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Review

New advances in the molecular biology of hepatitis C virus infection: towards the identication of new treatment targets
Alexander Ploss,1 Jean Dubuisson2
1

Center for the Study of Hepatitis C, Laboratory for Virology and Infectious Diseases, The Rockefeller University, New York, USA 2 Molecular and Cellular Virology of Hepatitis C, Center for Infection and Immunity of Lille (CIIL), Universite Lille Nord de France, Institut Pasteur de Lille, Lille, France Correspondence to Professor Alexander Ploss, The Rockefeller University, Center for the Study of Hepatitis C, Laboratory for Virology and Infectious Diseases, 1230 York Avenue, Box 64, New York 10065, USA; aploss@rockefeller.edu Professor Jean Dubuisson, Molecular & Cellular Virology of Hepatitis C, Center for Infection & Immunity of Lille (CIIL), Universite Lille Nord de France, Institut Pasteur de Lille, Lille, France; jean.dubuisson@ibl.fr

ABSTRACT Hepatitis C virus (HCV) causes chronic infection in almost 2% of the worlds population. If untreated, chronic carriers can develop severe liver disease including brosis, cirrhosis and hepatocellular carcinoma. Until recently, hepatitis C was treated with a combination of pegylated interferon and ribavirin, a treatment which was only partially effective and was plagued with side effects. In 2011 two inhibitors of the virally encoded NS3/4 protease have become part of standard therapy, which have improved treatment rates but can exacerbate the problematic side effects. While the addition of these rst directly acting antivirals (DAAs) marks a milestone in anti-HCV therapy, new and improved combinations of drugs are desperately needed. New generations of drugs will have to address genetic variability of HCV and issues of viral resistance. Furthermore, combination therapies have to be tailored to effectively cure patient populations that have traditionally been hardest to treat, including patients with cirrhosis, those receiving liver transplants and individuals who are co-infected with HIV or hepatitis B virus. Since the discovery of HCV a plethora of experimental tools have been developed which enabled detailed analysis of various aspects of the viral life cycle and the interaction of HCV with its human host. Such studies have revealed a growing list of targets for therapeutic intervention, some of which will be discussed in this review.

Hepatitis C remains a global epidemic. At least 130 million people have chronic hepatitis C and are at risk of developing severe and if untreated fatal liver disease. However, epidemiological data are incomplete and the actual number in the USA alone may be twice as high as currently estimated.1 Hepatitis C is caused by hepatitis C virus (HCV), a positive sense, single-stranded RNA virus of the Flaviviridae family. HCV has a high propensity for establishing a chronic infection. It has been estimated that in chronically infected people approximately 1012 viral particles are generated every day.2 This remarkable replicative tness in combination with the highly error prone polymerase results in a tremendous genetic diversity, translating into readily acquired viral resistance.3 Recent improvements in the standard of care therapy, now a combination of pegylated interferon (peg-IFN), ribavirin and one of two drugs interfering with the virally encoded
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NS3/4a protease, and promising data from clinical trials of even more effective compounds have raised the hope that HCV infection can be managed efciently in countries with adequate medical infrastructure. Addition of the rst-generation protease inhibitors to peg-IFN/ribavirin therapy has boosted HCV clearance from around 50% to 60e70% in some clinical trial cohorts.4e7 However, the current treatment is not equally effective for all of the seven HCV genotypes and all patient cohorts, has to be administered in a frequent and cumbersome dosing regimen over several months and causes considerable side effects, which require close medical supervision. The current goal for HCV therapy is to achieve sustained virological response in most patients with an efcient combination of directly acting antivirals (DAAs) not leading to the emergence of escape mutations. New generations of drugs will help to come closer to this goal but likely multiple drugs with diverse modes of action will be needed to cure HCV with an all oral, IFN-free cocktail. Removing IFN from the standard is desirable as peg-IFN causes substantial side effects, needs to be administered via injections and the response prole in patients is heterogeneous depending on the host background. The rst proof of concept recently emerged from clinical trials demonstrating that combinations of orally administered DAAs with different mechanisms of action can result in cure of chronic HCV infection.8 9 While these results are encouraging additional drugs with distinct targets may be needed to achieve a sustained virological response in optimally all patients. In this review we will discuss different aspects of the HCV life cycle and highlight putative new drug targets.

