2 protein was shown to be responsible for this inhibition [START_REF] Ji | Suppression of short interfering RNA-mediated gene silencing by the structural proteins of hepatitis C virus[END_REF]. Consistent with this observation, coprecipitation assays demonstrated that E2 binds to Argonaute-2 (Ago-2), a member of the RISC machinery, thus providing a basis for the suppression of RNAi by the virus. HCV core protein was shown to inhibit RNAi induced by shRNAs but not by synthetic siRNAs in various mammalian cells [START_REF] Chen | HCV core protein interacts with Dicer to antagonize RNA silencing[END_REF][START_REF] Wang | Hepatitis C virus core protein is a potent inhibitor of RNA silencing-based antiviral response[END_REF]. These authors provided evidence that core directly interacts with Dicer which notably processes dsRNAs into siRNAs. Through deletion analysis, the N-terminal domain of core was shown to be responsible for inhibition of Dicer and RNA silencing activity. Thus, HCV core protein may abrogate host cell RNAi-mediated defense by suppressing the ability of Dicer to process precursor dsRNAs into siRNAs. It has been proposed that since pre-miRNA are also cleaved by Dicer, the inhibition of RNAi by HCV core may suppress the maturation of miRNAs in the host liver, thus influencing the expression of certain classes of gene regulatory molecules leading to liver pathologies including hepatocarcinogenesis [START_REF] Wang | Hepatitis C virus core protein is a potent inhibitor of RNA silencing-based antiviral response[END_REF].

Cellular models for HCV investigations

Cellular models for investigating in vitro HCV infection necessitate two major actors: an appropriate cell type and a practical form of the virus. HCV genome replication occurs £ To the best of their knowledge, authors have no real or perceived conflicts of interests.

primarily within hepatocytes, although other cell types have been shown to be permissive as well although to a much lower extent, including peripheral blood mononuclear cells [START_REF] Cribier | In vitro infection of blood mononuclear cells by hepatitis C virus[END_REF][START_REF] Lerat | Specific detection of hepatitis C virus minus strand RNA in hematopoietic cells[END_REF], biliary cells [START_REF] Loriot | Permissiveness of human biliary epithelial cells to infection by hepatitis C virus[END_REF] and enterocytes [START_REF] Deforges | of hepatitis C virus proteins in epithelial intestinal cells in vivo[END_REF]. Hence, this review will focus on primary adult, fetal or immortalised hepatocytes and Huh-7 hepatoma cells and derivatives which have been largely used in recent developments. On the other hand, although the most natural form of the virus is present in the blood of infected patients as HCVser LVP [START_REF] Andre | Characterization of low-and very-low-density hepatitis C virus RNAcontaining particles[END_REF][START_REF] Kanto | Density analysis of hepatitis C virus particle population in the circulation of infected hosts: implications for virus neutralization or persistence[END_REF][START_REF] Miyamoto | Extraordinarily low density of hepatitis C virus estimated by sucrose density gradient centrifugation and the polymerase chain reaction[END_REF][START_REF] Prince | Visualization of hepatitis C virions and putative defective interfering particles isolated from lowdensity lipoproteins[END_REF][START_REF] Thomssen | Density heterogeneities of hepatitis C virus in human sera due to the binding of beta-lipoproteins and immunoglobulins[END_REF], several surrogate forms of HCV virions have been developed including, transfected partial or full length HCV genomes, viruslike particles (HCV-LP) [START_REF] Baumert | Hepatitis C virus structural proteins assemble into viruslike particles in insect cells[END_REF], pseudotyped retroviral particles (HCVpp) (Bartosch et al., 2003;[START_REF] Hsu | Hepatitis C virus glycoproteins mediate pH-dependent cell entry of pseudotyped particles[END_REF][START_REF] Mckeating | Diverse hepatitis C virus glycoproteins mediate viral infection in a CD81-dependent manner[END_REF], or cell culture particles (HCVcc) [START_REF] Cai | Robust production of infectious hepatitis C virus (HCV) from stably HCV cDNA-transfected hepatoma cells[END_REF][START_REF] Lindenbach | Complete replication of hepatitis C virus in cell culture[END_REF][START_REF] Wakita | Production of infectious hepatitis C virus in tissue culture from a cloned viral genome[END_REF][START_REF] Zhong | hepatitis C virus infection in vitro[END_REF]. Recent data suggest that the nature of the virus is an important point to take into account regarding for example the cell entry process [START_REF] Farquhar | Primary hepatocytes as targets for hepatitis C virus replication[END_REF]. Several cellular models combining cells of various origin and various forms of virus have been developed in recent years. They all represent valuable tools for investigating HCV biology and antiviral therapeutics.

HCV-like particles (HCV-LP)

The production of HCV-LP in insect cells using recombinant baculovirus expressing the cDNA of the HCV structural proteins (HCV-J strain, genotype 1b), has been first reported by [START_REF] Baumert | Hepatitis C virus structural proteins assemble into viruslike particles in insect cells[END_REF]. These studies demonstrated that HCV core and envelope proteins E1 and E2 were sufficient for viral particle assembly, presumably from the ER, and that HCV RNA was selectively incorporated into the particles. These particles exhibited a density of 1.14 to 1.22 g/cm 3 in sucrose density gradients (depending on studies), and a morphology (double-shelled particles 35 to 49 nm in diameter) similar to that of HCV particles detected in HCV infected chimpanzee liver. HCV-LPs were not secreted from cells but rather extracted from intracellular compartment, and were poorly infectious. This last point is a great advantage for the use of these particles in immunotherapeutic strategies (see section 7). A number of papers have been published on the use of these particles to investigate the structure and function of core and envelope proteins [START_REF] Choi | Hepatitis C virus core protein is efficiently released into the culture medium in insect cells[END_REF][START_REF] Clayton | Analysis of antigenicity and topology of E2 glycoprotein present on recombinant C virus-like particles[END_REF][START_REF] Owsianka | Functional analysis of hepatitis C virus E2 glycoproteins and virus-like particles £ To the best of their knowledge, authors have no real or perceived conflicts of interests. reveals structural dissimilarities between different forms of E2[END_REF] as well as the HCV-host cell interactions and virion entry, notably the interaction with and activation of dendritic cells (Barth et al., 2005). HCV-LP binding to and entry in cells is inhibited with anti-E1 or anti-E2 antibodies, demonstrating the critical role of E1 and E2 proteins in this process [START_REF] Triyatni | Interaction of hepatitis C virus-like particles and cells: a model system for studying viral binding and entry[END_REF]. In addition, these authors reported that HCV-LPs binding to several human hepatic cells (primary hepatocytes, HepG2, HuH7, and NKNT-3) and T-cells (Molt-4) was dose-and calcium-dependent but CD81-independent. Interestingly, preincubation of HCV-LPs with VLDL, LDL, or HDL inhibited their binding to cells, while preincubation of cells with VLDL, LDL, HDL, or anti-LDL-R antibody did not. Barth et al. demonstrated that binding of the HCV envelope glycoproteins to specific heparan sulfate structures on host hepatocarcinoma cells is critical for initiation of viral infection. This suggests that this interaction is a target for the antiviral host immune systems in responses to HCV infection in vivo [START_REF] Barth | Viral and cellular determinants of the hepatitis C virus envelope-heparan sulfate interaction[END_REF]. Conflicting results were reported on the role of the 5'-NTR on virion assembly and morphology in insect cells: the 5'-NTR of HCV genotype 1a is implicated in the assembly process of HCV structural proteins in insect cells, although no evidence for a direct interaction between the 5'-NTR and structural proteins has been reported [START_REF] Girard | Effect of the 5' non-translated region on self-assembly of hepatitis C virus genotype 1a structural proteins produced in insect cells[END_REF]. However, Zhao et al. reported that HCV 5'-NTR is not required for the assembly of HCV-like particles in insect £ To the best of their knowledge, authors have no real or perceived conflicts of interests. cells, that is, the core and envelope proteins are sufficient for viral particle formation [START_REF] Zhao | No requirement of HCV 5'NCR for particles assembly in insect cells[END_REF].

Other versions of HCV-like particles were engineered. Using a recombinant Semliki Forest Virus (SFV) subreplicon expressing genes encoding HCV structural proteins (genotype 1a), Blanchard et al. succeeded in producing HCV-LPs in mammalian BHK-21 cells [START_REF] Blanchard | Hepatitis C virus-like particle morphogenesis[END_REF]. Ezelle et al. used a recombinant Vesicular Stomatitis Virus (VSV) vector expressing HCV core, E1, and E2 (genotype 1b) to generate HCV-LPs possessing properties similar to the ultrastructural properties of HCV virions [START_REF] Ezelle | Generation of hepatitis C virus-like particles by use of a recombinant vesicular stomatitis virus vector[END_REF]. Both versions of SFV-and VSV-derived HCV-LPs were used to investigate the signal peptide peptidase processing of core protein, the interaction of structural proteins with the ER, and their role in HCV particle assembly and morphogenesis [START_REF] Ait-Goughoulte | Core protein cleavage by signal peptide peptidase is required for hepatitis C virus-like particle assembly[END_REF]Blanchard et al., 2003a;Blanchard et al., 2003;[START_REF] Ezelle | Generation of hepatitis C virus-like particles by use of a recombinant vesicular stomatitis virus vector[END_REF][START_REF] Hourioux | Core protein domains involved in hepatitis C virus-like particle assembly and budding at the endoplasmic reticulum membrane[END_REF].

HCV pseudotyped particle (HCVpp)

Pseudotyped particles have been constructed on the basis of vesicular stomatitis virus or influenza virus with chimeric E1 and E2 proteins [START_REF] Flint | Functional analysis of cell surface-expressed hepatitis C virus E2 glycoprotein[END_REF][START_REF] Lagging | Functional role of hepatitis C virus chimeric glycoproteins in the infectivity of pseudotyped virus[END_REF]Matsuura et al., 2001). These proteins had to be modified in their transmembrane domain to allow transport and assembly on the plasma membrane of host cells. However, these modifications affected the conformation and function of the E1-E2 complex, so that their use has been controversial. In 2003, Bartosch et al. generated highly infectious HCVpp by assembling the full length E1/E2 glycoproteins on retroviral core proteins derived from mouse leukemia virus (MLV). These particles were produced in 293T cells following transfection with three expression vectors coding for E1/E2, the MLV gag-pol core proteins and a packaging-competent MLV-derived genome encoding GFP as a marker. HCVpp harvested from cell supernatant were purified and shown to contain both E1 and E2 glycoproteins as well as the gag-pol core MLV proteins. Although E1 and E2 are supposed to be sequestered in the ER, FACS analysis revealed that a small fraction of these proteins reaches the cell surface, the typical site of MLV budding, thus suggesting that in this system HCVpp do indeed budd from the cell surface. Long-term expression of GFP was demonstrated suggesting that infection of target cells leads to integration of genomic DNA in the host. HCVpp generated from functional E1/E2 of different genotypes (1 to 6) exhibited different infectivity, although the reason for these differences are not clearly understood (Lavillette et al., 2005a). However, HCVpp generated with only either E1 or E2 exhibited strongly reduced infectivity. HCVpp were shown to infect primary human hepatocytes and several hepatocarcinoma cell lines including Huh-7, HepG2, and Hep3B. Infection of hepatocarcinoma cells (i.e. Huh-7) with these particles was used to investigate the HCV entry step in host cells and data confirmed the role of CD81 in this process (Bartosch et al., 2003;Bartosch et al., 2003a;[START_REF] Hsu | Hepatitis C virus glycoproteins mediate pH-dependent cell entry of pseudotyped particles[END_REF]Lavillette et al., 2005a;[START_REF] Mckeating | Diverse hepatitis C virus glycoproteins mediate viral infection in a CD81-dependent manner[END_REF]Zhang et al., 2004). This Huh-7/HCVpp model was also used to evaluate the neutralising activity of antibodies raised in the serum of chronically infected patients (see section 7). Receptor competition assays indicated that CD81 is used by HCVpp of all different genotypes, whereas siRNA silencing assays revealed that the level of SR-BI needed for efficient infection varies between genotypes. HCV E1 is believed to mediate fusion, while E2 has been shown to bind members of the cellular receptor complex, including CD81 and SR-BI. Three putative CD81 interaction sites on HCV E2 have been previously localized in three regions : region 1 474-492; 2 522-551; and 3, 612-619. Rothwangl et al. generated a series of alanine substitutions in E2 within these three regions to identify those residues that are critical for viral entry [START_REF] Rothwangl | Dissecting the role of putative CD81 binding regions of E2 in mediating HCV entry: putative CD81 binding region 1 is not involved in CD81 binding[END_REF]. The effect of each mutation was tested by challenging Huh-7 and Hep3B cells with HCVpp. Although most HCVpp mutants harboring £ To the best of their knowledge, authors have no real or perceived conflicts of interests. modifications within region 1 displayed greatly reduced infectivity (< 50% of wt HCVpp), the corresponding E2 proteins retained their capacity to bind soluble CD81, exhibited normal conformation, association with E1 and incorporation into HCVpp, suggesting that region 1 is not involved directly in the binding to CD81. In contrast, several mutants in regions 2 and 3 exhibited strongly reduced or no binding to CD81, suggesting a critical role of these regions in receptor binding, while the structure of the corresponding proteins was apparently correct as assessed by immunoprecipitation with appropriate antibodies. The lack of upstream peptides in proteins E1 and E2 is likely to affect their anchoring to the ER membrane, and in turn their glycosylation, folding, and heterodimerization, eventually leading to reduced infectivity. Using HCVpp, Bian et al. [START_REF] Bian | HCV envelope protein function is dependent on the peptides preceding the glycoproteins[END_REF]) demonstrated that at least 14 amino acids from the core C-terminus are required for E1 function and that at least 12 amino acids from the E1 C-terminus are required for E2 function. These peptides appear to influence the glycosylation of E1 and E2. Furthermore, the peptides upstream of E1 and E2 can be exchanged by those from another genotype.

