New targets for antiviral therapy of chronic hepatitis C

Transcription

1 Liver International ISSN REVIEW ARTICLE New targets for antiviral therapy of chronic hepatitis C Sandra Bühler and Ralf Bartenschlager Department of Infectious Diseases, Molecular Virology, Heidelberg University Heidelberg, Germany Keywords cyclophilin A direct acting antiviral (DAA) mir-122 NS3 protease NS5A protein NS5B RNA-dependent RNA polymerase phosphatidylinositol-4-kinase III alpha STAT-C sustained viral response Abbreviations DAA, direct acting antivirals; HCV, hepatitis C virus; PEG-IFN, pegylated interferon; RBV, ribavirin; SVR, sustained virological response. Correspondence Ralf Bartenschlager, MD, Department of Infectious Diseases, Molecular Virology Heidelberg University Im Neuenheimer Feld Heidelberg Germany Tel: Fax: ralf_bartenschlager@med. uni-heidelberg.de Received 27 September 2011 Accepted 7 October 2011 DOI: /j x Abstract Until recently, chronic hepatitis C caused by persistent infection with the hepatitis C virus (HCV) has been treated with a combination of pegylated interferon-alpha (PEG-IFNa) and ribavirin (RBV). This situation has changed with the development of two drugs targeting the NS3/4A protease, approved for combination therapy with PEG-IFNa/RBV for patients infected with genotype 1 viruses. Moreover, two additional viral proteins, the RNAdependent RNA polymerase (residing in NS5B) and the NS5A protein have emerged as promising drug targets and a large number of antivirals targeting these proteins are at different stages of clinical development. Although this progress is very promising, it is not clear whether these new compounds will suffice to eradicate the virus in an infected individual, ideally by using a PEG-IFNa/RBV-free regimen, or whether additional compounds targeting other factors that promote HCV replication are required. In this respect, host cell factors have emerged as a promising alternative. They reduce the risk of development of antiviral resistance and they increase the chance for broadspectrum activity, ideally covering all HCV genotypes. Work in the last few years has identified several host cell factors used by HCV for productive replication. These include, amongst others, cyclophilins, especially cyclophilina (cypa), microrna-122 (mir-122) or phosphatidylinositol-4-kinase III alpha. For instance, cypa inhibitors have shown to be effective in combination therapy with PEG-IFN/RBV in increasing the sustained viral response (SVR) rate significantly compared to PEG-IFN/RBV. This review briefly summarizes recent advances in the development of novel antivirals against HCV. Chronic hepatitis C is a main risk factor for the development of serious liver disease including cirrhosis and hepatocellular carcinoma (1, 2). An estimated million people are persistently infected with the causative agent, the hepatitis C virus (HCV) that was molecularly cloned about 22 years ago (3). With the implementation of blood tests to exclude HCV-containing samples, the incidence of HCV has dropped tremendously. However, most infections are asymptomatic and therefore diagnosed either by chance or at a time when chronic liver disease has already developed. Because of its high genetic variability, HCV is classified into seven different genotypes (4), which are predictive for successful therapy: infections with genotype 2 and 3 viruses are treated with a combination of pegylated interferonalpha (PEG-IFNa) with ribavirin (RBV) leading to a sustained viral response (SVR), i.e. absence of viral RNA 6 months or more after cessation of therapy) of about 85%. Although success rates are much lower in case of infections with genotype 1 and 4 viruses, the recent approval of the first HCV-specific directly acting antivirals (DAA) that are given in a triple combination with PEG-IFNa/RBV has increased cure rates in genotype 1 naïve patients from around 55% to around 75%, at least under conditions of standardized clinical trials (5). Given the large number of additional DAA that are currently being tested in clinical trials, it is expected that this number will increase further. Moreover, serious efforts are being made to develop an IFNa-free therapy to reduce the numerous side effects caused by the systemic administration of this cytokine. This review briefly summarizes the molecular aspects of the viral prime targets including host cell factors that are required for efficient HCV replication. Hepatitis C virus genome organization and replication cycle Hepatitis C virus is a small enveloped virus with a single stranded RNA genome of positive polarity belonging to the family Flaviviridae, genus Hepacivirus. Seven different genotypes are known that are differentiated based on a nucleotide sequence diversity of around 35% (4) John Wiley & Sons A/S 9