HCV ENTRY
Viral entry relies on a ne interplay between a viral particle and a host cell. This process is initiated by binding to an attachment factor present at the plasma membrane of target cells, which helps to concentrate virions on the cell surface. After this initial step, the viral particle interacts with more specic receptors, which actively promote virus entry by inducing conformational changes of the viral particle or by activating signalling pathways or internalisation of the virion. Currently, HCV entry is viewed as a complex multistep process
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since a series of specic cellular entry factors have been shown to be essential in the early steps of the HCV life cycle. These molecules include the scavenger receptor class B type I (SRB1, ofcially designated as SCARB1),10 the tetraspanin CD81,11 tight junction proteins, claudin-1 (CLDN1)12 and occludin (OCLN)13 14 and the receptor tyrosine kinases epidermal growth factor receptor (EGFR) and ephrin receptor A2 (EphA2)15 (gure 1). HCV dependence on a series of specic entry factors likely contributes to its liver tropism. However, restriction factors such as EWI-2wint, a CD81 partner expressed in non-hepatic cells, can also contribute to HCV tropism by blocking HCV entry in non-hepatic cells.16 An additional level of complexity in HCV entry is also added by the hybrid nature of the viral particle. Indeed, HCV virion is tightly associated with lipoproteins to form a complex particle that has been called lipoviroparticle17 and lipoprotein components have been shown to play some role in HCV entry (reviewed in Popescu and Dubuisson18). the hybrid nature of the viral particle, apolipoproteins such as apoE, apoB, apoC1, apoC2 and apoC3 can also be found in association with HCV virion (reviewed in Andre et al25 and Bartenschlager et al26). Furthermore, lipoprotein lipase, an enzyme known to hydrolyse triglyceride from triglyceriderich lipoproteins, can modulate HCV entry.27 Finally, a recent characterisation of cell cultureproduced particles indicates that their composition resembles the one of very-low-density lipoproteins (VLDLs) and low-density lipoproteins (LDLs) with cholesteryl esters accounting for almost half of the total HCV lipids.28 Thus, HCV particles possess a unique lipid composition that is very distinct from any other virus characterised so far.