Blanchard et al. studied the cellular mechanisms of HCV endocytosis using HCVpp [START_REF] Rouille | Subcellular localization of hepatitis C virus structural proteins in a cell culture system efficiently replicates the virus[END_REF]. The data obtained with siRNA-mediated clathrin heavy chain depletion and with chlorpromazine (an inhibitor of clathrin-coated pit formation at the plasma membrane) suggest that HCV enters target cells via this pathway, followed by a fusion step within an acidic endosomal compartment. Several reports [START_REF] Dreux | High density lipoprotein inhibits hepatitis C virus-neutralizing antibodies by cell entry via activation of the scavenger receptor BI[END_REF][START_REF] Voisset | High density lipoproteins facilitate hepatitis C virus entry through the receptor class B type I[END_REF][START_REF] Voisset | High-density lipoproteins reduce the neutralizing effect of hepatitis C virus (HCV)-£ To the best of their knowledge, authors have no real or perceived conflicts of interests. infected patient antibodies by promoting HCV entry[END_REF] demonstrated that HCVpp entry in Huh-7 and subsequent endocytosis is accelerated by the presence of HDL (high density lipoprotein), although these lipoproteins do not interact directly with E1/E2. Notably, the time lag between HCV particles binding and internalisation, which is likely to reflect the receptor complex assembly, is reduced by HDL. The authors suggest that by interacting with SR-BI (an HDL as well as HCV receptor) HDL significantly increase CD81 recrutement that leads eventually to HCV internalisation. Thus, HDL likely acts by reducing the exposition of HCV to neutralising antibodies. The authors suggest that the HDL/SR-BI interaction interferes with antibodies blocking E2 binding to CD81, but not those antibodies targeting E1. Drugs inhibiting SB-BI activity (BLT4) or silencing of this receptor by siRNA strongly reduced the HDL-mediated effects on HCV entry. von Hahn et al. observed that oxidized (but not native) low-density lipoprotein (oxLDL), a ligand of SR-BI, inhibited HCVpp (gen 1a, 1b, 2a) entry in hepatoma (Hep3B, Huh-7 or HepG2/CD81) cells with a IC50 of 1.5 µg/mL, that is within the range of oxLDL in human plasma [START_REF] Von Hahn | Oxidized low-density lipoprotein inhibits hepatitis C virus cell entry in human hepatoma cells[END_REF]. However, binding of soluble E2 to SR-BI or CD81 was not affected by oxLDL, suggesting that oxLDL does not act as a simple receptor blocker. At the same time, oxLDL incubation altered the biophysical properties of HCVpp, suggesting a ternary interaction of oxLDL with both virus and target cells. HCVpp systems have been widely used in the screening of anti-viral compounds and in immunological studies (see below section 6 and 7). Yoo et al. and Dash et al. were the first to report the replication of transcripts of a putative full-length cDNA clone of hepatitis C virus type 1 (HCV-1) after transfection in Huh7 and HepG2 cell lines [START_REF] Dash | Transfection of HepG2 cells with infectious hepatitis C virus genome[END_REF][START_REF] Yoo | Transfection of a differentiated human hepatoma cell line (Huh7) with in vitro-transcribed hepatitis C virus (HCV) RNA and establishment of a long-term culture persistently infected with HCV[END_REF]. However, in both cases the HCV genome lacked the 3'-NTR region which appears to be mandatory for viral genome replication (Kolykhalov et al., 2000;[START_REF] Yanagi | In vivo analysis of the 3' untranslated region of the hepatitis C virus after in vitro mutagenesis of an infectious cDNA clone[END_REF]. Hence, the use of these replicons has been controversial [START_REF] Bartenschlager | Replication of hepatitis C virus[END_REF]. Only non structural proteins are involved in the control of virus genome replication. Thus, to construct a sub-genomic replicon system, Lohman et al. deleted the entire structural region of a cloned HCV consensus genome Con1 (genotype 1b) and inserted a selectable gene marker (neomycin phosphotransferase, £ To the best of their knowledge, authors have no real or perceived conflicts of interests.

HCV subgenomic and genomic replicon systems

Neo) downstream of the HCV IRES; the translation of NS proteins was controlled by the IRES of the encephalomyocarditis virus which was located downstream of the Neo gene [START_REF] Lohmann | of subgenomic hepatitis C virus RNAs in a hepatoma cell line[END_REF]. This construct was transfected in Huh-7 cells and selection with G418 allowed to isolate resistant colonies. Positive colony examination revealed a very high level of HCV RNA synthesis with 1000 to 5000 copies per cell. Although this high level of replication did not apparently affect cell morphology, more extensive analysis revealed some alterations of the ER that were linked to the presence of non structural proteins [START_REF] Gosert | Identification of the hepatitis C virus RNA replication complex in Huh-7 cells harboring subgenomic replicons[END_REF]. In addition, the rate of viral RNA replication was dependent on the cell cycle, thus emphasizing the occurrence of functional interactions between the virus and the host cell [START_REF] Pietschmann | Tissue culture and animal models for hepatitis C virus[END_REF]. Extensive analysis demonstrated that both viral and host cell determinants were important for the maintenance of a high level of replication. Among these, some mutations in all NS proteins (either individually or in combination) have been shown to confer a better efficiency to the replicon [START_REF] Blight | Efficient initiation of HCV RNA in cell culture[END_REF][START_REF] Friebe | Sequences in the 5' nontranslated region of hepatitis C virus required for RNA replication[END_REF][START_REF] Guo | Effect of alpha interferon on the hepatitis C virus replicon[END_REF][START_REF] Krieger | Enhancement of hepatitis C virus RNA replication by cell culture-adaptive mutations[END_REF][START_REF] Lohmann | Mutations in hepatitis C virus RNAs conferring cell culture adaptation[END_REF]. These mutations appear not to reduce the cytopathic potential of the replicon, nor to affect the enzyme activity of NS3 and NS5B proteins. It is likely that they confer adaptation to the host cell. Interestingly, several mutations have been idenfied in the ISDR (interferon sensitivity-determining region) of NS5A [START_REF] Bartenschlager | Hepatitis C virus molecular clones and their replication capacity in vivo and in cell culture[END_REF]. On the other hand, the host cell seems to contribute to the efficiency of replicon as evidenced by the strong changes in replication rate as a function of the number of passages of the cell line [START_REF] Lohmann | Viral and determinants of hepatitis C virus RNA replication in cell culture[END_REF]. Significant progress has been made recently in the development of efficient high throughput screening (HTS) HCV replication systems [START_REF] Pietschmann | Tissue culture and animal models for hepatitis C virus[END_REF], as well as replicons expressing green fluorescent protein [START_REF] Ikeda | Efficient of a full-length hepatitis C virus genome, strain O, in cell culture, and development of a luciferase reporter system[END_REF][START_REF] Moradpour | Insertion of green fluorescent protein into nonstructural protein 5A allows direct visualization of functional hepatitis C virus replication complexes[END_REF]. Kim et al. used a subgenomic HCV replicon (Huh7/Rep-Feo) derived from a chimpanzee infectious clone (strain HCV-N, genotype 1b) [START_REF] Kim | A cellbased, high-throughput screen for small molecule regulators of hepatitis C virus replication[END_REF]. Huang et al. developed another form of HTS subgenomic replicon (PLN5 and PLNCP54) based on Huh-7 cells and the HCV Con1b sequence [START_REF] Huang | Discovery characterization of substituted diphenyl heterocyclic compounds as potent and s elective inhibitors of hepatitis C virus replication[END_REF]. Replicon PLNCP54 included NS2 sequence allowing identification of compounds inhibiting NS2/3 cleavage through differential screening with PLN5. Each plasmid carried a firefly luciferase reporter under the control of the poliovirus IRES. The use of these replicons allows basic and applied studies on viral protein expression and subcellular localisation, the role of cis-acting RNA elements in replication, the antiviral effect of interferon, as well as on the screening of antiviral molecules targeting the IRES, protease (NS2/NS3, NS3/NS4), helicase (NS3) and polymerase (NS5B).

HCV production in Huh-7 cell culture models (HCVcc)

Apparently, the major reason for the lack of virus particle production in full length replicon systems is that adaptative mutations that increase RNA replication are deleterious for virus particle assembly or release. In other words, high levels of RNA and protein production do not necessarily lead to efficient virus production. In contrast to replicon of genotype 1, replicon JFH1 (genotype 2a) generated from the serum of a patient with a fulminant infection exhibited an unusual high rate of RNA replication (20 times greater than that of Con-1) when transfected in Huh-7 cells, without requiring adaptative mutations [START_REF] Kato | Efficient replication of the genotype 2a hepatitis C virus subgenomic replicon[END_REF]. When a full length genome HCV RNA JFH1 (2a) or intragenomic or intergenomic chimera (structural/non structural proteins) between J6 (2a)/JFH1, H77(1a)/JFH1 or S52(3a)/JFH1 were transfected in Huh-7-derived cells, notably the Huh-7.5 clone, several groups observed that infectious HCV particles (HCVcc) were secreted in the extracellular medium [START_REF] Gottwein | Robust hepatitis C genotype 3a cell culture releasing adapted intergenotypic 3a/2a (S52/JFH1) viruses[END_REF][START_REF] Lindenbach | Complete replication of hepatitis C virus in cell culture[END_REF][START_REF] Lindenbach | Cell culture-grown hepatitis C virus is infectious in vivo and can be recultured in vitro[END_REF][START_REF] Pietschmann | Construction and characterization of infectious intragenotypic and intergenotypic hepatitis C virus chimeras[END_REF][START_REF] Rouille | Subcellular localization of hepatitis C virus structural proteins in a cell culture system efficiently replicates the virus[END_REF][START_REF] Wakita | Production of infectious hepatitis C virus in tissue culture from a cloned viral genome[END_REF][START_REF] Yi | Production of infectious genotype 1a hepatitis C virus (Hutchinson strain) in cultured human hepatoma cells[END_REF][START_REF] Zhong | hepatitis C virus infection in vitro[END_REF]. The data suggest that determinants within the structural proteins have a major impact on kinetic and efficiency £ To the best of their knowledge, authors have no real or perceived conflicts of interests.

of virus assembly and release. Sucrose density gradient analysis of HCVcc particles revealed that core, E1, E2 and HCV RNA co-sedimented at the same density, approximately 1.15-1.17. Using anti-E2 antibodies, spherical HCV particles of 50 to 65 nm could be visualised by immunoelectronic microscopy. These particles were shown to be infectious for Huh-7 cells as well as in vivo for chimpanzees. Interestingly, virions recovered from these animals exhibited a greater specific infectivity and lower density (i.e. 1.09) than those recovered in cell culture, suggesting increased association of virions with lipoproteins [START_REF] Andre | Hepatitis C virus particles and lipoprotein metabolism[END_REF][START_REF] Lindenbach | Cell culture-grown hepatitis C virus is infectious in vivo and can be recultured in vitro[END_REF]. CD81 specific antibodies as well as serum from chronically infected patients significantly inhibited HCVcc-mediated infection of Huh-7 cells. Interestingly, confocal imaging of Huh7.5 cells infected with either J6/JFH1, or S52/JFH1 revealed that in HCVinfected cells lipid droplets are redistributed from the entire cytoplasm to a perinuclear area and colocalized with core. However, no difference in lipid content between infected and noninfected cells was detected. Recently, full-length HCV DNA constructs (genotypes 1a, 1b, and 2a) in HCV-ribozyme expression plasmids were shown to generate extracellular HCVcc particles when transfected into Huh-7.5 cells [START_REF] Heller | An in vitro model of hepatitis C virion production[END_REF][START_REF] Kato | Production of infectious hepatitis C virus of various genotypes in cell cultures[END_REF]. As found for HCVpp entry [START_REF] Rouille | Subcellular localization of hepatitis C virus structural proteins in a cell culture system efficiently replicates the virus[END_REF], HCVcc entry was reported to be pH-dependent and sensitive to concanamycin A and NH 4 Cl [START_REF] Tscherne | Time-and temperature-dependent activation of hepatitis C virus for low-pH-triggered entry[END_REF].