2 The replication cycle of HCV starts with the entry of the virus into its main target cell, the hepatocyte. Viral entry is a highly complex multi-step process initiated by binding of the envelope glycoproteins E1 and E2 to different host cell molecules that either trap virus particles on the cell surface (e.g. heparan sulphate, low-densitylipoprotein receptor) or contribute to virus entry (CD81, occludin, scavenger receptor class B type I, claudin) [reviewed in (6)]. Virus spread occurs either via extracellular particles entering cells in a clathrin-dependent manner or via cell-to-cell spread, which appears to have different entry molecule requirements (7 10). After genome release into the cytosol, the viral RNA is translated via the internal ribosome entry site (IRES) residing in the 5 nontranslated region (NTR). Translation generates a polyprotein that is cleaved by viral and cellular proteases into at least 10 different proteins (Fig. 1A). The first third of the genome encodes the proteins that are major constituents of virus particles, i. e. core, E1 and E2, or that are required for assembly and release of infectious particles, i.e. the viroporin p7 and the NS2 protease (11 15). At least five nonstructural (NS) proteins constitute the HCV replicase [reviewed in (16)]: (i) NS3, a multi-functional enzyme with an amino-terminal serine protease domain and a carboxy-terminal RNA helicase/ntpase domain (Fig. 1B). (ii) NS4A, a co-factor of the NS3 protease forming a stable heterodimeric NS3/4A complex (Fig. 1B). (iii) NS4B, the presumed central organizer of the HCV replicase complex and a main inducer of intracellular membrane rearrangements. (iv) NS5A, an RNA-binding phosphoprotein required both for RNA replication and assembly of infectious virus particles (Fig. 1B). Fig. 1. (A) A schematic representation of the hepatitis C virus (HCV) genome organization is shown in the top. The 5 NTR containing the internal ribosome entry site, IRES and the 3 NTR are indicated with thin lines and their proposed secondary structures. The polyprotein is given in orange with borders between the viral proteins indicated by vertical lines. Shown below is the structure of a selectable subgenomic replicon. It is composed of the HCV 5 NTR containing the IRES, the selectable marker neo encoding for the neomycin phosphotransferase, which for cloning purposes is amino-terminally fused to 16 codons of the core coding region (C), the IRES of the encephalomyocarditis virus (EMCV), which directs translation of the HCV NS3 to NS5B polyprotein, and the 3 NTR. (B) 3D structures of the three main viral drug targets: NS3/4A, NS5A and NS5B. The left panel shows a ribbon diagram of the crystal structure of full length NS3 in complex with the central NS4A protease activation domain (yellow). The protease (prot) domain (cyan) is located in the left. Side chain atoms of the catalytic triad amino acids (his-57, asp-81, ser-139) are represented as magenta spheres. Subdomains I, II and III of the helicase are coloured in silver, red, and blue respectively. Conserved sequence motifs involved in helicase activity are coloured in green. The blue stick structure represents the C-terminal segment of the helicase domain, which occupies the catalytic site of the protease subdomain. Note the linker that is part of subdomain I (given in silver) connecting the protease and the helicase domain. A ribbon diagram of the NS5A dimer (64) associated to a membrane via the N-terminal amphipathic a-helix is shown in the middle panel. The three domains are given. Domains 1 (D1) of the two monomers form an RNA-binding cleft oriented towards the cytosol. D1 is composed of two subdomains (IA and IB); they are coloured in magenta and pink in one monomer and cyan and ice blue in the other monomer respectively. The zinc atoms of the zinc-binding motif in subdomain IA are shown as orange spheres. Domains 2 and 3 are intrinsically unfolded. The panel in the right shows the ribbon diagram of the NS5B RNA-dependent RNA polymerase catalytic domain (65, 66) complexed with UTP (stick structure in yellow) and Mn ions (magenta sphere). The triphosphate moiety of a nucleotide bound to the priming site is indicated by the stick structure in grey. The carboxy-terminal membrane anchor is not shown. The fingers, palm and thumb subdomains are coloured in blue, red and green respectively. The thumb subdomain b-loop contributing to template binding is given in orange. Shown structures are based on the following Protein Data Bank accession codes: 1CU1 for NS3, 1GX6 for NS5B, 1R7E for NS5A N-terminal membrane anchor, and 1ZH1 for D1 structure John Wiley & Sons A/S