HCV entry factors

The initial approach to identify HCV receptors was based on nding cellular partners of HCV envelope glycoprotein E2 expressed at the plasma membrane of target cells. This led to the identication of CD81 and SRB1 as putative entry factors.10 11 CD81 belongs to a family of proteins, called tetraspanins, which are involved in many cell functions such as adhesion, morphology, proliferation and differentiation. After its initial identication, the role of CD81 in HCV entry was extensively conrmed in different functional models (reviewed in Bartosch and Dubuisson29). CD81 does not seem to play a role in the early phase of HCV entry.30 Rather, it seems to prime HCV envelope proteins for low pH-dependent fusion.31 SRB1 is a multiligand receptor that was rst identied as a major receptor for high-density lipoproteins (HDLs). This receptor plays a crucial role in selective lipid uptake and bidirectional transfer of free cholesterol. The role of SRB1 in HCV entry was rst suggested by its ability to mediate E2 binding and HVR1 is essential for this interaction.10 However, this interaction could be non-essential since deletion of HVR1 is not lethal.32 Due to the hybrid nature of the HCV particle, one cannot exclude an interaction between the lipoprotein component and SRB1.33 Later on, the involvement of SRB1 in HCV entry was also extensively conrmed in vitro (reviewed in Bartosch and Dubuisson29) and in vivo.34 35 Although its exact role remains to be determined, the lipid transfer activity of SRB1 seems to be required for HCV cell entry.36 More recently, the tight-junction proteins CLDN1 and OCLN have been identied as two essential entry factors for HCV by using iterative complementary DNA library screening approaches to identify cellular factors that confer susceptibility to HCV pseudoparticle infection.12 14 Tight junctions are membrane components that control paracellular diffusion and restrict the diffusion of membrane proteins and lipids at the apical or basolateral pole of cells.37 CLDN1 and OCLN are two transmembrane components of the tight junctions. It is worth noting that other members of the CLDN family, CLDN6 and CLDN9, can replace CLDN1 in HCV entry38 39 but are only expressed at very low levels in the liver. CLDN1 and OCLN do not seem
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HCV particle
The HCV RNA genome, core and the envelope glycoproteins, E1 and E2, are the known viral components of the virion. HCV genome interacts with the core to form the nucleocapsid that is surrounded by a lipid membrane, called the viral envelope, in which are anchored the envelope glycoproteins. These envelope glycoproteins are type I transmembrane proteins which form a noncovalent heterodimer within infected cells, whereas they assemble as large covalent complexes stabilised by disulphide bonds on the viral particle.19 The presence of disulphide bridges between HCV envelope glycoproteins suggests that lateral proteineprotein interactions assisted by disulphide bond formation might play an active role during the budding process of HCV particles. HCV envelope glycoproteins are key determinants for HCV entry. They play a role in receptor binding, and they mediate the fusion process between the viral envelope and an endosomal host cell membrane. E2 glycoprotein interacts with receptors or co-receptors on target cells.10 20 Furthermore, based on similarities with envelope proteins of the Flaviviridae family, E2 has also been proposed to be the fusion protein that will interact with lipids of the target membrane and a putative structural model has been proposed.21 This model reveals the distribution of E2 amino acids among three distinct domains (DI, DII and DIII) with hypervariable region 1 (HVR1) extending the N-terminus of DI. This model indicates that the DI domain contains determinants for CD81 interaction and the DII domain contains a putative fusion loop. Much less information is available on E1. Besides its involvement in E2 folding,22 it seems to also contribute to the fusion process.23 HCV envelope glycoproteins are highly glycosylated. Most HCV glycans are highly conserved and some of them have been shown to play a role in virus entry or in protection from antibody neutralisation.24 Importantly, due to
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to directly interact with HCV particles and they are probably involved in a late post-binding step. However, CLDN1 might potentially play its role by interacting with CD81.40 Recently, a functional RNAi kinase screen identied EGFR and EphA2 as host cofactors for HCV entry.15 These receptor tyrosine kinases mediate HCV entry by regulating CD81eCLDN1 coreceptor associations and viral glycoprotein-dependent membrane fusion. More recently, NiemannPick C1-like 1 (NPC1L1) cholesterol absorption receptor has also been shown to play a role in HCV entry, probably at the fusion step.41 evidence that E2 glycoprotein can interact with SRB1 and CD81. Recently, it has been proposed that HCV virion interacts rst with SRB1.49 However, it remains to be determined whether SRB1 and CD81 form a co-receptor complex or whether the virus is transferred from SRB1 to CD81. Interestingly, CD81 mediates virus binding, triggers activation of the signalling pathways likely required during the virus life cycle50 and primes HCV envelope proteins for low pH-dependent fusion.31 Currently, there is no evidence indicating that the HCV particle directly interacts with CLDN1 or OCLN. However, it has been shown that CLDN1 and CD81 can interact at the plasma membrane,40 51 suggesting that CLDN1 might be a partner of CD81 in a HCV receptor complex. The exact role of OCLN in HCV entry remains to be determined. The proposed mechanism of HCV entry is summarised in gure 1. Single particle imaging during viral entry in Huh7.5 cells indicates that after initial binding the HCV particle binds cells on lopodia and reaches the cell body by a mechanism that relies on retrograde actin transport.52 After trafcking at the cell surface, the virions are internalised by clathrinmediated endocytosis.52 53 After internalisation, the virion is transported to Rab5a-positive early endosomes along actin stress bres, where fusion seems to take place.39 52 Generally, virus endocytosis is mediated by receptors. However, the exact role of

Mechanisms of entry
As for many viruses, HCV attachment to hepatocytes seems to be mediated by heparan sulphate proteoglycans (reviewed in Zeisel42). HCV envelope glycoproteins have been proposed to be involved in this process.43 44 However, due to the hybrid nature of the virion, one cannot exclude that lipoprotein components, like apoE, could be involved in this early step. Furthermore, the LDL receptor (LDLR) has also been proposed to play a role in the early phase of HCV entry.45e47 However, HCVeLDLR interaction seems to involve a non-productive entry pathway that can potentially lead to viral particle degradation as suggested recently.48 After the initial attachment to the cell surface, the viral particle will interact with specic entry factors. There is