Human hepatocarcinoma and immortalized hepatocyte (IHH)-based systems

Several lines of immortalized human hepatocytes have been generated. PH5CH cell lines (clones PH5CH1 to PH5CH8) were produced by immortalisation of primary human hepatocytes by the large T antigen of the simian virus SV40 [START_REF] Ikeda | Human hepatocyte clonal cell lines that support persistent replication of hepatitis C virus[END_REF]. HuS-E and HuS-T cell lines were generated by immortalisation of primary human hepatocytes with vectors expressing hTERT (telomerase) and either E6/E7 genes of human papiloma virus or the large T of SV40, respectively [START_REF] Aly | Serum-derived hepatitis C virus infectivity in interferon regulatory factor-7-suppressed human primary hepatocytes[END_REF]. Immortalized human hepatocytes (IHH) were generated by transfecting primary human hepatocytes with an HCV core (genotype 1a) expression plasmid. These cells exhibited a significant telomerase activity [START_REF] Ray | Hepatitis C virus core protein promotes of primary human hepatocytes[END_REF]. FLC4 is a cell line derived from a human hepatocellular carcinoma [START_REF] Aoki | A human liver cell line exhibits efficient translation of HCV RNAs produced by a recombinant expressing T7 RNA polymerase[END_REF] ; these cells were maintained in culture for long periods in a radial flow bioreactor. All these cell lines were shown to express some hepatocyte phenotypic markers (as assessed by RT-PCR analysis) as previously observed with other immortalized hepatocyte preparations or hepatocellular carcinoma cells.

PH5CH, FLC4 and HuS-E were shown to be sentitive to HCV infection and permissive to viral genome replication after being inoculated with serum from chronically infected patients (HCVser). Anti-CD81 antibodies and IFN were shown to block infection in these models. PH5CH and FLC4 cell lines, but not HuS-E, were shown to produce virions in the extracellular medium for a long period (4 to 8 weeks) after infection. Hyper variable region quasispecies analysis in FLC4 cells versus inoculum revealed a different pannel of sequences in the cellular viral RNA as compared with the inoculum RNA, suggesting adaptation of HCV to the conditions prevailing in these cells [START_REF] Aizaki | Production and release of infectious hepatitis C virus from human liver cell cultures in the three-dimensional radial-flow bioreactor[END_REF][START_REF] Murakami | Dynamic behavior of hepatitis C virus quasispecies in a long-term culture of the threedimensional radial-flow bioreactor system[END_REF]. When FLC4 cells were transfected with HCV H77 (1a) RNA and cultured in a bioreactor, HCV RNA was detected in the extracellular medium almost two months posttransfection and remained at a level of 10 5 -10 6 copies/day for another three months, consistent with the relative amount of core protein (13pg/day). Extracellular medium analysis revealed the presence of virus-like particles that sedimented in the range 1.03-1.06, that is, close to the range in which HCV particles sediment from human serum (1.03-1.12) [START_REF] Andre | Characterization of low-and very-low-density hepatitis C virus RNAcontaining particles[END_REF]. Finally, when the medium collected 93-95 days post transfection was incubated with naive FLC4 cells both HCV RNA and core protein were detected in the extracellular medium between 15 and 30 days post infection.

£ To the best of their knowledge, authors have no real or perceived conflicts of interests.

IHH were transfected by electroporation with the HCV H77 (1a) RNA. HCV RNA and protein expression were detected in cell cultures for up to 12 days, and the cultures were discontinued for lack of growth after 2 weeks. Similarly, IHH transfected with HCV RNA JFH1 exhibited signs of virus replication as assessed by intracellular RNA and localization of NS3 protein by immunofluorescence. The appearance of virus-like particles in H77 RNAtransfected IHH indicated that HCV 1a replicates and assembles as virus particles in these cells. Moreover, the presence of HCV 5'-NTR was detected in culture medium of IHH cells transfected with HCV genome. Interestingly, medium from cultured cells was able to infect naive IHHs as assessed by HCV RNA acccumulation and immonudetection of NS proteins at day 4 post-infection. This infection was inhibited by serum from HCV-infected patient, displaying neutralizing activity against the vesicular stomatitis virus/HCV pseudotype. Interestingly, while JFH1 replicates with a higher efficiency than H77 at the RNA level in Huh-7 cells or its derivatives, similar levels of virus genome copies were detected in the supernatant of H77 and JFH1 RNA-transfected IHH cultures; the number of focus-forming units per milliliter (relative to H77 and JFH1) was also similar. This suggests an equal ability of IHH to respond to a wider range of HCV genotypes as compared to Huh-7 cells and derivatives. However, the production of HCV particles in these cells appears to be restricted to a short period not exceeding a few days. Interestingly, recent studies demonstrated that HCV infection triggers in these cells both IFN signaling pathway and an autophagic response [START_REF] Ait-Goughoulte | C virus genotype 1a growth and induction of autophagy[END_REF][START_REF] Kanda | Generation of infectious hepatitis C virus in immortalized human hepatocytes[END_REF][START_REF] Kanda | Hepatitis C virus infection induces the beta interferon signaling pathway in immortalized human hepatocytes[END_REF]. [START_REF] Carloni | Susceptibility of human liver cell cultures to hepatitis C virus infection[END_REF][START_REF] Iacovacci | Molecular characterization and dynamics of hepatitis C virus replication in human fetal hepatocytes infected in vitro[END_REF]. For this purpose, human fetal hepatocytes were grown in serum-free medium and innoculated with HCVser. Genomic and replicative viral RNA strands and viral proteins were detected in hepatocytes 5 days after inoculation. Virions were released from the infected cells into the medium and could be serially passaged three times into fresh liver cell cultures. More recently, Lazaro et al. [START_REF] Lazaro | Hepatitis C virus replication in transfected and serum-infected cultured human fetal hepatocytes[END_REF] characterized long-term, serum-free primary and passaged cultures of nontransformed hepatocytes from human fetal liver. After transfection of these hepatocytes with genotype 1a HCV (p90/HCVFLpU of genotype 1a) or infection with HCVser of genotypes 1, 2, and 3, medium analysis revealed up to 10 6 copies/ml of HCV RNA exhibiting a cyclic pattern; virus-like particles (density 1.17) were detected in the medium of transfected hepatocytes. Viral infection (from transfected cells) could be transmitted to naive cells, and the infection was shown to be inhibited by IFN. Evidence for cell death in infected hepatocytes suggests that HCV has some cytotoxic effects in this model. However, the authors could not establish whether cell death is associated with HCV levels in the medium.

Human fetal hepatocyte cultures (HFH)

Carloni et al. and Iacovacci et al. investigated the infection of human fetal hepatocytes by hepatitis C virus

Primary human hepatocyte cultures (PHH)

Primary human hepatocytes are prepared by collagenase perfusion of either human liver segments resected for medical purpose (metastasis, adenoma, hepatocellular carcinoma) or from livers that have been refuted for transplantation. Plating substrates and chemicallydefined media allow these cells to be maintained in primary culture for weeks with preservation of several hepatocyte phenotypic markers such as production of plasma proteins, blood coagulation factors, urea, expression of detoxications systems (cytochromes P450, conjugation enzymes, membrane transporters), response to physiopathological stimulii in terms of signaling pathway activation, etc (Guguen- [START_REF] Guguen-Guillouzo | Human hepatocyte cultures[END_REF][START_REF] Guillouzo | Liver cell models in in vitro toxicology[END_REF][START_REF] Hewitt | Primary hepatocytes: current understanding of the regulation of metabolic enzymes and transporter proteins, and pharmaceutical practice for the use of £ To the best of their knowledge, authors have no real or perceived conflicts of interests. hepatocytes in metabolism, enzyme induction, transporter, clearance, and hepatotoxicity studies[END_REF][START_REF] Pichard | Human hepatocyte culture[END_REF][START_REF] Pichard-Garcia | Use of long-term cultures of human hepatocytes to study cytochrome P450 gene[END_REF]. Ito et al. were the first to £ To the best of their knowledge, authors have no real or perceived conflicts of interests. demonstrate that hepatocytes isolated from an HCV-positive patient, could be cultured and continued to stably produce HCV infectious virions in their extracellular medium [START_REF] Ito | Cultivation of hepatitis C virus in primary hepatocyte culture from patients with chronic hepatitis C results in release of high titre infectious virus[END_REF]. Intracellular negative-strand RNA could be detected by a strand-specific RT-PCR method and the core protein could be detected by immunofluorescence microscopy. Fournier et al. were the first to demonstrate that hepatocytes isolated from non-infected patients were sensitive to HCVser infection and permissive to viral genome replication (Fournier et al., 1998). Since then, several groups have confirmed these findings. Although the performances of these primary culture models vary from one laboratory to another, depending on cell culture conditions, reported results are consistent and reproducible. HCV infection of primary human hepatocytes with natural different HCVser genotypes (from 1 to 5) has been assessed by detection of the replicative RNA strand, increased intracellular accumulation of genomic RNA, production of infectious virions, generation of new quasispecies in culture, and inhibition of replication by IFN [START_REF] Buck | Direct infection and replication of naturally occurring hepatitis C virus genotypes 1, 2, 3 and 4 in normal human hepatocyte cultures[END_REF][START_REF] Carriere | A novel, sensitive, and specific RT-PCR technique for quantitation of hepatitis C virus[END_REF][START_REF] Castet | Alpha interferon inhibits hepatitis C virus replication in primary human hepatocytes infected in vitro[END_REF][START_REF] Chong | Primary human hepatocytes in spheroid formation to study hepatitis C infection[END_REF]Fournier et al., 1998;[START_REF] Molina | The low-density lipoprotein receptor plays role in the infection of primary human hepatocytes by hepatitis C virus[END_REF][START_REF] Molina | Serum-derived hepatitis C virus infection of primary human hepatocytes is CD81 dependent[END_REF][START_REF] Rumin | Dynamic analysis of hepatitis C virus replication and quasispecies selection in long-term cultures of adult human hepatocytes infected in vitro[END_REF]. This model of primary and highly differentiated human hepatocytes infected with HCVser is clearly the one that most closely mimics the physiological situation. In addition, primary human hepatocytes were also shown to be sensitive to HCVpp [START_REF] Codran | Entry of hepatitis C virus pseudotypes into primary human hepatocytes by clathrin-dependent endocytosis[END_REF][START_REF] Meertens | The tight junction proteins claudin-1, -6, and -9 are entry cofactors for hepatitis C virus[END_REF][START_REF] Regeard | Entry of hepatitis C virus into primary human hepatocytes depends on the class B type I receptor[END_REF] and HCVcc (JFH1) infection [START_REF] Molina | Serum-derived hepatitis C virus infection of primary human hepatocytes is CD81 dependent[END_REF][START_REF] Reynolds | Hepatitis C virus receptor expression in normal and diseased liver tissue[END_REF]. Freshly isolated primary human hepatocytes have been recently used to evaluate comparatively the transcriptome profiling induced by core, NS3 and NS5A, or the HBV HBx protein as a control (via infection with adenovirus) [START_REF] Budhu | Induction of a unique gene expression profile in primary human hepatocytes by hepatitis C virus core, NS3 and NS5A proteins[END_REF].