3 (v) NS5B, the RNA-dependent RNA polymerase catalysing the amplification of the viral RNA genome (Fig. 1B). The newly synthesized positive-strand RNAs, which are transcribed from negative-strand RNA intermediates, serve as templates for RNA translation or for negative-strand RNA synthesis. Alternatively, positive-strand progeny is used for the assembly of infectious virus particles via a process that is tightly linked to cytosolic lipid droplets and the very-low-density lipoprotein (VLDL) pathway [reviewed in (17)]. Cell culture systems supporting self-replicating hepatitis C virus RNAs It took more than 10 years from the first molecular cloning of the HCV genome up to the establishment of the first robust cell culture system, which was initially based on self-replicating HCV mini-genomes, called replicons (18). They were derived from a molecular genotype 1b consensus genome by replacing the region encoding core to NS2 by the selectable marker neomycin phosphotransferase (neo) conferring resistance against the cytotoxic drug G418 (Fig. 1A). Synthetic RNA derived from the cloned DNA copy of such a selectable replicon was transfected into cells of the human hepatoma cell line Huh7 that were subsequently cultured in G418-containing medium. Only cells in which the replicon amplified to high levels could survive and, indeed, several G418-resistant cell colonies were obtained containing high amounts of viral RNA and proteins. The exceptionally high level RNA replication was attributable to the selection for both more permissive Huh7 cell clones and the accumulation of cell culture adaptive mutations enhancing RNA synthesis [reviewed in (19)]. Owing to its high efficiency, the replicon system became accepted as an important tool to study the molecular mechanisms of HCV RNA replication. Importantly, the replicon system provided the first functional cell-based platform for screening of antiviral agents targeting HCV RNA replication and for validation of compounds directed against recombinant viral enzymes. The major drawback of the HCV replicon system was its limitation to the intracellular steps of the viral replication cycle whereas production of infectious virus particles could not be achieved (20 22). This was because of an interference of replication enhancing mutations with the assembly process of HCV particles (22). A major breakthrough was therefore the identification of a genotype 2a isolate that was cloned from a patient with fulminant hepatitis and that was replicated to high levels without requiring adaptive mutations (23). This isolate, designated JFH-1 (an acronym derived from Japanese patient with fulminant hepatitis) supports virus production and cell culture-produced particles that are infectious in vivo (24). This new HCVcc (cell culture) system thus closes the gaps of the replicon system and recapitulates the complete viral replication cycle that can now by targeted at any step (see Table 1). Specifically targeted antiviral therapy for hepatitis C virus (STAT-C) Inhibitors of the NS3/4A protease As protease inhibitors were successfully developed for treatment of HIV infections, soon after the first molecular cloning of the HCV genome most efforts focused on the development of inhibitors of the HCV NS3 serinetype protease. In spite of its unfavourable overall Table 1. Hepatitis C virus (HCV) proteins and their use as antiviral drug targets HCV protein Function in the HCV replication cycle Development of inhibitor Core + 1; mini-cores? No Core Viral capsid protein; RNA-binding Yes; pre-clinical E1 Envelope glycoprotein Yes; e.g. neutralizing antibodies against E1, and E2; neutralizing E2 Envelope glycoprotein; receptor binding antibodies or compounds targeting cellular receptors [reviewed in (6)] p7 Viroporin; assembly and release Yes; various inhibitors [reviewed in (67)] NS2 Cysteine protease; assembly No NS3 Serine protease; helicase Yes; multiple compounds including two approved drugs [reviewed in (68)] NS4A Co-factor of the NS3 protease Yes, e.g. ACH-1095 ( NS4B Induction of membrane rearrangements; main Yes; e.g. clemizole (69) organizer of membranous HCV replication complex NS5A RNA replication and assembly Yes; two inhibitor classes (34, 35) NS5B RNA-dependent RNA polymerase Yes; NI and NNI [reviewed in (70)] 2012 John Wiley & Sons A/S 11

4 structural features, especially the rather shallow and solvent-exposed substrate-binding pocket, potent inhibitors could be developed. They were based on the observation that after cleavage the P-side product remains bound in the substrate-binding cleft and causes an autoinhibition [reviewed in (25)]. Thus, P-side derived peptidic inhibitors were developed, including the macrocyclic tripeptide BILN 2061 that was successfully tested in a clinical trial in Administration of BILN 2061 to patients infected with HCV genotype 1 for 2 days resulted in an impressive reduction of HCV RNA plasma levels, thus providing proof-of-concept that the NS3/4A protease is a valid drug target (26). However, owing to toxicity in animals this compound was not pursued further. As described above, two NS3 protease inhibitors have recently been approved: boceprevir (SCH ) and telaprevir (VX-950). Their use in combination with PEG-IFNa/RBV increases SVR to around 75% in naïve patients infected with genotype 1 virus. However, these drugs also cause side effects such as rash and anaemia leading, e.g. in case of telaprevir in around 15% of cases to discontinuation of therapy (27). Moreover, these first generation