Figure 1 Model of hepatitis C virus (HCV) entry. The HCV virion is tightly associated with lipoproteins to form a complex particle that has been called lipoviroparticle (LVP). It initiates its life cycle by binding to glycosaminoglycans (GAGs). Then the virus can follow either a productive or a non-productive pathway. In the non-productive pathway, the lipoprotein component of the viral particle interacts with low-density lipoprotein receptor (LDL-R) and the virion is rapidly internalised and sent to a degradation pathway. The productive pathway is a complex multistep process involving a series of specic cellular entry factors, which include scavenger receptor class B type I (SRB1), CD81, tightjunction proteins, claudin-1 (CLDN1) and occludin (OCLN), and epidermal growth factor receptor (EGFR) (see text for details). After binding to several components of the host cell, the HCV particle is internalised by clathrin-mediated endocytosis and fusion takes place in early endosomes.
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HCV co-receptors in endocytosis of the virus remains undetermined. It is worth noting that hepatocytes are polarised with separate apical and basolateral membrane domains, showing a specic organisation that is unique to the liver. It is therefore very likely that the specic architecture of the liver plays a major role in HCV propagation within this organ. Although cell polarity seems to play a role in HCV entry,50 some controversy remains on how this affects the entry process.54 HCV seems to induce depolarisation, at least in cell culture.55 As for other viruses, HCV can also spread, at least in cell culture, by direct cell-to-cell transfer.56 57 Although it seems to involve most HCV entry factors identied so far,58 the mechanism governing this process remains unknown. appear to be very potent in neutralising viral strains of different genotypes in vitro,60e62 their in vivo efcacy turns out to be much lower.63 64 This could be due to the fact that HCV can infect neighbouring cells by cell-to-cell contacts, an alternative transmission route that is resistant to neutralising antibodies.56 57 Other molecules targeting the viral particle have been shown to block HCV entry. Lectins such as cyanovirin-N and grifthsin bind to the high-mannose glycans present on the HCV particle and thereby inhibit HCV entry by blocking the interaction between E2 and CD81.65 66 Although such molecules can potentially inhibit cell-to-cell transmission,66 they are immunogenic and small lectin-like compounds could be better adapted to be used in clinical settings.67 Other small molecules targeting HCV envelope proteins have been described recently. The green tea polyphenol epigallocatechin-3-gallate has been shown to inhibit HCV entry by blocking its binding to target cells.68 69 A potent HCV-specic triazine inhibitor, called EI-1, has also been identied recently by screening a small molecule library.70 However, this compound, which targets HCV envelope proteins at a post-binding step, seems to be genotype specic. Amphipathic phosphorothioate oligonucleotides are known to mimic the amphipathic a helical hinge

HCV ENTRY AS A THERAPEUTIC TARGET

The complexity of the HCV entry process offers a large number of potential therapeutic targets (gure 2). HCV entry could potentially be inhibited by blocking either the viral particle or some of the cellular entry factors. Different strategies for the prevention of HCV infection have already been tested. HCVentry can be inhibited with monoclonal or polyclonal neutralising antibodies (reviewed in Edwards et al59). Although neutralising antibodies

apoE

apoB

LVP

nAbs, EI-1, lectins, EGCG, Virus entry A blocking Ab, ITX5061, ezetimibe, erlotinib

G Virion release

Entry factor(s)

Endocytic compartment
H+ H+ H+

B Receptor-mediated endocytosis

Fusion C Uncoating

NS3/4A protease inhibitors D Translation and polyprotein processing

F Virion assembly Inhibition of core dimerisation, E1/E2 folding, p7, NS2, NS5A inhibitors DGAT, MTP inhibitors E RNA replication NS3H, NS4B, NS5A, NS5B inhibitors miR122, CypA PI4KIII inhibitors

(+)

(+/-)