Comparative analysis of HCV cellular models

In order to be used with confidence for predictive antiviral effect in man, HCV cellular models must meet at least two major criteria: i) to mimic as closely as possible the in vivo situation in man at both the cellular and virion levels, ii) to be practical, reliable and adaptable to high throughput screening. When considering the panel of models described in section 4, it is clear that none of them appears to meet these two criteria (Table 1).

HCV-LP and HCVpp represent practical and flexible forms of virions and can be easily obtained in large amounts. These particles appear to be very different from the natural virions that circulate in the blood of patients and are almost exclusively used for assessing viral entry and neutralisation of infection by patient-derived antibodies. However, recent results obtained with the model of primary human hepatocytes infected by HCVser [START_REF] Molina | The low-density lipoprotein receptor plays role in the infection of primary human hepatocytes by hepatitis C virus[END_REF] do not confirm some of the data generated with HCV-LP or HCVpp. Notably, using these particles, several authors questioned the role of LDLR in the cellular entry of HCV. Triyatni et al. observed no inhibition of HCV-LP entry into MOLT4 cells by anti-LDLR antibodies [START_REF] Triyatni | Interaction of hepatitis C virus-like particles and cells: a model system for studying viral binding and entry[END_REF]. Bartosch et al. and Hsu et al. observed no inhibition of HCVpp infection of Huh-7 cells by LDL, or by anti-LDLR antibodies, respectively (Bartosch et al., 2003;[START_REF] Hsu | Hepatitis C virus glycoproteins mediate pH-dependent cell entry of pseudotyped particles[END_REF]. In contrast, LDLR was shown to play a part in the entry of HCVser in primary human hepatocytes [START_REF] Molina | The low-density lipoprotein receptor plays role in the infection of primary human hepatocytes by hepatitis C virus[END_REF]. It is likely that these differences result from the fact that HCV-LP and HCVpp do not fully mimic the natural virions, especially in terms of association with lipoproteins [START_REF] Andre | Characterization of low-and very-low-density hepatitis C virus RNAcontaining particles[END_REF][START_REF] Andre | Hepatitis C virus particles and lipoprotein metabolism[END_REF]. On the other hand, HCVcc particles exhibit a density of 1.15 to 1.17 g/ml, that is significantly greater than that characterizing infectious HCVser LVP in human plasma (<1.06 g/ml) suggesting that HCVcc exhibit minor association with lipoproteins and lipids. It was recently reported that CD81 was indispensable for infection of primary human hepatocytes with HCVser of different genotypes [START_REF] Buck | Direct infection and replication of naturally occurring hepatitis C virus genotypes 1, 2, 3 and 4 in normal human hepatocyte cultures[END_REF][START_REF] Molina | Serum-derived hepatitis C virus infection of primary human hepatocytes is CD81 dependent[END_REF]. However, while a soluble form of CD81 large extracellular loop is capable of preventing infection of Huh7.5 cells by JFH1-HCVcc, it did not prevent infection of cultured primary hepatocytes by HCVser £ To the best of their knowledge, authors have no real or perceived conflicts of interests. [START_REF] Molina | Serum-derived hepatitis C virus infection of primary human hepatocytes is CD81 dependent[END_REF]. In addition, soluble CD81 was much more effective in blocking HCVcc infection in Huh7.5 cells than in primary hepatocytes. These inconsistencies show that both the form of virus particle and the host cell are important determinants for HCV replication.

Subgenomic replicon systems appear to be very practical by combining simplicity, rapidity, robustness, and reproducibility. These systems lack the virion partner and are primarily used only for the screening of antiprotease and antipolymerase drugs under conditions that are far from the in vivo situation (see below). Although these replicons have been used to reveal drug resistance mechanisms [START_REF] Bartenschlager | The hepatitis C virus replicon system: from basic research to clinical application[END_REF], recent investigations for example on the effect of HCV protein E2 on the modulation of TJs in infected cells clearly emphasized the limitations of these models [START_REF] Benedicto | Hepatitis C virus envelope components alter of hepatocyte tight junction-associated proteins and promote occludin retention in the endoplasmic reticulum[END_REF].

The Huh-7.5/HCVcc models provide greater advantages as they can be used to investigate every aspects of HCV biology from cellular entry to secretion. Hence, these models are ideal candidates for specific testing of therapeutics targeting viral entry, assembly, and release, as well as neutralizing antibodies and other drugs acting on cellular entry, such as fusion inhibitors. However, all these systems too often incorrectly referred to as human hepatocytes, are based on hepatocellular carcinoma cells which although being of human origin are not hepatocytes. These cells are dedifferentiated and exhibit abnormal hepatic phenotype with deregulated proliferation, gene expression, signaling pathways, host anti-viral response, endogenous metabolism, etc. In addition, these systems are based on the replication of synthetic HCV RNAs expressed from selected or engineered clones. For example, JFH-1 is a rare virus isolated from a patient with fulminant hepatitis C [START_REF] Kato | Efficient replication of the genotype 2a hepatitis C virus subgenomic replicon[END_REF]. Moreover, these cells cannot be infected with natural HCVser. Another problem with hepatoma or other cell lines including immortalized human hepatocytes is that their proliferation may change the cell population by adaptation to both the virus and the therapeutic compounds being tested. This is obviously not the case in primary human hepatocytes and hepatocytes in vivo which are known to proliferate at a very low rate, especially in seriously damaged liver with fibrosis and cirrhosis [START_REF] Roskams | Liver stem cells and their implication in hepatocellular and[END_REF]. Such drastic difference in phenotype, notably proliferating versus quiescent cells (but this is not the only difference) may clearly lead to false or unappropriated anti-viral responses that will not be observed in vivo. A further problem encountered with cultured hepatoma cells (including HepG2, Hep3B and Huh-7) is their impaired poly(I-C)-and virus-activated IFN responses and the considerable variations they exhibit in the IFN response, notably in different Huh7 cell lines originating from different laboratories [START_REF] Keskinen | Impaired antiviral response in human hepatoma cells[END_REF][START_REF] Lanford | Antiviral effect and virus-host interactions in response to alpha interferon, gamma interferon, poly(i)-poly(c), tumor necrosis factor alpha, and ribavirin in hepatitis C virus subgenomic replicons[END_REF]Li et al. 2005). This suggests that studies carried out with these cells must be interpreted with caution. Notably, TLR3 and RIG-I signaling pathways have been shown to be defective respectively, in Huh7 cells [START_REF] Lanford | Antiviral effect and virus-host interactions in response to alpha interferon, gamma interferon, poly(i)-poly(c), tumor necrosis factor alpha, and ribavirin in hepatitis C virus subgenomic replicons[END_REF]Preiss et al., 2008) and in Huh7.5 cells because of a point mutation within the RIG-I CARD-like homology domain [START_REF] Sumpter | Regulating intracellular antiviral defense and permissiveness to hepatitis C virus RNA replication through a cellular RNA helicase, RIG-I[END_REF]. It is likely that these defects account for the high permissiveness of Huh-7.5 cells for HCV RNA replication. In contrast, the PH5CH8 immortalized cells that were established from non-neoplastic hepatocytes retain robust IFN responses to extracellular and intracellular poly(I-C) (Preiss et al., 2008). In this respect, these cells are likely to mimic normal hepatocytes in vivo, and thus may represent a valuable model for investigation of antiviral responses in hepatocytes.

Cultured fetal human hepatocytes appear to be as functional as adult hepatocytes in terms of natural virus infection, replication and release. However, some of their enzyme machinery are known to be different from those found in the adult, notably in terms of drug metabolizing enzymes [START_REF] Hines | The ontogeny of drug metabolism enzymes and implications for adverse drug events[END_REF]. Thus, care must be taken when using these cells for anti-viral screening because prodrug activation, if required, may be catalytically inactive or not expressed, and drug metabolism which limits drug availability may be absent.

Primary adult human hepatocyte models infected with natural HCVser of any genotype closely mimic the in vivo situation. In addition, these models offer wide possibilities £ To the best of their knowledge, authors have no real or perceived conflicts of interests. to investigate antiviral drugs: i) the cells can be maintained in a differentiated phenotype, ii) they can be cultured in such a way as to maintain their drug metabolizing capacities [START_REF] Pichard-Garcia | Use of long-term cultures of human hepatocytes to study cytochrome P450 gene[END_REF], iii) they can be infected with HCVser of any genotype (including HCVpp and HCVcc particles), iv) the innate immune response is fully preserved, in contrast to what is observed in Huh-7 cells and derivatives, and other hepatoma cell lines [START_REF] Keskinen | Impaired antiviral response in human hepatoma cells[END_REF][START_REF] Lanford | Antiviral effect and virus-host interactions in response to alpha interferon, gamma interferon, poly(i)-poly(c), tumor necrosis factor alpha, and ribavirin in hepatitis C virus subgenomic replicons[END_REF]Li et al., 2005;[START_REF] Sumpter | Regulating intracellular antiviral defense and permissiveness to hepatitis C virus RNA replication through a cellular RNA helicase, RIG-I[END_REF] ; indeed, fetal and adult human hepatocytes produce IFNβ and ISGs in response to IFN and HCV infection [START_REF] Buck | Direct infection and replication of naturally occurring hepatitis C virus genotypes 1, 2, 3 and 4 in normal human hepatocyte cultures[END_REF][START_REF] Castet | Alpha interferon inhibits hepatitis C virus replication in primary human hepatocytes infected in vitro[END_REF][START_REF] Lazaro | Hepatitis C virus replication in transfected and serum-infected cultured human fetal hepatocytes[END_REF] a process that is likely to account for the low level of viral infectivity in these cells ; iv) finally, hepatocytes can be prepared from different patients so that the contribution of the interindividual variability of the host biology to antiviral drug response can be evaluated, in contrast to hepatoma cell lines of immortalized hepatocytes that are issued from a single individual; indeed previous analysis show that such diversity exists in terms of natural anti-viral response, response to IFN, receptor expression, etc. (Chevaliez & Pawlotsky, 2007a).

Metabolic phosphorylation of nucleoside analogs is an essential step for the pharmacological activity of these compounds against NS5B (see section 6). The question of whether the rate and extent of nucleoside phosphorylation are similar or different in hepatoma cell lines and primary human hepatocytes has to be addressed in any investigation aimed at screening antiviral molecules. Indeed, significant differences have been noted between different hepatoma cell lines as well as between hepatoma cell lines and primary human hepatocytes [START_REF] Hernandez-Santiago | Metabolism of the anti-hepatitis C virus nucleoside beta-D-N4-hydroxycytidine in different liver cells[END_REF][START_REF] Klumpp | Hepatitis C virusencoded enzymatic activities and conserved RNA elements in the 3' nontranslated region are essential for virus replication in vivo[END_REF][START_REF] Paeshuyse | Comparative in vitro anti-hepatitis C virus activities of a selected series of polymerase, protease, and helicase inhibitors[END_REF]. Interestingly, it has been reported that the phosphorylation of some nucleoside analog derivatives that is observed in primary human hepatocytes does not occur in replicon cells (Huh-7) either because the compounds cannot enter the cells or because of a deficient conversion [START_REF] Ma | Characterization of the metabolic activation of hepatitis C virus nucleoside inhibitor beta-D-2'-Deoxy-2'-fluoro-2'-C-methylcytidine (PSI-6130) and identification of a novel active 5'-triphosphate species[END_REF]. This clearly emphasizes the risk of false negative in the screening of anti-HCV compounds using solely the replicon systems. Drug metabolism plays a major role in controling the therapeutic effects of drugs. It is clear that the battery of drug metabolizing enzymes (including cytochromes P450 and conjugation enzymes) and transporters are expressed at very low level or quite differently in the hepatoma cell lines as compared with differentiated hepatocytes [START_REF] Hewitt | Primary hepatocytes: current understanding of the regulation of metabolic enzymes and transporter proteins, and pharmaceutical practice for the use of £ To the best of their knowledge, authors have no real or perceived conflicts of interests. hepatocytes in metabolism, enzyme induction, transporter, clearance, and hepatotoxicity studies[END_REF][START_REF] Jover | Re-expression of C/EBP alpha induces CYP2B6, CYP2C9 and CYP2D6 genes in HepG2 cells[END_REF]. Thus, false responses may be obtained as a consequence of either the absence of prodrug activation or the absence of drug metabolism. The same caveat arises with immortalized hepatocyte cell lines. This suggests that antiviral candidates that require a metabolic activation (other than phosphorylation) may be tested negative in cell lines. On the other hand, drugs that are rapidly biotransformed in hepatocytes to inactive metabolites may eventually appear to be inactive in vivo but will be tested as positive in cell lines (in which no inactivation takes place). Another important aspect to consider is that HCV chronic patients are (and will be in the future) polymedicated so that drug-drug interactions have to be anticipated [START_REF] Thompson | Directly acting antivirals the treatment of patients with hepatitis C infection: a clinical development update addressing key future challenges[END_REF]. Primary human hepatocyte cultures are the only in vitro system that allows one to predict such interactions with full confidence [START_REF] Gomez-Lechon | Hepatocytes--the choice to drug metabolism and toxicity in man: in vitro variability as a reflection of in vivo[END_REF][START_REF] Hewitt | Primary hepatocytes: current understanding of the regulation of metabolic enzymes and transporter proteins, and pharmaceutical practice for the use of £ To the best of their knowledge, authors have no real or perceived conflicts of interests. hepatocytes in metabolism, enzyme induction, transporter, clearance, and hepatotoxicity studies[END_REF][START_REF] Pichard | Human hepatocyte culture[END_REF]. Last but not least, mounting evidence suggests that host genes significantly modulate HCV infection and replication (Ikeda & Kato, 2007;[START_REF] Moriishi | Host factors involved in the replication of hepatitis C virus[END_REF][START_REF] Randall | Cellular cofactors affecting hepatitis C virus infection and replication[END_REF][START_REF] Sheikh | Hepatitis C virus infection: molecular pathways to metabolic syndrome[END_REF]. Since gene expression is highly dependent on the differentiation status of cells, this provides a further and strong argument in favor of the use of primary human hepatocytes for investigating HCV biology.