DAA require rather complex treatment regimens such as strict time schemes when the drug must be taken (three times a day every 7 9 h), or a high load of pills, thus reducing adherence. A main problem of the recently approved DAA telaprevir and boceprevir is the rapid selection for drug resistant HCV variants, which is a particular concern for patients with poor response to PEG-IFNa/RBV (27, 28). Importantly, several of these mutations confer cross resistance to other protease inhibitors [reviewed in (29)]. For instance, the following resistance mutations have been reported with telaprevir (numbers in parenthesis refer to fold shift of the IC 50 of the mutant as compared to the wild type): V36A/M/C (3.5- to 7-fold); T54A/S (6- to 12-fold); R155K/T/Q (8.5- to 11-fold); V36A/M + R155K/T (57- to 71-fold); A156V/T (74- to 410-fold); and V36A/M+A156V/T (>781-fold) [reviewed in (29)]. Interestingly, in case of R155K, only one nucleotide change is required for this amino acid substitution with genotype 1a, although two nucleotide changes are required with genotype 1b. This explains why the R155K variant is frequently found in treated patients with a genotype 1a virus infection whereas in genotype 1b patients this variant is virtually absent. As shown with the R155K mutation, which drastically reduces replication capacity when introduced into a subgenomic replicon, resistance mutations often impair viral fitness (30). However, during therapy second site mutations are selected that restore fitness, explaining why the R155K primary mutation is frequently found in association with V36M in case of genotype 1a viruses. Obviously proper management protocols have to be established to monitor HCV drug resistance. Moreover, guidelines have been drafted that recommend strict stop rules in case of patients with viral breakthrough and (proven) adherence to therapy. The main concern is the selection for highly replication competent variants that are resistant to the used drug. These variants are probably persistent for a very long time and therefore difficult to eradicate. Moreover, in principle these variants can be transmitted to other individuals thus acquiring a drug resistant isolate. Inhibitors of the NS5B RNA-dependent RNA polymerase Another important drug target for development of DAA is the NS5B RNA-dependent RNA polymerase (Fig. 1B). Inhibitors of this enzyme can be classified into two groups: Nucleosidic inhibitors (NI) and non-nucleosidic inhibitors (NNI). NIs are nucleotide analogues and incorporated by the NS5B polymerase into the nascent RNA. Owing to the lack of a proper 3 OH-group that is used as acceptor for the 5 phosphate group of the incoming NTP, incorporated NIs cause chain termination. As the active site of the polymerase is highly conserved between different genotypes, NIs are likely to act across different genotypes. NIs also have a high genetic resistance barrier and only a few resistance mutations were found in vitro. For example, the resistant variant S282T was selected in cells treated with the NI RG7128. The position of this amino acid substitution is in close vicinity to the catalytic site of the NS5B polymerase and is conserved across all genotypes with the exception of the genotype 4a isolate ED43 [reviewed in (31)]. Importantly, the resistance mutation S282T causes a dramatic loss of viral fitness without having much effect on drug activity. It is therefore unlikely that this mutation will be selected in patient populations. Indeed, resistance was not observed in patients treated with RG7128 monotherapy for 2 weeks (32). Several NIs are currently under development in phase II clinical trials (e.g. RG7128 by Roche and Pharmasset; PSI-7977 by Pharmasset; IDX184 by Idenix). Moreover, because of the superior resistance profile, a clinical trial has been started by combining two NIs with or without RBV to determine whether or not PEG-IFNa-free therapy is possible using NIs alone. In contrast to NI, NNIs of the NS5B polymerase have a low genetic resistance barrier. Substances of this class bind to one of at least four allosteric sites within NS5B leading to inhibition of the enzyme and thus a block of viral RNA synthesis. Because of their rather low clinical efficacy and the rapid selection for resistant variants, the clinical use of NNIs may be limited. Nevertheless, several NNIs are currently being tested in phase II clinical trials and it remains to be determined what role this class of compounds might play in the future [reviewed in (33)]. Inhibitors of the NS5A replicase protein Attributable to its poorly characterized role in HCV RNA replication and the lack of enzymatic activity, NS5A has long been neglected as a primary drug John Wiley & Sons A/S