Membraneous web

p7 C E1 E2 NS2 NS3 4A 4B 5A 5B

Structural Proteins

Non-structural

Nucleus
Figure 2 The hepatitis C virus (HCV) life cycle, intervention points and selected inhibitor classes. (A) The HCV lipoviroparticle (LVP) engages numerous entry factors prior to receptor-mediated endocytosis (B). Following membrane fusion and uncoating (C) the RNA genome is translated in a continuous polyprotein and processed by viral and host proteases in the 10 mature viral proteins (D). (E) Several non-structural proteins and host factors form a membranous replication complex (membranous web), in which the positive sense HCV RNA genome is amplied via a negative strand intermediate. HCV assembly (F) and release (G) is closely linked to very-low-density lipoprotein biogenesis and egress. Ab, antibody; apo, apolipoprotein; CypA, cyclophillin A; DGAT, diacylglycerol acyltransferase; EGCG, epigallocatechin-3-gallate; miR122, micro RNA 122; MTP, microsomal triglyceride transfer protein; nAb, neutralizing antibody; PI4KIIIa, phosphatidyl inositol 4-kinase IIIa. Directly activating antivirals are in red and host-targeting antivirals are in purple.
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domains of fusion proteins on many enveloped viruses, and such compounds have also been shown to inhibit HCV at a post-binding step.71 Peptides, derived or not from HCV glycoproteins, can also exhibit anti-HCV activity at a post-binding step or by inactivating the viral particle.72e74 To overcome the high variability of the viral envelope proteins, the well conserved cellular entry factors may be a more promising target for inhibition of HCV entry. Blockade of CD81, SR-B1 and CLDN1 with monoclonal antibodies has been shown to be a very efcacious way to prevent HCV infection in vitro and in vivo in a genotype independent manner.35 75 76 Such antibodies could be developed for liver transplant patients, but the cost of producing these proteins remains high. Small molecules targeting HCV receptors could therefore be a cheaper alternative. The most advanced inhibitor of HCV entry is ITX 5061, a small molecule compound that was initially identied to promote HDL levels in animals and patients by targeting SRB1.77 The recent identication of EGFR, EphA2 and NPC1L1 as HCV entry factors also opens new opportunities for the development of entry inhibitors.15 41 Indeed, licensed molecules such as erlotinib (an EGFR inhibitor) and ezetimibe (an NPC1L1 inhibitor) have been shown to impair HCV entry. Finally, clathrin-dependent endocytosis is another potential target for the development of anti-HCV molecules.78 79 Although targeting cellular factors is a promising antiviral approach, interactions of blocking agents with cellular proteins could interfere with the natural function of these host proteins and potentially induce unwanted side effects. structural (NS) proteins. Dimerised NS2 in conjunction with the N-terminal domain of NS3 is thought to cleave the NS2/NS3 junction. NS3 mediates cleavage of NS4A from itself and NS4B, after which NS4A associates with the N-terminus of NS3. The resulting NS3/4A protease complex facilitates cleavage at the NS4B/5A and NS5B junctions in trans. After translation, the HCV proteins are associated with membranes derived from the endoplasmic reticulum (ER). NS3 through NS5B comprise the replication machinery, which replicates the positive sense RNA genome through a negative strand intermediate. Nascent RNA genomes are translated to produce new viral proteins, serve as new/additional RNA templates for further RNA replication and are assembled to infectious virions. The HCV replication complex is subcellularly located in the so-called membranous web (gure 2), an accumulation of ER-derived membrane vesicles and lipid droplets (LDs), which is induced by NS4B possibly in combination with NS5A (reviewed in Bartenschlager80). The advent of subgenomic and full-length HCV replicase constructs, HCV replicons and the infectious HCV cell culture system allowed assessment of the dependence of HCV replication on host factors. Targeted and genome-wide siRNA screens revealed numerous cellular host factors putatively promoting or restricting HCV replication.52 88e98 Unfortunately, independent studies are often minimally overlapping and the relevance of many of these interactions to HCV biology remains to be demonstrated. Strong experimental evidence has been provided for the critical role of cyclophilin A (CypA) in HCV RNA replication and probably also virion assembly.99 100 CypA probably interacts directly with NS5A to exert its effect, through its peptidyl-prolyl isomerase activity, on maintaining the proper structure and function of the HCV replicase. Another host factor critical for HCV genome replication, which was independently identied by several groups,88 93 94 97 98 is phosphatidylinositol 4 kinase IIIa (PI4KIIIa). PI4KIIIa is recruited to the membranous web via interaction with NS5A. The lipid kinase activity of PI4KIIIa activated by NS5A binding appears to contribute to the integrity of the membranous replication complex.97