However, primary human hepatocyte culture models suffer from several major drawbacks. First, procurement and use of human tissue face ethical concerns that require necessary autorisations from regulatory agencies and informed consents of donors or donor's family. Adult human liver tissue suitable for the isolation of hepatocytes is obtained either from patients undergoing surgical liver resection for the removal of different types of lesions £ To the best of their knowledge, authors have no real or perceived conflicts of interests.

(metastasis, biliary cyst, etc.), or from brain-dead organ donors whose liver has been refuted for transplantation (tissue traumatism, high level of steatosis, presence of a tumor, etc.). Since the liver tissue is strongly sensitive to ischemia and deteriorates rapidly after death, human hepatocyte isolation requires an appropriate coordination and logistics between the laboratory, surgeons, and anatomopathologists. The samples are transported to the laboratory as quickly as possible but often arrive late in the afternoon or in the evening meaning that the initial part of the work including hepatocyte isolation and plating is often overnight. Second, availability of human liver is limited and unpredictable. Indeed, there may be several weeks between the obtention of one liver sample of adequate quality and the next one, while in other cases, several liver samples may be obtained within the same day meaning that at least two teams have to work in parallel. Third, the quality of the tissue is variable from one liver sample to another due to the health conditions of the donor, nature of lesion, pre-surgery treatment or to the duration of transportation. For instance, a level of steatosis greater than 60% or presurgery chemotherapy (which is very toxic to the liver) results in poor quality and yield of cells. The amount of tissue is also variable from one liver sample to another (i.e. from 50 grams to 500 grams or more) making difficult to standardize the isolation procedure. Thus, the amount of hepatocytes isolated from a liver sample may therefore vary between a few millions to billions of cells. Fourth, the phenotype of hepatocytes has a tendency to exhibit more or less drastic changes immediately after cells isolation and plating, due to adaptation to the culture conditions. This process is often referred to as de-differentiation. Indeed, a number of phenotypic markers may be lost more or less rapidly after plating. Within the last decades, however, extensive investigations have allowed to design culture conditions that considerably limit this de-differentiation process by using chemically defined culture media, specific cell substrates (such as collagen sandwich or matrigel), specific cytokine/growth factor cocktails, co-cultures with other liver cell types, or 3D-cell configurations [START_REF] Hewitt | Primary hepatocytes: current understanding of the regulation of metabolic enzymes and transporter proteins, and pharmaceutical practice for the use of £ To the best of their knowledge, authors have no real or perceived conflicts of interests. hepatocytes in metabolism, enzyme induction, transporter, clearance, and hepatotoxicity studies[END_REF]. Fifth, another problem is the high level of autofluorescence of primary human hepatocytes in culture which makes extremely difficult, if not impossible, to investigate intracellular events such as nuclear translocation of proteins, or to visualize intracellular molecules or proteins, including notably HCV proteins by immunofluorescence-based methods. Thus, in most reports, HCV infection is mainly investigated by RT-PCR measurement of viral RNA. Sixth, and as a consequence of all the drawbacks listed above, it is evident that these cultures are quite inappropriate for high throughput screening explorations. Therefore, primary human hepatocyte cultures should be used as a secondary screening system to confirm, invalidate, or complement data obtained with the other more practical systems described above.

Cellular models for the development of anti-HCV agents

The current therapy for HCV infection is a combination including pegylated interferon-α (IFN) and a nucleoside analog ribavirin (Chevaliez & Pawlotsky, 2007b). This therapy allows permanent eradication of circulating HCV virions (undetectable level of viral RNA in the blood) in only approximately 50% of patients infected with HCV of genotype 1, while the success of this therapy is greater (80% of patients cured) with other genotypes such as 2 and 3. In addition, IFN/ribavirin treatment is associated with many side effects (including depression, headache, fever, hemolytic anemia) that not only worsen the patient's quality of life but also lead to non-compliance with prescribed treatment in a significant number of cases [START_REF] Hayashi | Antiviral therapy for chronic hepatitis C: past, present, and future[END_REF][START_REF] Shepherd | Interferon (pegylated and non-pegylated) and ribavirin for the treatment of mild chronic hepatitis C: a systematic review and economic evaluation[END_REF][START_REF] Younossi | The effects of HCV infection and management on health-related quality of life[END_REF]. It is therefore evident that new therapeutic approaches that combine new viral targets, better efficiency and safety are crucially needed. Excellent reviews have been published in this respect [START_REF] Neyts | Selective inhibitors of hepatitis C virus replication[END_REF][START_REF] Soriano | Emerging drugs for hepatitis C[END_REF][START_REF] Stauber | Drugs in development for hepatitis C[END_REF][START_REF] Wakita | HCV research and anti-HCV drug discovery: toward the next generation[END_REF]. In this section, we are going to summarize a number of experiments and investigations that have been carried out £ To the best of their knowledge, authors have no real or perceived conflicts of interests. using the HCV cellular models described in section 4. We shall focus on the use of these models in the screening of new chemical entities and RNA interference strategies. The use of such models for vaccine research and for the characterization of the neutralising activity of antibodies from chronically infected patients will be discussed in section 7.

HCV targeting by new chemical entities

Most pharmaceutical research on new chemical entities against HCV focus on inhibitors of viral entry, E2 binding inhibitors, NS2/NS3/NS4A protease inhibitors, NS5A inhibitors, NS3 helicase inhibitors, IRES inhibitors, NS5B polymerase inhibitors, inhibitors of virus assembly, inhibitors of virus release [START_REF] Neyts | Selective inhibitors of hepatitis C virus replication[END_REF][START_REF] Soriano | Emerging drugs for hepatitis C[END_REF][START_REF] Stauber | Drugs in development for hepatitis C[END_REF][START_REF] Wakita | HCV research and anti-HCV drug discovery: toward the next generation[END_REF]. Of these, inhibitors of NS3/4A and NS5B are the most extensively developed therapeutic molecules since these enzymes play a highly strategic role in the virus life [START_REF] Thompson | Directly acting antivirals the treatment of patients with hepatitis C infection: a clinical development update addressing key future challenges[END_REF]. Huh-7/HCVpp models, subgenomic replicons, Huh-7/HCVcc replicon systems, immortalized hepatocytes and primary human hepatocytes have been used in this respect. Several reviews aimed at describing the use of the subgenomic replicons and the Huh-7.5/HCVcc systems for the screening of anti-HCV drugs have been published [START_REF] Bartenschlager | The hepatitis C virus replicon system: from basic research to clinical application[END_REF][START_REF] Bartenschlager | Hepatitis C virus molecular clones and their replication capacity in vivo and in cell culture[END_REF][START_REF] Horscroft | Replicon cell culture system as a valuable tool in antiviral drug hepatitis[END_REF][START_REF] Pietschmann | Tissue culture and animal models for hepatitis C virus[END_REF]. We have therefore decided to focus the present review on the most recently published data (since 2007 in most of cases). A list of potential anti-HCV compounds is shown in Table 2. This list is by no means extensive but merely intented to provide examples of recently characterized inhibitors using various HCV cellular models. In general, these investigations are based on the analysis of viral markers such as HCV RNA and antigens as indicators of treatment efficacy; occasionally, viral genome analysis of resistant cellular clones allows to identify/confirm the target of antiviral compounds. Of note, the use of primary human hepatocyte infected with HCVser is still quite limited. Some of the inhibitors previously discovered in cellular systems are now being tested in patients [START_REF] Ali | Selected replicon variants with low-level in vitro resistance to the hepatitis C virus NS5B polymerase inhibitor PSI-6130 lack cross-resistance with R1479[END_REF][START_REF] Paeshuyse | Comparative in vitro anti-hepatitis C virus activities of a selected series of polymerase, protease, and helicase inhibitors[END_REF].

Targeting NS3/4A protease is particularly important because this viral enzyme not only orchestrates the processing of viral proteins, but also because it participates to the virusmediated inhibition of the host response by cleaving TRIF and MAVS (see section 3). This has been refered to as the "double whammy" effect [START_REF] Farley | A double whammy for hep C[END_REF]. Several types of NS3 serine protease inhibitors have been discovered. These include substrate-based peptide inhibitors targeting the active site of the enzyme, strong electrophiles located at the position of the scissile amide bond, phenanthrenequinones, thiazolidines, benzoylamides [START_REF] Trahtenherts | Inhibition of hepatitis C virus RNA replicons by peptide aptamers[END_REF]. BILN 2061 was the first HCV protease inhibitor to enter clinical trials, and early results were highly promishing showing a rapid and significant decline in plasma HCV RNA virus load in all treated patients infected with HCV genotype 1 [START_REF] Lamarre | An NS3 protease inhibitor with antiviral effects in humans infected with hepatitis virus[END_REF]. Unfortunately, further development of the compound had to be stopped due to cardiac toxicity [START_REF] Reiser | Antiviral efficacy of NS3-serine protease inhibitor BILN-2061 in patients with chronic genotype 2 and 3 hepatitis C[END_REF]. In recent studies, a comparative analysis of the ability of two potent macrocyclic inhibitors of NS3/4A protease (TMC435350 and TMC380765, Table 2) to inhibit HCV RNA replication and to restore IFNβ production was carried out using a genome length NNeoC-5B/2-3 cell replicon system [START_REF] Liang | Antiviral suppression vs restoration of RIG-I signaling by hepatitis C protease and polymerase inhibitors[END_REF]. The results showed that the concentrations of inhibitor necessary to restore the RIG-I signaling pathway leading to IFN production were much greater (2 orders of magnitude at least) than those necessary to inhibit the viral genome replication, in spite of the finding that the level of full-length MAVS protein expression was recovered. No definitive explanation has been provided for these observations. It is suggested that the rescue of the host response by NS3/4A inhibitors may not contribute significantly to their therapeutic effect. However, it reminds to be verified that similar observations are observed in primary hepatocytes infected with HCVser.

£ To the best of their knowledge, authors have no real or perceived conflicts of interests.

NS5B inhibitors are classified into nucleoside and nonnucleoside inhibitors [START_REF] Beaulieu | Non-nucleoside inhibitors of the HCV polymerase: progress in the discovery and development of novel agents for the treatment HCV infections[END_REF][START_REF] Pauwels | Binding-site identification and genotypic profiling of hepatitis C virus inhibitors[END_REF]. Nucleosidic inhibitors compete with the natural ribonucleoside triphosphate substrates at the catalytic site of NS5B, while nonnucleosidic inhibitors exhibit different chemical structures and inhibit the initiation and/or elongation step by targeting different sites of the enzyme, including domains close to the active site and allosteric sites. Several new compounds have been tested including derivatives of nucleosides, naphthalenones, benzothyiadiazepines, pyridazinones, and benzodiazepines.