5 target. However, by using replicon-based screens and subsequent intensive chemical refinements of primary hits, a highly active compound (BMS ) characterized by its symmetric structure was identified (34). This compound has an amazingly high potency with antiviral efficacy in the picomolar range. In fact, it has been calculated that one inhibitor molecule can block NS5A molecules, arguing that BMS might exert a dominant negative phenotype. This high potency was well recapitulated in a phase I clinical trial with chronic hepatitis C patients; a single application of 100 mg BMS reduced viral load around fold within 24 h after application (34). The mode of action of this drug class is unknown and is an area of intense research (34 36). Biochemical studies suggest that the compound directly binds to NS5A and resistance analyses have identified mutations in domain I of NS5A, arguing for direct inhibition of NS5A function(s) (Fig. 1B). However, because of the multiple roles of this protein in the HCV replication cycle and the numerous cellular interaction partners, viral replication might be indirectly blocked, e.g. by inhibiting an interaction with an important host cell factor. Alternatively, oligomeric complexes might form within an infected cell and binding of the compound to one NS5A molecule may disturb formation of such a large complex, which would explain the dominant negative phenotype of the compound. The NS5A inhibitors, like NNIs and NS3 protease inhibitors, cause rapid selection for antiviral resistance. Several resistance mutations have been identified residing either in domain 1 (e.g. Y93H/C/W) or in the linker region connecting the amino-terminal amphipathic a-helix with domain 1 (35). Although some of these mutations reduce the antiviral efficacy of BMS around 1800-fold (Y93C) or even 3400-fold (L31V), because of the extremely high potency of this compound, the EC 50 is still in the nanomolar range even for these resistance mutations. Inhibitors targeting host cell factors required for hepatitis C virus replication As HCV is an obligate intracellular parasite, its replication relies heavily on the host cell environment. Thus, cell factors promoting HCV replication (so-called dependency factors) are an alternative target for antiviral therapy. It is assumed that cellular targets might be superior, because they should lower the risk for selection of resistance mutants. Moreover, host cell factors are well conserved and therefore, drugs interfering with such factors should be active across different genotypes. The most advanced cellular drug targets for therapy of chronic hepatitis C are cyclophilin A (CypA) and micro-(mi)rna-122. Cyclophilins are molecular chaperones catalysing the cis-trans isomerization of proline residues and are therefore called peptidyl-prolyl cis-trans-isomerases (PPIases). Seven different Cyp isoforms have been identified in humans. Pharmacological inhibition of cyclophilins by the immunosuppressive drug cyclosporine A (CsA), causes profound inhibition of HCV replication (37). The underlying mechanism by which cyclophilins contribute to viral replication is still not known. It is assumed that CypA binds to NS5A and might induce a conformational change leading to an activation of the viral replicase (38 41). There is also evidence that CypA might also be required for NS2 folding or activity, but this remains to be determined (38, 42). The inhibition of replicons by CsA on one hand, and the strong immunosuppressive effect of this drug on the other hand, led to the development of CsA analogues that retained CypA binding, but lost binding to calcineurin, which is responsible for the immuno-suppressive effect. One of these derivatives, DEBIO-025 (alisporivir) has shown great promise in clinical trials in combination with PEG-IFNa/RBV (43, 44). Moreover, a recent pilot study with genotype 3-infected patients suggests that even short-term monotherapy might be sufficient to achieve SVR (45). In hepatocytes, the accumulation of unfolded proteins in the endoplasmic reticulum (ER) causes ER stress and the unfolded protein response (UPR), mediated by the ER-resident stress sensors ATF-6, IRE1 and PERK. UPR-responsive genes are involved in the fate of ERstressed cells. Cells carrying HCV subgenomic replicons exhibit in vitro ER stress and suggest that HCV inhibits the UPR. In vivo, liver from patients with untreated CHC exhibit in vivo hepatocyte ER stress and activation of the three UPR sensors without apparent induction of UPR-responsive genes (46). This lack of gene induction may be explained by the inhibiting action of HCV per se (as suggested by in vitro studies) and/or by our finding of the localized nature of hepatocyte ER stress. Autophagy is a regulated process that can be involved in the elimination of intracellular microorganisms and in antigen presentation. Some in vitro studies have shown an altered autophagic response in HCV-infected hepatocytes. In vivo, autophagy is altered in hepatocytes from CHC patients, likely caused by a blockade of the last step of the autophagic process (47). It has been reported that the addition of an inhibitor of cyclophilins, cyclosporine A, reduced the activity of autophagy induced by nutrition starvation (48). Interestingly, Ke et al. described a new role of autophagy in the regulation of innate immunity during HCV infection (49). HCV may encode an NS3/4A independent activity that triggers autophagy to limit the production of IFNB. Therefore, cyclophilin inhibitors might act as a potent anti-autophagy agent and limit both inhibition of innate antiviral response and HCV replication (50). MicroRNAs (mirnas) are a class of small non coding RNA molecule of nucleotides that control gene expression by targeting mrnas for transcriptional repression or cleavage. They are involved in the regulation of crucial cellular mechanisms such as 2012 John Wiley & Sons A/S 13