HCV RNA TRANSLATION AND REPLICATION

The HCV genome contains a single open reading frame, which is anked by 59 and 39 non-translated regions (NTRs). These NTRs contain highly structured RNA elements that are critical for genome translation and HCV RNA replication (reviewed in Bartenschlager80). It was demonstrated that two sites in the HCV 59 untranslated region (UTR) interact with a liver-specic microRNA, miR122.81 82 These interactions were shown to be critical for efcient HCV RNA replication, presumably by protecting 59 terminal viral sequences from nucleolytic degradation or from inducing innate immune responses to the RNA terminus,83 resulting in greater viral RNA abundance in infected cultured cells and in the liver of infected chimpanzees.84 The 59 NTR contains an internal ribosomal entry site, which initiates translation of the HCV genome into a single continuous polyprotein (gure 2). Viral and host encoded proteases process the viral polyprotein into the 10 mature proteins, core, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B. Signal peptidase85 and signal peptide peptidase86 87 facilitate cleavage of the structural proteins, core, E1, E2 and the p7/NS2 junction. Cis-cleaving and transcleaving viral autoproteases process the nonGut 2012;61(Suppl 1):i25ei35. doi:10.1136/gutjnl-2012-302048

TARGETING HCV NON-STRUCTURAL PROTEINS: DAAS

HCV proteins with obvious enzymatic activities were the rst to be targeted for antiviral drug development (gure 2). In 2011, two inhibitors of the NS3/4A protease, telaprevir and boceprevir, have been approved for treatment of HCV infection. Addition of one of two inhibitors to standard of care therapy has substantially increased sustained virological response rates in patients infected with HCV genotype 1, reaching 70% in some clinical trial cohorts.4e7 However, this rst generation of proteases aggravates the side effects
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of peg-IFN and ribavirin therapy, and is less effective against non-genotype 1 viruses. Both shortcomings are likely to be addressed by secondgeneration protease inhibitors. The HCV replication can also be efciently inhibited with allosteric and active site inhibitors interfering with the enzymatic activity of the HCV RNA-dependent RNA polymerase, NS5B. Some of the most promising chain-terminating nucleoside analogues have a very high barrier of resistance and are broadly active against multiple genotypes as the active site of the RNA polymerase is highly conserved across genotypes (reviewed in Membreno and Lawitz101) and are likely to become an important part of DAA cocktails in the future. Nonnucleoside inhibitors interfere with the enzymatic function of the RNA-dependent RNA polymerase through binding to one of at least four allosteric sites within NS5B. Non-nucleoside inhibitors have a low genetic resistance barrier, exhibit only limited clinical efcacy (reviewed in Membreno and Lawitz101) and thus their role for future therapy remains to be determined. Other enzymatic targets, including the NS2/3 autoprotease and an RNA helicase encoded in the C-terminal region of NS3, have been less well explored. NS2 is a dimeric cysteine protein cleaving a single junction at the NS2/3 boundary, which is required for RNA replication of full-length HCV replicons, in the infectious tissue culture system, and in chimpanzees (reviewed in Lorenz102). It is conceivable that, not NS2 itself but the cleaved Nterminus of NS3 is necessary for the onset of replication. Whether NS2 has a direct role in the replication of HCV genomes is unclear. Dimerisation of NS2 and the NS2eNS3 interaction could be targeted to disturb NS2eNS3 processing and in turn virus replication. Our understanding of the multifunctional NS3 protease/helicase has