Treatments of replicon systems with antiviral molecules directed against NS3/4 or NS5B generally lead to the emergence of mutants with reduced sensitivity to the treatment. The duration of treatment required to observe the emergence of such mutants varies considerably with the nature of the antiviral molecule. For instance, Ali et al. showed that short term (less than one month) treatment of replicon did not generate mutants to PSI-6130, NM107 or R1479 (two other nucleoside analogs) while resistant variants emerged during treatment with a non nucleotide analog NNI-1 [START_REF] Ali | Selected replicon variants with low-level in vitro resistance to the hepatitis C virus NS5B polymerase inhibitor PSI-6130 lack cross-resistance with R1479[END_REF]. In contrast, long-term treatments (several months) resulted in the generation of mutants (notably S282T) with reduced sensitivity to PSI-6130 and its prodrug R7128. Interestingly, while these two molecules generate the same active species, wide differences in resistance and nature of mutations were observed presumably due to cell-dependent ability to generate the active species. These results suggest that the extent of resistance observed during a drug treatment is not only linked to the virus adaptation through the selection of mutants, but also to the host cell through the selection of cell populations with a modified nucleoside metabolism. A panel of HCV NS3 mutants confering resistance to protease inhibitors (BILN2061, VX950 and ITMN191) were investigated by transient replication assay in a genotype 1b N strain replicon [START_REF] He | Relative replication capacity and selective advantage profiles of protease inhibitor-resistant C virus (HCV) NS3 protease mutants in the HCV genotype 1b replicon system[END_REF]. The results showed that the selective advantage conferred by drug-resistant mutants depends on their relative drug susceptibility, relative replication capacity compared to the wild-type virus, and the concentration of drug. Different protease inhibitors caused unique selective advantage profiles that might correlate with their resistance profiles. Bassit et al. evaluated combinations of nucleoside analogues beta-D-2'-C-methylcytidine (2'-C-MeC; NM-107) or beta-D-2'-deoxy-2'-fluoro-2'-C-methyleytidine (PSI-6130) with IFNα2b or triple combination with ribavirin using the Huh-7 subgenomic HCV replicon (Clone B) [START_REF] Bassit | Combinations of 2'-Cmethylcytidine analogues with interferon-alpha2b and triple combination with ribavirin in the hepatitis C virus replicon system[END_REF]. Double and triple combinations of 2'-C-MeC or 2'-F-C-MeC with IFNα2b or of IFNα2b plus ribavirin with either 2'-C-MeC or 2'-F-C-MeC demonstrated synergistic antiviral effects.

Other inhibitors have been investigated in addition to those described above. Some interferon variants display EC 50 (the concentration that inhibits HCV replication by 50%) significantly smaller than that relative to the routinely used IFNα2b [START_REF] Erickson | Antiviral potency analysis and functional of consensus interferon, interferon-alpha2a and pegylated interferonalpha2b against hepatitis C virus infection[END_REF][START_REF] Escuret | Novel alpha interferon (IFN-alpha) variant with improved inhibitory activity against hepatitis C virus genotype 1 replication compared to IFN-alpha2b therapy in a subgenomic replicon system[END_REF]. Using an HTS replicon system, Huang et al. screened a library of 230,000 compounds (P. [START_REF] Huang | Discovery characterization of substituted diphenyl heterocyclic compounds as potent and s elective inhibitors of hepatitis C virus replication[END_REF]. They defined hits as compounds with an EC 50 below 10 µM and a CC 50 (the concentration that inhibits cell viability by 50%) of 10-fold over the EC 50 . Two diphenyl heterocyclic compounds were discovered R706 and R803 that contained a dichloroacetamide moiety (Table 2). Both inhibited HCV replication at protein and RNA levels. However, it was not possible to identify a target for these compounds of either viral or cellular origin. Interestingly, R803 was shown to be able inhibit HCV RNA replication that was refractory to IFN-mediated inhibition, and to induce only few resisitant clones with respect to NS3/4 or NS5B inhibitors.

Medicinal herbs or natural compounds are being used increasingly in the search for safe and efficient drug candidates for hepatitis C virus infection. Lee et al. investigated the anti-HCV effect of compounds from Mori Cortex Radicis [START_REF] Lee | Inhibition of HCV replicon cell growth by 2-arylbenzofuran derivatives isolated from Mori Cortex Radicis[END_REF]. The screening of a library of extracts from medicinal plants (173 species) for compounds with anti-HCV affinity led to the selection of a methanol extract from Mori Cortex Radicis. Fractionation of £ To the best of their knowledge, authors have no real or perceived conflicts of interests.

the extract by monitoring antiviral activity with a replicon model revealed five compounds including mulberroside C, moracin P, O, and M and mulberrofuran K that behaved as potent inhibitors of the replicon assay. This was confirmed by NS3 helicase inhibitory activity for moracin P, and O. Using the Huh-7.5/JFH1 HCVcc system, Nahmias et al. observed that naringenin, an abundant flavonoid in citrus fruits, is an inhibitor of VLDL secretion and of the MTTP activity as well as the transcription of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase and acyl-coenzyme A:cholesterol acyltransferase 2 in infected cells [START_REF] Nahmias | Apolipoprotein B-dependent hepatitis C virus secretion is inhibited by the grapefruit naringenin[END_REF]. Most interestingly, these authors observed that naringenin reduces HCV secretion in Huh-7.5 infected cells by 80%. Supplementing HCV patient diet with this compound should therefore be an option that deserves to be tested in the future. However, this compound is a potent inhibitor of several human liver cytochrome P450s [START_REF] Breinholt | In vitro investigation of cytochrome P450-mediated metabolism of dietary flavonoids[END_REF][START_REF] Brill | In vitro Interactions with Repeated Grapefruit Juice Administration Peel or not to Peel?[END_REF] and its use will have to be tighly controlled. Several compounds have been shown to inhibit HCV entry or E2-CD81 interaction including cyanovirin-N, arbidol, a peptide of lactoferrin (C-s-3-33), serum amyloid A (Abe et al., 2007;[START_REF] Cai | Human serum amyloid A protein inhibits hepatitis C virus entry into cells[END_REF][START_REF] Helle | Cyanovirin-N inhibits hepatitis C virus entry by binding to envelope protein glycans[END_REF][START_REF] Lavie | Serum amyloid A has antiviral activity against hepatitis C virus by inhibiting virus entry in a cell culture system[END_REF][START_REF] Pecheur | Biochemical mechanism of hepatitis C virus inhibition by the broad-spectrum antiviral arbidol[END_REF]. Using the model of primary human hepatocyte infected with HCVser (genotypes 1, 2 and 3), Molina et al. demonstrated that the human recombinant soluble LDLR (the LDL binding domain consisting of the 7 repeats) is a potent inhibitor of HCV entry [START_REF] Molina | The low-density lipoprotein receptor plays role in the infection of primary human hepatocytes by hepatitis C virus[END_REF]. This peptide which mimics the LDLR binding site apparently competes with membrane bound LDLR and thus inhibits HCVser binding to hepatocytes.

The screening of most of these inhibitors has necessarily been carried out with a limited number of HCV cellular systems, notably for many of these with genotype 1b subgenomic replicon systems. One may therefore wonder whether the candidate anti-HCV compounds will be as efficient on the other HCV genotypes and subtypes; the existence of multiple quasispecies that prevail in vivo further amplifies this concern (Le [START_REF] Guillou-Guillemette | Genetic diversity of the hepatitis C virus: impact and issues in the therapy[END_REF][START_REF] Paeshuyse | Comparative in vitro anti-hepatitis C virus activities of a selected series of polymerase, protease, and helicase inhibitors[END_REF]. In this respect, cyanovirin-N, an inhibitor of HCV entry offers a promishing new contribution to the arsenal of anti-HCV drugs (Helle et al., 2006). Using the Huh-7/HCVpp and Huh-7.5/JFH1 HCVcc systems, these authors showed that by interacting with N-linked glycans associated with HCV envelope glycoprotein E2, this compound is likely to inhibit the E2-CD81 interaction and cellular entry of HCV of various genotypes. Interestingly, a comparative analysis of the anti-HCV activities of a selection of compounds with distinct molecular targets (NS3, helicase and NS5B inhibitors, cyclophilin binding compounds, IFNs) was reported using several genotype 1b replicon systems using Huh-5-2, Huh-9-13, Huh-Mono and Huh-6 cells [START_REF] Paeshuyse | Comparative in vitro anti-hepatitis C virus activities of a selected series of polymerase, protease, and helicase inhibitors[END_REF]. These authors observed no significant difference in the response of the different replicon cell lines to the different inhibitors. A recent study demonstrated the utility of chimeric replicons for broadspectrum activity determination of HCV inhibitors. Herlihy et al. created a panel of intergenotypic chimeric replicons harboring NS5B sequences from different genotypes (2b, 3a, 4a, 5a, 6a) [START_REF] Herlihy | Development of intergenotypic chimeric replicons to determine the broad-spectrum antiviral activities of hepatitis C virus polymerase inhibitors[END_REF]. Stable cell lines were generated with replicationcompetent chimeras in order to evaluate the antiviral activity of HCV nonnucleoside polymerase inhibitors (NNIs) that target different regions of the protein. Very wide ranges of inhibitory effects of selected compounds were evidenced against NS5B chimeric replicons harboring non-genotype 1b sequences. Similarly, Huang et al. observed widely variable responses (> 30-fold shift in EC50) of genotypes 1 and 2a to inhibitor R803 [START_REF] Huang | Discovery characterization of substituted diphenyl heterocyclic compounds as potent and s elective inhibitors of hepatitis C virus replication[END_REF]. In addition, and as indicated above, HCV variants resistant to selective inhibitors are expected to emerge. Thus, a drug should be effective against several (if not all) genotypes and have a high barrier to resistance selection to be used in antiviral therapy against HCV. Alternatively, when the tested compound exhibits a genotype-dependent action, analysis of intergenotypic replicon systems or the use of human hepatocytes infected with serum derived HCV of different genotypes may provide additional hints regarding its mechanism of action.

£ To the best of their knowledge, authors have no real or perceived conflicts of interests.

Host proteins that cooperate with the virus represent other classes of therapeutic targets (Ikeda & Kato, 2007;[START_REF] Moriishi | Host factors involved in the replication of hepatitis C virus[END_REF][START_REF] Sheikh | Hepatitis C virus infection: molecular pathways to metabolic syndrome[END_REF] (Table 2). In contrast to viral inhibitors, cellular protein inhibitors have the advantage of being unsensitive to the virus genetics (genotype and subtype), although their use may lead to side effects. The use of cellular models that are as close as possible to the differentiated human hepatocyte should be mandatory in this respect. Using an HTS subgenomic HCV replicon (Huh7/Rep-Feo) and OR6 cells stably expressing the full-length genotype 1 replicon ORN/C-5B/KE9 as a secondary screening system, Kim et al. screened an appropriately formatted library of 2568 bioactive molecules including FDA-approved drugs [START_REF] Kim | A cellbased, high-throughput screen for small molecule regulators of hepatitis C virus replication[END_REF]. Corticosteroids (triamsinolone, dexamethasone, prednisolone and methyl prednisolone) were found to be promoters of HCV replication, irrespective of their immunomodulatory effect, as well as some PPARγ agonists (notably troglitazone, but not clofibrate). In contrast, phosphodiesterase inhibitors (trequinsin), MAP38 inhibitors (SB 203580), and HMG-CoA reductase inhibitors (atorvastatin, simvastatin, and fluvastatin -while the inhibitory effect of lovastatin and pravastatin was weaker ) as well as other compounds demonstrated antiviral activity in these systems. Cholesterol synthesis inhibitors reduce the production of mevalonate, a precursor in isoprenoid and geranylgenranyl pyrophosphate synthesis. It has been reported that geranylgeranylation inhibition, rather than cholesterol synthesis inhibition per se is responsible for the inhibition of HCV RNA replication [START_REF] Kapadia | Hepatitis C virus RNA replication is regulated by host geranylgeranylation and fatty acids[END_REF][START_REF] Ye | Disruption of hepatitis C virus RNA replication through inhibition of host protein geranylgeranylation[END_REF]. Indeed, the anti-HCV effect of the statins was rescued by addition of geranylgeraniol. Because the HCV genome does not encode a geranylgeranylated protein, it is likely that a host geranylgeranylated protein must play an important role in HCV replication. Wang et al FBL2 as such a protein required for HCV replication through formation of an active complex with NS5A [START_REF] Wang | Identification of FBL2 as a geranylgeranylated cellular protein required for hepatitis C virus RNA replication[END_REF]. In contrast, using primary human hepatocytes infected with HCVser we reported that another statin, squalestatin, increased and not decreased the entry and genome replication of HCV most likely through up-regulation of LDL receptor [START_REF] Molina | The low-density lipoprotein receptor plays role in the infection of primary human hepatocytes by hepatitis C virus[END_REF]. This opposite effect of squalestatin as compared to other statins is most likely due to the fact that this compound which blocks squalene synthesis, a step downstream of farnesyl pyrophosphate, does not interefere with isoprenoid and geranylgenranyl pyrophosphate synthesis. As any other statins squalestatin induces the expression of the LDLR and this is likely the reason for its stimulating effect on hepatocyte infection by HCVser. On the other hand, and interestingly, 25-hydroxycholesterol inhibited LDLR expression and HCV genome replication in the same model [START_REF] Molina | The low-density lipoprotein receptor plays role in the infection of primary human hepatocytes by hepatitis C virus[END_REF].