6 development, cell differentiation, proliferation and apoptosis. The importance of the mirnas machinery in HCV replication has been recently described in various studies [reviewed in (51)]. Recently, micro-(mi)r-122 was identified as an essential HCV dependency factor (52). Expression of this micro-rna is liver-specific. Its mode of action is still under debate, but mir-122 appears to promote HCV replication in several ways. First, it was shown to enhance HCV RNA translation by enhancing the association of ribosomes with the viral RNA at an early initiation stage (53); second, it may mask the 5 triphosphate end of the HCV genome, thus impairing recognition by intracellular RNA sensors such as RIG-I (54, 55); third, mir-122 promotes RNA replication by an as yet unknown mechanism. Importantly, studies in the chimpanzee animal model demonstrated proof-of-concept that sequestration of mir-122 with chemically modified antagonists dampens HCV replication indicating that mirnas might be a novel therapeutic target (56). Another, more recently identified HCV dependency factor is phosphatidylinositol 4-kinase type III-a (PI4KIII-a) that has been identified in several independent sirna-based screenings (9, 57 60). It was shown that PI4KIII-a is required for structural integrity of the membranous HCV replication complex. The enzyme is recruited to the sites of viral replication via an interaction with domain I of NS5A triggering an activation of PI4KIII-a, which in turn leads to a massive accumulation of PI4-phosphate at intracellular membranes where HCV RNA replication occurs (57, 61). Pharmacological inhibition of PI4KIII-a results in a massive decrease of HCV RNA replication making this kinase a promising target for therapeutic intervention. Concluding remarks The ideal future therapy of chronic hepatitis C should fulfil at least four criteria. First, it should be IFN-free to reduce side effects and contraindications; second, it should impose a high barrier of drug resistance; third, it should require only short treatment duration; forth, SVR should be as high as possible, ideally greater than 90%. Initial clinical studies have shown that IFNa-free therapy is possible in principle (62, 63). Gane and colleagues have conducted a clinical trial (INFORM-1) based on a combination of two DAA: (1) RG7128, a NI with a high resistance barrier and (2) Danoprevir, a protease inhibitor with a low resistance barrier (62). Patients infected with genotype 1 viruses were treated for 14 days and showed a marked decrease in viral load. Viral breakthrough could not be detected. The combination therapy was well-tolerated by patients and discontinuation was not observed. Together with other clinical data this study shows great promise that IFNa-free therapy of chronic hepatitis C is not a fiction, but might become reality in the not too distant future. Acknowledgements We are grateful to Francois Penin for providing the 3D structures shown in Figure 1B. Work in the authors laboratory was supported by the Deutsche Forschungsgemeinschaft, FOR1202, TP1 and the European Union, European Training Network on (+)RNA Virus Replication and Antiviral Drug Development (EUVIRNA), grant number: Conflicts of interest The authors have not declared any conflicts of interests. References 1. Levrero M. Viral hepatitis and liver cancer: the case of hepatitis C. Oncogene 2006; 25: Marcellin P, Asselah T, Boyer N. Fibrosis and disease progression in hepatitis C. Hepatology 2002; 36: S Choo QL, Kuo G, Weiner AJ, et al. Isolation of a cdna clone derived from a blood-borne non-a, non-b viral hepatitis genome. Science 1989; 244: Kuiken C, Simmonds P Nomenclature and numbering of the hepatitis C virus. Methods Mol Biol 2009; 510: Asselah T, Marcellin P New direct-acting antivirals combination for the treatment of chronic hepatitis C. Liver Int 2011; 31(Suppl. 