New targets for antiviral therapy of chronic hepatitis C

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New targets for antiviral therapy of chronic hepatitis C

Liver International

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Special Issue: Proceedings of the 5th Paris Hepatitis Conference. International Conference of the Management of Patients with Viral Hepatitis: Special Edition Hepatitis C
Volume 32, Issue Supplement s1, pages 9–16, February 2012
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

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-IFN[alpha]) and ribavirin (RBV). This situation has changed with the development of two drugs targeting the NS3/4A protease, approved for combination therapy with PEG-IFN[alpha]/RBV for patients infected with genotype 1 viruses. Moreover, two additional viral proteins, the RNA-dependent 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-IFN[alpha]/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 broad-spectrum 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 130–180 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 interferon-alpha (PEG-IFN[alpha]) 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-IFN[alpha]/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 IFN[alpha]-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].
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-density-lipoprotein 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, 8, 9, 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, 12, 13, 14, 15].




Figure 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 [alpha]-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 β-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.
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At least five nonstructural (NS) proteins constitute the HCV replicase [reviewed in [16]]:
  1. NS3, a multi-functional enzyme with an amino-terminal serine protease domain and a carboxy-terminal RNA helicase/NTPase domain (Fig. 1B).
  2. NS4A, a co-factor of the NS3 protease forming a stable heterodimeric NS3/4A complex (Fig. 1B).
  3. NS4B, the presumed central organizer of the HCV replicase complex and a main inducer of intracellular membrane rearrangements.
  4. NS5A, an RNA-binding phosphoprotein required both for RNA replication and assembly of infectious virus particles (Fig. 1B).
  5. 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 [21, 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).
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 antibodies or compounds targeting cellular receptors [reviewed in [6]]
E2 Envelope glycoprotein; receptor binding
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 (http://www.achillion.com/HCV-overview)
NS4B Induction of membrane rearrangements; main organizer of membranous HCV replication complex Yes; e.g. clemizole [69]
NS5A RNA replication and assembly Yes; two inhibitor classes [34, 35]
NS5B RNA-dependent RNA polymerase Yes; NI and NNI [reviewed in [70]]


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 serine-type protease. In spite of its unfavourable overall 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 auto-inhibition [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 2003. 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 503034) and telaprevir (VX-950). Their use in combination with PEG-IFN[alpha]/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-IFN[alpha]/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 IC50 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-IFN[alpha]-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 target. However, by using replicon-based screens and subsequent intensive chemical refinements of primary hits, a highly active compound (BMS-790052) 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 10–100 NS5A molecules, arguing that BMS-790052 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-790052 reduced viral load around 1000-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, 35, 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 [alpha]-helix with domain 1 [35]. Although some of these mutations reduce the antiviral efficacy of BMS-790052 around 1800-fold (Y93C) or even 3400-fold (L31V), because of the extremely high potency of this compound, the EC50 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, 39, 40, 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-IFN[alpha]/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 ER-stressed 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 20–22 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 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-[alpha] (PI4KIII-[alpha]) that has been identified in several independent siRNA-based screenings [9, 57, 58, 59, 60]. It was shown that PI4KIII-[alpha] 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-[alpha], 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-[alpha] 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 IFN[alpha]-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 IFN[alpha]-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: 264286.

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