Lipid metabolism is likely to play an important role in HCV RNA replication. Using replicon systems, Leu et al. and Kapadia et al. reported that polyunsaturated fatty acids (PUFAs) such as arachidonic acid, docosahexanoic acid and eicosapentaenoic acid inhibit HCV replication by a mechanism that appears to be independent of their antagonist effect on LXR (for example T0901317 an agonist of LXR had no effect on HCV replication), while linoleic and linolenic acids reduced HCV RNA levels only slightly, and saturated fatty acids enhanced HCV RNA levels [START_REF] Kapadia | Hepatitis C virus RNA replication is regulated by host geranylgeranylation and fatty acids[END_REF][START_REF] Leu | Anti-HCV activities of selective polyunsaturated fatty acids[END_REF]. Interestingly, using primary human hepatocytes infected with JFH1/HCVcc we observed that linoleic and linolenic acids inhibit the replication of HCV RNA by 50 to 80% (Molina & Maurel, unpublished results). Sphingolipid biosynthesis pathway is another locus of anti-HCV strategy. Together with cholesterol, these lipids as well as glycosphingolipids enter into the composition of lipid rafts, the suspected site of HCV replication [START_REF] Shi | Hepatitis C virus RNA replication occurs on a detergent-resistant membrane that cofractionates with caveolin-2[END_REF]. Screening a natural product library with an HCV1b HTS replicon system, Sakamoto et al reported that inhibitors of serine palmitoyltransferase (the first step enzyme in sphingolipid biosynthesis) such as myriocin or NA255, inhibitors of dihydroceramide synthase (a further step in this pathway) such as fumonisin B1, and inhibitors of glycosphingolipid (GSL) synthesis such as PPMP all inhibited HCV replication [START_REF] Sakamoto | Host sphingolipid biosynthesis as a target for hepatitis C virus therapy[END_REF]. The mechanism of this effect is £ To the best of their knowledge, authors have no real or perceived conflicts of interests. not yet clear but could involve the NS5B-membrane web association.

Another pathway that is likely to play a major part in HCV replication is the GTP biosynthesis pathway. The first enzyme involved in this pathway is the inosine monophosphate dehydrogenase (IMPDH). Inhibitors of this enzyme such as ribavirin, mizoribine, micophenolic acid and VX-497 (in association with ribavirine) were all shown to inhibit HCV replicon systems (Henry et al., 2006;[START_REF] Naka | Mizoribine inhibits hepatitis C RNA replication: effect of combination with interferon-alpha[END_REF][START_REF] Zhou | The effect of ribavirin and IMPDH inhibitors on hepatitis C virus subgenomic replicon RNA[END_REF].

The N-glycosylation pathway that controls proper folding of proteins has also been identified as a possible locus of anti-HCV action. Derivatives of deoxynojirimycin (DNJ) iminosugars, which are inhibitors of α-glucosidase, have been shown to prevent the correct folding and assembly of E1 and E2 HCV glycoproteins [START_REF] Branza-Nichita | Antiviral effect of N-butyldeoxynojirimycin against bovine viral diarrhea virus correlates with misfolding of E2 envelope proteins and impairment of their association into E1-E2 heterodimers[END_REF]. By using the Huh-7/HCVpp model Chapel et al. showed that the inhibition of α-glucosidases by iminosugars results in the misfolding and misassembly of HCV glycoprotein pre-budding complexes et al., 2007). This leads to a reduction in the incorporation of E1/E2 complexes into HCVpp and a subsequent reduction of infectivity of the secreted particles. This suggests the potential usefulness of DNJ derivatives in targeting HCV infection.

Several problems must be anticipated when screening chemical libraries with cellular models, including protein binding and cytotoxicity of compounds. It has been demonstrated that drugs including certain antiviral compounds show markedly reduced activity in vivo or in cellular models because of their natural tendency to bind serum proteins [START_REF] Molla | Human serum attenuates the activity of protease inhibitors toward wild-type and mutant human immunodeficiency virus[END_REF]. To evaluate this possibility, the inhibitory effect of the antiviral candidates should be measured in cells cultured in media containing increasing concentrations of human serum [START_REF] Huang | Discovery characterization of substituted diphenyl heterocyclic compounds as potent and s elective inhibitors of hepatitis C virus replication[END_REF]. Many small molecules are cytotoxic, and hepatoma replicon cell lines are highly sensitive to cytotoxic or cytostatic agents. This may generate false positives. Indeed, cytotoxic effects can be mistaken for antiviral effects by decreasing the end-point signal by reducing cell number, while not affecting the rate of HCV replication. Primary human hepatocytes are generally less sensitive to drug toxic effects [START_REF] Huang | Discovery characterization of substituted diphenyl heterocyclic compounds as potent and s elective inhibitors of hepatitis C virus replication[END_REF], because they express higher level of detoxication enzymes [START_REF] Jover | Re-expression of C/EBP alpha induces CYP2B6, CYP2C9 and CYP2D6 genes in HepG2 cells[END_REF]. It is therefore mandatory to run cytoxicity tests in parallel to antiviral tests in all antiviral analysis using cellular systems.

Finally, the possibility that in vitro models do not correctly predict drug effects in vivo cannot be ruled out (Henry et al., 2006). Indeed, the case of various immunosuppressors provides a typical example in this respect. Cyclosporin A and mycophenolic acid were shown to inhibit HCV replication in replicon or immortalised cell lines [START_REF] El-Farrash | In vitro infection of immortalized primary hepatocytes by HCV genotype 4a and inhibition of virus replication by cyclosporin[END_REF]Henry et al., 2006;[START_REF] Nakagawa | Specific inhibition of hepatitis C virus replication by cyclosporin A[END_REF]. However, treatment of liver transplanted recipients with immunosuppressors does not eradicate the virus but in contrast activates re-infection of the grafted liver [START_REF] Pollard | Calcineurin inhibition and disease recurrence in the hepatitis C viruspositive liver transplant recipient[END_REF]. is likely that the suppressive effect of these drugs on the host immune response (which is obviously absent from the HCV replication cellular models) largely dominates and actually masks the decrease in viral load resulting from their antiviral activity.

HCV targeting by RNA interference and related approaches

Given the critical role played by miRs in the control of both HCV infection and replication, and host gene regulation, therapeutic strategies using RNAi offer promishing possibilities [START_REF] Watanabe | Therapeutic application of RNA interference for hepatitis C virus[END_REF]. In addition and in contrast to the long and high cost development of new chemical entities, designing and validating an siRNA that targets a gene is a relatively fast process. Synthetic siRNA or shRNA use the same machinery as miRs to downregulate their target genes. HCV targeting by RNAi strategies can be envisaged both on HCV RNA itself (for instance the 5'-NTR) and on host genes that are mandatory for viral infection and/or replication (for instance the CD81 receptor). Subgenomic £ To the best of their knowledge, authors have no real or perceived conflicts of interests. replicons, HCVcc replicon systems and primary human hepatocytes have been used in this respect, siRNAs or shRNAs being either transfected directly or expressed after transduction of host cells with lentiviruses. Almost all regions of HCV (genotypes 1 and 2) have been targeted by RNAi including the 5'-NTR, E2, core, NS3, NS4B, NS5A, NS5B and the 3'-NTR using both subgenomic replicon and Huh-7/HCVcc systems [START_REF] Korf | Inhibition of HCV subgenomic replicons by siRNAs derived from plasmids with opposing U6 and H1 promoters[END_REF][START_REF] Watanabe | Intracellular-diced dsRNA has enhanced efficacy for silencing HCV RNA and overcomes variation in the viral genotype[END_REF] (Table 2). In most of cases, 80 to 95% inhibition of both HCV genome replication and protein synthesis was obtained with low concentrations (in the µM-nM of siRNA. However, as observed with anti HCV drugs, the virus is likely to escape or resist to siRNAs through mutations of the sequences which are specifically targeted. Several strategies have been tested to tentatively overcome this problem with convincing results (Watanabe et al., The 5'-NTR which displays the greatest homology between different genotypes has been targeted in many studies. However, this region is highly structured and, as a rather reluctant to hybridization. Another strategy is based on the use of long dsRNA that can be degraded by Dicer in a number of siRNAs that can be used to target multiple sites of a heterogeneous population of viral genomes. Thus, siRNA raised against the of HCV 1b were able to inhibit the replication of HCV 2a [START_REF] Watanabe | Intracellular-diced dsRNA has enhanced efficacy for silencing HCV RNA and overcomes variation in the viral genotype[END_REF]. Another strategy consists to target different regions of HCV genome by combining several siRNAs [START_REF] De Francesco | Challenges and successes in developing new therapies for hepatitis C[END_REF][START_REF] Korf | Inhibition of HCV subgenomic replicons by siRNAs derived from plasmids with opposing U6 and H1 promoters[END_REF].

Ribozymes and DNAzymes have also been used to inhibit HCV replication [START_REF] Bhindi | Brothers in arms: DNA enzymes, short interfering RNA, and the emerging wave of small-molecule nucleic acid-based gene-silencing strategies[END_REF][START_REF] Usman | Nuclease-resistant synthetic ribozymes: developing a new of therapeutics[END_REF]. Jarczak et al. combined hairpin ribozymes targeting the 3'-NTR and siRNAs targeting the 5'-NTR of HCV with significant inhibition [START_REF] Jarczak | Hairpin ribozymes in with siRNAs against highly conserved hepatitis C virus sequence inhibit RNA replication and protein translation from hepatitis C virus subgenomic replicons[END_REF]. Trans-splicing ribozymes have been used to repair mutant RNAs responsible for human genetic diseases and cancers [START_REF] Ryu | Comparative analysis of intracellular trans-splicing ribozyme activity against hepatitis C virus internal ribosome entry site[END_REF]. These authors designed a series of such ribozymes to target the HCV IRES region, that is, to replace HCV transcripts with a new RNA exerting or not anti-HCV activity. Gonzalez-Carmona et al. tested a series of hammerhead ribozymes targeting the 5'-NTR of HCV [START_REF] Gonzalez-Carmona | Hammerhead ribozymes with cleavage site specificity for NUH and NCH display significant anti-hepatitis C viral effect in vitro and in recombinant HepG2 and CCL13 cells[END_REF]. Ribozyme (Rz1293) targeting the GCA 348 cleavage site in the HCV loop IV displayed a significant inhibitory activity (>70%).

are prefered to ribozymes because they are easier to prepare, less sensitive to chemical and enzymatic degradation and easier to deliver into cells. Roy et al. designed DNAzymes targeting different regions of HCV IRES of different genotypes (Roy et al., The RNA cleavage and translation inhibitory activities revealed that the majority of DNAzymes efficiently and specifically inhibited HCV RNA translation. Interestingly, DNAzymes exhibited genotype-specific activities, some of them targeting genotype1b and others targeting genotype 2a, more efficiently. Trepanier et al. designed a series of 2'-Omethyl-modified DNAzymes to target highly-conserved RNA sequences located within the HCV core-E1 coding region [START_REF] Trepanier | Reduction in intracellular HCV RNA and virus protein expression in human hepatoma cells following treatment with 2'-Omethyl-modified anti-core deoxyribozyme[END_REF]. One of these, Dz858-4-Ome, produced a decrease of 63, 87 and 84% of intracellular HCV RNA, core protein and HCV antigen expression, respectively, 6 h post-transfection. DNAzymes can thus be used as selective and effective inhibitors of HCV RNA replication and represent potent therapeutic agents against HCV infection.

of cell factors that are mandatory for HCV infection and replication is another potent RNAi strategy because these genes are not (or much less) subjected to mutations. Such a strategy has been used successfully by targeting La, eIF2B, hVAP-33, proteasome a-subunit 7 and Hu antigen R proteins, as well as proteins interacting with the NTRs of HCV (Zhang et al., 2004). We reported on the downregulation of CD81 in primary human hepatocytes by RNAi strategy. Our results showed a significant reduction of HCVser entry and replication in a genotype-independent manner [START_REF] Molina | Serum-derived hepatitis C virus infection of primary human hepatocytes is CD81 dependent[END_REF]. Henry et al., combined shRNAs directed to IRES, NS5B and CD81. Interestingly, although these various shRNAs were active on their own, their combination resulted in an additive effect (Henry et £ To the best of their knowledge, authors have no real or perceived conflicts of interests. al., 2006). Using a systematic RNAi screening and the Huh-7.5/HCVcc (genotype 1) model, Randall et al. identified a panel of host genes that regulate HCV replication, either by direct interaction with the viral genome or by controling signaling pathways [START_REF] Randall | Cellular cofactors affecting hepatitis C virus infection and replication[END_REF].