1): Zeisel MB, Fofana I, Fafi-Kremer S, Baumert TF. Hepatitis C virus entry into hepatocytes: molecular mechanisms and targets for antiviral therapies. J Hepatol 2011; 54: Timpe JM, McKeating JA. Hepatitis C virus entry: possible targets for therapy. Gut 2008; 57: Blanchard E, Belouzard S, Goueslain L, et al. Hepatitis C virus entry depends on clathrin-mediated endocytosis. J Virol 2006; 80: Trotard M, Lepere-Douard C, Regeard M, et al. Kinases required in hepatitis C virus entry and replication highlighted by small interference RNA screening. FASEB J 2009; 23: Coller KE, Berger KL, Heaton NS, et al. RNA interference and single particle tracking analysis of hepatitis C virus endocytosis. PLoS Pathog 2009; 5: e Steinmann E, Penin F, Kallis S, et al. Hepatitis C virus p7 protein is crucial for assembly and release of infectious virions. PLoS Pathog 2007; 3: e Griffin SD, Beales LP, Clarke DS, et al. The p7 protein of hepatitis C virus forms an ion channel that is blocked by the antiviral drug, Amantadine. FEBS Lett 2003; 535: Jones CT, Murray CL, Eastman DK, Tassello J, Rice CM. Hepatitis C virus p7 and NS2 proteins are essential for production of infectious virus. J Virol 2007; 81: Jirasko V, Montserret R, Appel N, et al. Structural and functional characterization of nonstructural protein 2 for its role in hepatitis C virus assembly. J Biol Chem 2008; 283: Boson B, Granio O, Bartenschlager R, Cosset FL. A concerted action of hepatitis C virus p7 and nonstructural John Wiley & Sons A/S

7 protein 2 regulates core localization at the endoplasmic reticulum and virus assembly. PLoS Pathog 2011; 7: e Poenisch M, Bartenschlager R. New insights into structure and replication of the hepatitis C virus and clinical implications. Semin Liver Dis 2010; 30: Bartenschlager R, Penin F, Lohmann V, Andre P. Assembly of infectious hepatitis C virus particles. Trends Microbiol 2011; 19: Lohmann V, Körner F, Koch JO, et al. Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science 1999; 285: Bartenschlager R, Sparacio S. Hepatitis C virus molecular clones and their replication capacity in vivo and in cell culture. Virus Res 2007; 127: Pietschmann T, Lohmann V, Kaul A, et al. Persistent and transient replication of full-length hepatitis C virus genomes in cell culture. J Virol 2002; 76: Blight KJ, McKeating JA, Rice CM. Highly permissive cell lines for subgenomic and genomic hepatitis C virus RNA replication. J Virol 2002; 76: Pietschmann T, Zayas M, Meuleman P, et al. Production of infectious genotype 1b virus particles in cell culture and impairment by replication enhancing mutations. PLoS Pathog 2009; 5: e Kato T, Date T, Miyamoto M, et al. Efficient replication of the genotype 2a hepatitis C virus subgenomic replicon. Gastroenterology 2003; 125: Wakita T, Pietschmann T, Kato T, et al. Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat Med 2005; 11: Bartenschlager R. The NS3/4A proteinase of the hepatitis C virus: unravelling structure and function of an unusual enzyme and a prime target for antiviral therapy. J Viral Hepatitis 1999; 10: Lamarre D, Anderson PC, Bailey M, et al. An NS3 protease inhibitor with antiviral effects in humans infected with hepatitis C virus. Nature 2003; 426: McHutchison JG, Manns MP, Muir AJ, et al. Telaprevir for previously treated chronic HCV infection. N Engl J Med 2010; 362: Bacon BR, Gordon SC, Lawitz E, et al. Boceprevir for previously treated chronic HCV genotype 1 infection. N Engl J Med 2011; 364: Halfon P, Locarnini S. Hepatitis C virus resistance to protease inhibitors. J Hepatol 2011; 55: Zhou Y, Muh U, Hanzelka BL, et al. Phenotypic and structural analyses of hepatitis C virus NS3 protease Arg155 variants: sensitivity to telaprevir (VX-950) and interferon alpha. J Biol Chem 2007; 282: Pawlotsky JM. Treatment failure and resistance with direct-acting antiviral drugs against hepatitis C virus. Hepatology 2011; 53: Le Pogam S, Seshaadri A, Ewing A, et al. RG7128 alone or in combination with pegylated interferon-alpha2a and ribavirin prevents hepatitis C virus (HCV) replication and selection of resistant variants in HCV-infected patients. J Infect Dis 2010; 202: Vermehren J, Sarrazin C. New hepatitis C therapies in clinical development. Eur J Med Res 2011; 16: Gao M, Nettles RE, Belema M, et al. Chemical genetics strategy identifies an HCV NS5A inhibitor with a potent clinical effect. Nature 2010; 465: Lemm JA, O Boyle D, Liu M, et al. Identification of hepatitis C virus NS5A inhibitors. J Virol 2010; 84: Asselah T. NS5A inhibitors: a new breakthrough for the treatment of chronic hepatitis C. J Hepatol 2011; 54: Watashi K, Hijikata M, Hosaka M, Yamaji M, Shimotohno K. Cyclosporin A suppresses replication of hepatitis C virus genome in cultured hepatocytes. Hepatology 2003; 38: Kaul A, Stauffer S, Berger C, et al. Essential role of cyclophilin A for hepatitis C virus replication and virus production and possible link to polyprotein cleavage kinetics. PLoS Pathog 2009; 5: e Yang F, Robotham JM, Nelson HB, et al. Cyclophilin A is an essential cofactor for hepatitis C virus infection and the principal mediator of cyclosporine resistance in vitro. J Virol 2008; 82: Chatterji U, Lim P, Bobardt MD, et al. HCV resistance to cyclosporin A does not correlate with a resistance of the NS5A-cyclophilin A interaction to cyclophilin inhibitors. J Hepatol 2010; 53: Hanoulle X, Badillo A, Wieruszeski JM, et al. Hepatitis C virus NS5A protein is a substrate for the