improved substantially but the NS3 helicase function is one of the least developed drug targets. The NS3 helicase can separate RNA and DNA duplexes and can displace nucleic acid bound proteins in an ATP-dependent process (reviewed in Frick103). The exact role of the helicase in HCV replication is incompletely understood, but a functional helicase is indispensable for HCV RNA replication in cell culture.104 HCV RNA harbouring point mutations in the NS3 ATPase do not result in viraemia in experimentally inoculated chimpanzees.105 Few HCV helicase inhibitors have advanced from early discovery stages because of limited specicity and high toxicity. However, new high throughput screening modalities that measure nucleic acid stimulated ATP hydrolysis and displacement of a molecular beacon bound to DNA or RNA may be suitable to identify more promising lead compounds (reviewed in Belon and Frick106). Several non-enzymatic targets provide suitable intervention points (gure 2). The exact function of NS4B remains somewhat enigmatic but has recently achieved more attraction as a drug target. NS4B is critical for HCV RNA replication and has
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been shown to be necessary and sufcient for the induction of membranous replication complexes. The precise mechanism of how NS4B triggers the formation of these specic, vesicular membrane rearrangements and facilitates HCV RNA replication remains opaque. Several portions of NS4B may be involved in this process: it was demonstrated that conserved elements in the C-terminal portion of NS4B are required for the establishment of functional HCV replication complexes.107 Furthermore, NS4B contains adjacent N-terminal amphipathic helices, 4BAH1 and 4BAH2, which are essential for RNA replication.108 109 NS4B has nucleotide binding and NTPase activity and can bind to HCV RNA, with a preference for the 39 terminus of the negative strand, the presumed region where progeny plus-strand genomes are initiated.110 Clemizole was previously identied in a small molecule screen as a potent inhibitor of HCV RNA replication by blocking the binding of viral RNA to NS4B.110 Clemizole may be particularly attractive as an antiviral lead compound as it has presumably two viral targets, the NS4B protein and the 39 -end of the negative strand, which may create a higher barrier for developing resistance mutations. Furthermore, clemizole operates highly synergistically with NS3 protease inhibitors,111 and has a long track record of good tolerability in patients as it was originally prescribed in the 1950s and 1960s as an antihistamine. More recently, a new class of small-molecule inhibitors was identied that prevents HCV replication by preventing either 4BAH2 oligomerisation or 4BAH2 membrane association.109 These advances establish clear proof of concept for the utility of NS4B as an antiviral drug target. The dimeric phosphoprotein NS5A can bind viral RNA and has been proposed as an important regulator of the RNA replication and virion assembly of HCV. Changes in the phosphorylation state are thought to mediate the switch between NS5A functions in the life cycle.112 Due to the lack of apparent enzymatic activity and consequently suitable assay systems it was challenging to devise screens targeting NS5A. Nonetheless, a chemical genetics strategy led to the discovery of the rst NS5A inhibitor potently reducing HCV viral load in vitro and in vivo.113 Single amino acid mutations, which map to the interface of the NS5A dimer, are sufcient to confer resistance to this inhibitor class. Nonetheless, pharmacological inhibition of NS5A has proven to lead to remarkable reductions in viral load,113 indicating that this compound class may become a key component of next-generation anti-HCV therapies.