Proteins that exhibited a strong potential for HCV replication included CD81, DDX3X (dead box helicase), SEC11L1 and HM13 (signal peptidases), ATF6 (unfolded protein response), VAPA and VPS35 (vesicular traffic), ACTN1 (cytoskeleton), and various kinases that bind NS5A including PDPK1, RAF1, Interestingly, inhibition of ATF6 expression stimulated HCV replication. Similarly, Superkova et al. screened a panel of siRNAs that preferentially target human protein kinases using Huh-7 and a selectable tricistronic HCV genotype 1b subgenomic replicon expressing firefly luciferase gene as reporter [START_REF] Supekova | Identification of human kinases involved in hepatitis C virus replication by small interference RNA screening[END_REF]. They identified three kinases, Csk, Jak1, and Vrk1 which promote HCV replication as assessed by viral RNA and protein levels. Interestingly downregulating Fyn, a kinase that is negatively regulated by Csk, induced HCV replicon, suggesting that Csk mediates its effect on HCV replication through Fyn. In another study, Ariumi et al. hypothesized that kinases such as the ataxia-telangiectasia mutated kinase (ATM), ATM-and Rad3-related kinase (ATR), poly(ADP-ribose) polymerase 1 (PARP-1), and checkpoint kinase 2 (Chk2) that control the host response to genomic stress might affect HCV replication [START_REF] Ariumi | The DNA sensors ataxia-telangiectasia mutated kinase and checkpoint kinase 2 are for hepatitis C virus RNA replication[END_REF]. Using Huh-7 cells and subgenomic and full length genotype 1b replicon as well as the genotype 2a HCVcc system, they observed that shRNA-mediated downregulation of ATM or Chk2 significantly inhibited HCV replication. NS3-NS4A interacts with ATM and NS5B interacts with both ATM and Chk2, suggesting that the ATM signaling pathway is critical for HCV RNA replication. Using the Huh-7.5/JFH1 HCVcc system, Yang et al. reported that expression of fatty acid synthase (FASN), a cytosolic protein, increases in the extracellular medium of HCV-infected cells [START_REF] Yang | Fatty acid synthase is up-regulated during hepatitis C virus infection and regulates hepatitis C virus entry and production[END_REF]. Whether this increase coincides with increased lipogenesis is presently unknown. Interestingly, shRNAs directed to FASN mRNA, as well as C75 a pharmacological inhibitor of FASN blocked HCV RNA synthesis in the replicon (genotype 1b) and Huh-7.5/HCVcc (2a) systems. This coincided with a reduction of CLDN1 (but not CD81) expression. These results provide an interesting link between HCV infection and the lipid metabolic disorder (steatosis) that appears to be induced by this infection.

Cellular models for the development of anti-HCV vaccine and the characterization of neutralizing antibody activity

Although HCV-LPs derived from insect cells are noninfectious, they display some properties of the native virions and are able to confer protection in animal models. Indeed, virus-LPs have been used successfully as vaccines in papilloma, rotavirus, and Norwalk virus infections [START_REF] Baumert | Hepatitis C virus-like particles synthesized in insect cells as a potential vaccine candidate[END_REF]. strategies for HCV vaccine development have been tested using insect cell-derived HCV-LPs. Such particles were shown to be highly immunoreactive to anti-HCV antibodies in serum of patients infected with various HCV genotypes. Moreover, HCV-LPs derived from insect cells were safe and immunogenic in different animal models including BALB/c and HLA-A 2.1 transgenic (AAD) mice, and two non human primates baboon and chimpanzees [START_REF] Baumert | Hepatitis C virus-like particles synthesized in insect cells as a potential vaccine candidate[END_REF][START_REF] Elmowalid | Immunization with hepatitis C virus-like particles results in control hepatitis C virus infection in chimpanzees[END_REF][START_REF] Jeong | Immunization with hepatitis C virus-like particles induces humoral and cellular immune responses in nonhuman primates[END_REF][START_REF] Murata | Immunization with hepatitis C virus-like particles protects mice from recombinant hepatitis C virus-vaccinia infection[END_REF][START_REF] Steinmann | Inhibition of hepatitis C virus-like particle binding to target cells by antiviral antibodies in acute and chronic hepatitis C[END_REF]. Mice immunized with VSV-HCV-C/E1/E2 generated cell-mediated immune responses to all of the HCV structural proteins, and humoral responses notably to E2 were also readily evident. Thus, engineered VSVs expressing HCV Core, E1, and E2 and/or HCV-LPs represent useful tools in vaccine and immunotherapeutic strategies designed to address HCV infection [START_REF] Ezelle | Generation of hepatitis C virus-like particles by use of a recombinant vesicular stomatitis virus vector[END_REF]. Jeong et al. and Elmowalid et al. characterized the immunogenicity and induction of immunity by HCV-LPs in baboons and chimpanzees [START_REF] Elmowalid | Immunization with hepatitis C virus-like particles results in control hepatitis C virus infection in chimpanzees[END_REF] £ To the best of their knowledge, authors have no real or perceived conflicts of interests. [START_REF] Jeong | Immunization with hepatitis C virus-like particles induces humoral and cellular immune responses in nonhuman primates[END_REF]. Animals were immunized with HCV-LPs or HCV-LPs plus AS01B adjuvant. After immunization, they developed an HCV-specific immune response and proliferative lymphocyte responses against core, E1, and When submitted to infectious HCV inoculum, one chimpanzee developed transient viremia with low HCV RNA titers (10 3 to 10 4 copies per ml) in the third and fourth weeks. Other animals exhibited high levels of viremia (10 4 to 10 5 copies per ml) which however became undetectable by week 10 after infection. After infection, all animals demonstrated a significant increase in peripheral and intrahepatic T cell and proliferative responses against the HCV structural proteins. These responses correlated with the decrease in HCV RNA levels. Interestingly, when nonimmunized chimpanzees were infected with the same HCV inoculum, three developed infection with viremia in the range of 10 5 to 10 6 copies per ml. These results show that HCV-LP immunization induces HCV-specific cellular immune responses that can control HCV challenge in primate models.

The factors leading to spontaneous clearance of HCV or to viral persistence are elusive. Understanding virus-host interactions that enable acute HCV clearance represent a major step in the development of more effective therapeutic and prophylactic strategies. In this respect, several groups used HCV particle infection systems to monitor the neutralizing activity of antibodies collected from acutely or chronically infected patients. Steinman et al developed an HCV-LP-based model system using hepatoma cell lines Huh-7 and HepG2 et al., 2004). They observed that cellular HCV-LP binding was specifically inhibited by antibodies from patients with acute or chronic hepatitis C in a dose-dependent manner. Interestingly, the presence of neutralizing antibodies was not associated with viral clearance in vivo. Using a library of homologous overlapping envelope peptides covering the entire HCV envelope, they identified an epitope in the N-terminal E2 region as a target of human neutralizing antibodies. Using a sensitive neutralization assay based on infectious HCVpp, it was reported that sera from haemodialysis infected patients (genotype 1b) either inhibited or enhanced HCVpp (E1/E2 genotype 1b or 1a) infectivity (Lavillette et al., 2005). The inhibitory effect inversely correlated with the HCV RNA level in the serum suggesting that inhibitory antibodies may be involved, at least in part, in the control of virus replication in vivo. However, using a similar system, Meunier et al. observed that the presence of antibodies is not necessarilly required to clear the virus [START_REF] Meunier | Evidence for cross-genotype neutralization of hepatitis C virus pseudo-particles and of infectivity by apolipoprotein C1[END_REF]. On the other hand, enhancement of infectivity was observed preferentially with serum from patients or chimpanzee with sustained high replication rate, as well as with serum from non infected individuals. Apolipoprotein C1 alone or as a component of HDL was shown to be responsible for this enhancement [START_REF] Meunier | Evidence for cross-genotype neutralization of hepatitis C virus pseudo-particles and of infectivity by apolipoprotein C1[END_REF]. Owsianka et al. used HCVpp to assess the reactivity and neutralizing capability of antisera and monoclonal antibodies raised against portions of the HCV E2 envelope protein [START_REF] Owsianka | Monoclonal antibody AP33 defines a broadly neutralizing epitope on the hepatitis C virus E2 envelope glycoprotein[END_REF]. Rabbit antisera raised against either HVR1 or ectodomain of E2 showed limited and strain specific neutralization. By contrast, the monoclonal antibody (MAb) AP33 (recognizing E2 residues 412-423, a region highly conserved across different genotypes) demonstrated potent neutralization (inhibition of CD81 binding) of infectivity against HCVpp carrying E1E2 representative of all genotypes tested. Identification of neutralizing antibodies is likely to be of significant benefit to future vaccine and therapeutic antibody development. Boo et al. investigated the capacity of antibodies in patients submitted to antiviral therapy to neutralize HCVpp [START_REF] Boo | Neutralizing antibodies in patients with chronic hepatitis C infection treated with (Peg)-interferon/ribavirin[END_REF]. No correlation was observed between baseline viral load, quasispecies population, neutralizing antibody levels and the response of patients to treatment with PEG-IFN and ribavirin. More recently Haberstroh et al. using HCVpp in kinetic and fusion assays observed that after binding of the virus to the target cell, neutralizing antibodies in the majority of HCV-infected individuals target an early step of HCV entry, most likely related to HCV-CD81, SR-BI interactions as well as membrane fusion (Haberstroh et al., £ To the best of their knowledge, authors have no real or perceived conflicts of interests. 2008). Furthermore, they identified viral epitopes in E1 and E2 representing potential targets for neutralizing antibodies.

Conclusion

A number of in vitro cellular models have been generated to investigate different aspects of HCV including cell entry, replication, assembly, excretion, host antiviral and IFN responses and viral resistance to treatments. These models are based on a range of host cells and a range of forms of virion particles. All these models exhibit advantages and disadvantages at the biological and experimental levels. On the one hand, investivations aimed at basic research should favor the systems that most closely mimic the in situation for the step of virus biology that is the object of the study. On the other hand, investigations aimed at applied therapeutics and pharmacology should first be based on the most adapted HTS systems for the screening of chemical libraries or other antiviral agents. A number of new chemical entities and new therapeutic strategies (RNAi) have been discovered and identified with these cellular models. In many cases, it will be necessary to subsequently confirm the efficacy of selected positive candidates with the model of primary human hepatocytes infected with natural HCVser, using different hepatocyte preparations (from different liver donors) and different serum samples (from different infected patients with different load and genotype). This model will even allow predicting pharmacokinetics of the new chemical entities as well as possible drug interactions and liver toxicity. These last points are important because it is now clearly anticipated that in the near future anti-HCV treatments will involved a series of different drugs/agents.

Legends for figures

Figure 1. The various steps of HCV entry in hepatocytes. Refer to text (sections 2.2. and 2.3.) for detail.

  (Fig 1, step 7). This process eventually leads to the release of the nucleocapsid into the cytosol (Fig 1, step 8).

Figure 2 .

 2 Figure 2. HCV genome replication, assembly and release. Refer to text (sections 2.3 and 2.4) for detail.