peptidyl-prolyl cis/trans isomerase activity of cyclophilins A and B. J Biol Chem 2009; 284: Ciesek S, Steinmann E, Wedemeyer H, et al. Cyclosporine A inhibits hepatitis C virus nonstructural protein 2 through cyclophilin A. Hepatology 2009; 50: Flisiak R, Horban A, Gallay P, et al. The cyclophilin inhibitor Debio-025 shows potent anti-hepatitis C effect in patients coinfected with hepatitis C and human immunodeficiency virus. Hepatology 2008; 47: Crabbe R, Vuagniaux G, Dumont JM, et al. An evaluation of the cyclophilin inhibitor Debio 025 and its potential as a treatment for chronic hepatitis C. Expert Opin Investig Drugs 2009; 18: Patel H, Heathcote EJ. Sustained virological response with 29 days of Debio 025 monotherapy in hepatitis C virus genotype 3. Gut 2011; 60: Asselah T, Bièche I, Mansouri A, et al. In vivo hepatic endoplasmic reticulum stress in patients with chronic hepatitis C. J Pathol 2010; 221: Rautou PE, Cazals-Hatem D, Feldmann G, et al. Changes in autophagic response in patients with chronic hepatitis C virus infection. Am J Pathol 2011; 178: Carreira RS, Lee Y, Ghochani M, et al. Cyclophilin D is required for mitochondrial removal by autophagy in cardiac cells. Autophagy 2010; 6: Ke PY, Chen SS. Activation of the unfolded protein response and autophagy after hepatitis C virus infection suppresses innate antiviral immunity in vitro. J Clin Invest 2011; 121: Estrabaud E, De Muynck S, Asselah T. Activation of unfolded protein response and autophagy during HCV infection modulates innate immune response. J Hepatol 2011; 55: Asselah T, Estrabaud E, Bieche I, et al. Hepatitis C: viral and host factors associated with non-response to pegylated interferon plus ribavirin. Liver Int 2010; 30: Jopling CL, Yi M, Lancaster AM, Lemon SM, Sarnow P. Modulation of hepatitis C virus RNA abundance by a liver-specific MicroRNA. Science 2005; 309: John Wiley & Sons A/S 15

8 53. Henke JI, Goergen D, Zheng J, et al. microrna-122 stimulates translation of hepatitis C virus RNA. EMBO J 2008; 27: Jopling CL, Schutz S, Sarnow P. Position-dependent function for a tandem microrna mir-122-binding site located in the hepatitis C virus RNA genome. Cell Host Microbe 2008; 4: Machlin ES, Sarnow P, Sagan SM. Masking the 5 terminal nucleotides of the hepatitis C virus genome by an unconventional microrna-target RNA complex. Proc Natl Acad Sci USA 2011; 108: Lanford RE, Hildebrandt-Eriksen ES, Petri A, et al. Therapeutic silencing of microrna-122 in primates with chronic hepatitis C virus infection. Science 2010; 327: Reiss S, Rebhan I, Backes P, et al. Recruitment and activation of a lipid kinase by hepatitis C virus NS5A is essential for integrity of the membranous replication compartment. Cell Host Microbe 2011; 9: Vaillancourt FH, Pilote L, Cartier M, et al. Identification of a lipid kinase as a host factor involved in hepatitis C virus RNA replication. Virology 2009; 387: Borawski J, Troke P, Puyang X, et al. Class III phosphatidylinositol 4-kinase alpha and beta are novel host factor regulators of hepatitis C virus replication. J Virol 2009; 83: Berger KL, Cooper JD, Heaton NS, et al. Roles for endocytic trafficking and phosphatidylinositol 4-kinase III alpha in hepatitis C virus replication. Proc Natl Acad Sci USA 2009; 106: Berger KL, Kelly SM, Jordan TX, Tartell MA, Randall G. Hepatitis C virus stimulates the phosphatidylinositol 4-kinase III alpha-dependent phosphatidylinositol 4-phosphate production that is essential for its replication. J Virol 2011; 85: Gane EJ, Roberts SK, Stedman CA, et al. Oral combination therapy with a nucleoside polymerase inhibitor (RG7128) and danoprevir for chronic hepatitis C genotype 1 infection (INFORM-1): a randomised, double-blind, placebo-controlled, dose-escalation trial. Lancet 2010; 376: Zeuzem S, Asselah T, Angus P, et al. Efficacy of the protease inhibitor BI , polymerase inhibitor BI , and ribavirin in patients with chronic HCV infection. Gastroenterology 2011; 141: Tellinghuisen TL, Marcotrigiano J, Rice CM. Structure of the zinc-binding domain of an essential component of the hepatitis C virus replicase. Nature 2005; 435: Bressanelli S, Tomei L, Roussel A, et al. Crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus. Proc Natl Acad Sci USA 1999; 96: Lesburg CA, Cable MB, Ferrari E, et al. Crystal structure of the RNA-dependent RNA polymerase from hepatitis C virus reveals a fully encircled active site. Nat Struct Biol 1999; 6: Griffin S. Inhibition of HCV p7 as a therapeutic target. Curr Opin Investig Drugs 2010; 11: Raney KD, Sharma SD, Moustafa IM, Cameron CE. Hepatitis C virus non-structural protein 3 (HCV NS3): a multifunctional antiviral target. J Biol Chem 2010; 285: Einav S, Gerber D, Bryson PD, et al. Discovery of a hepatitis C target and its pharmacological inhibitors by microfluidic affinity analysis. Nat Biotechnol 2008; 26: Legrand-Abravanel F, Nicot FIzopet J. New NS5B polymerase inhibitors for hepatitis C. Expert Opin Investig Drugs 2010; 19: John Wiley & Sons A/S