TARGETING HCV NON-STRUCTURAL PROTEINS: HOST-TARGETING ANTIVIRALS

HCV requires numerous host factors during its replicative cycle. Due to the minimal sequence variation between individuals and the lack of resistance mutations to inhibitors, targeting such host factors is being considered as a valuable
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addition to treatment with DAAs (gure 2). Of those host-targeting antivirals CypA inhibitors are most advanced. Originally, the immunosuppressive compound ciclosporin was shown to inhibit HCV replication by interfering with CypAs functions.114 115 Subsequently, the non-immunosuppressive analogue of cyclosporine, Debio-025 (alisporivir), was identied, which can bind to CypA and inhibit viral replication.116 Alisporivir is effective against a broad range of HCV genotypes and resulted in clinical trials with a low incidence of adverse events. SiRNA mediated knock-down of PI4KIIIa expression and subsequent reconstitution demonstrate that PI4KIIIa clearly plays a critical role in the HCV replication cycle. Interpretation of pharmacological inhibition experiments is hampered by the pleiotropic actions of commonly used kinase inhibitors such as wortmannin or PIK93. Currently pharmacological inhibitors that interfere with adequate specicity with PI4KIIIa function and thus would minimise off-target toxicity have not been reported on. Furthermore, it has to be kept in mind that potent inhibition of PI4KIIIa will also interfere with its physiological functions and may result in detrimental outcomes for the hostda common caveat when targeting any host factor. Thus, it remains to be determined whether this kinase is also a suitable antiviral target for the treatment of HCV and whether it is possible to design a potent inhibitor with tolerable side effects in vivo. MicroRNA122 is an unusual but nonetheless highly attractive viral drug target. Recently, a locked nucleic acid-modied anti-sense oligonucleotide (SPC3649 or miravirsin) binding to the 59 end of miR-122 was developed which potently antagonises a broad spectrum of HCV genotypes in vitro.117 Importantly, SPC3649 can functionally inactivate miRNA-122 in vivo, resulting in a drastically reduced viral load in chronically infected chimpanzees without exhibiting any signicant side effects except for a profound decrease in serum cholesterol levels.84 Thus SPC3649 holds promise as a new therapeutic intervention approach with a high barrier to resistance and a tolerable sideeffect prole. surrounded by a phospholipid monolayer that harbours numerous proteins.120 LDs are normally distributed throughout the cytoplasm in uninfected cells, whereas they accumulate in the perinuclear region upon HCV infection. Importantly, LDs appear to play a central role in HCV assembly since all the viral proteins as well as the viral genome accumulate in close proximity of this organelle (reviewed in Popescu et al121). The core protein plays an essential role in this process since it directly interacts with LDs122 123 and it is responsible for LD relocalisation.124 Furthermore, when the coreeLD association is prevented, virus assembly is drastically impaired.125 126 Interestingly, the coreeLD association can also be inuenced by endogenous proteins. Indeed, diacylglycerol acyltransferase-1 (DGAT1), an enzyme involved in LD morphogenesis, has recently been demonstrated to interact with core and to enable coreeLD association and virus production.127 In addition to the core protein, some viral nonstructural proteins like NS5A and NS3 are also found around LDs in HCV-infected cells.126 128 The coreeLD association is essential for the recruitment of these other viral proteins and for virus production.112 129 130 Importantly, NS5A emerges as a central player in the transition between replication and assembly. Indeed, although the C-terminal domain of this protein is dispensable for replication, it plays a major role in HCV assembly.112 129 130 A model has been proposed in which NS5A is maintained in the functional replicase via association with host factors, but dissociates upon phosphorylation, leading to viral particle assembly.131 132 Indeed phosphorylation of NS5A at a cluster of serine residues in its C-terminal domain is important for the interaction of NS5A with core112 129 and thus the initiation of the assembly process. Besides the structural proteins and the viral components of the replication complex, the remaining HCV proteins, p7 and NS2, are also essential for HCV morphogenesis.133e135 NS2 interacts with E1, E2 and p7 and these proteins seem to travel together to the assembly site.136e139 It has been proposed that E1E2 heterodimer, NS2 and p7 form a functional unit, which migrates close to the LDs.138 Besides their role in helping in the transport of HCV envelope glycoproteins to the assembly site, p7 and NS2 might also play additional functions during the assembly process.133 134 140 However, it remains to be determined how they contribute to the late steps of the assembly process. Following accumulation of all the viral components close to the LDs, virion assembly can start. This process can be divided into three steps: nucleocapsid formation, budding and maturation to the infectious particle.141 Importantly, the VLDL assembly pathway is closely related to the infectious particle production. Some reports suggested that both ApoB and microsomal triglyceride transfer protein (MTP) are involved in HCV assembly.141 142 The role of MTP in HCV assembly is in contradiction with the observation that HCV can inhibit the expression of this protein.143
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HCV ASSEMBLY
The HCV assembly process is the least understood step in the virus life cycle. HCV morphogenesis presents the peculiarity of the double role of the NS proteins in both replication and assembly. Another major specic feature of HCV is that its life cycle, and the assembly step in particular, is intricately connected to the lipid metabolism (reviewed in Targett-Adams et al118). Structural and NS proteins of HCV interact with components of the lipid metabolism at different levels. A major feature in HCV infection is the profound change in the intracellular distribution of LDs (reviewed in McLauchlan119). LDs are organelles which function as a deposit of triacylglycerides and cholesteryl esters
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However, initial upregulation of MTP could favour assembly and propagation of HCV while downregulation in the chronic phase could cause impairment of triglyceride secretion and excessive lipid accumulation.144 The virion has a VLDL-like and LDL-like lipid composition and is associated with apoE, which is essential for the infectious virus assembly.28 141 142 145 However, the detailed mechanisms leading to this lipoviroparticle assembly remain to be determined.

SUMMARY/PERSPECTIVE
Improved therapies for chronic HCV infection are desperately needed. Current standard of care therapy consisting of peg-IFN, ribavirin and an NS3/4A inhibitor is ineffective or only partially effective, and often contraindicated due to side effects in multiple patient populations. These patient populations include those whose condition has not responded to previous treatment, patients suffering from advanced liver disease (brosis and cirrhosis) and those with HCV co-infected with HIV or HBV. For the short term it remains a priority to develop IFN-free therapies, which effectively eliminate the virus. IFN is effective in the majority of patients, especially those with a favourable interleukin-28B polymorphism, which was previously shown to strongly correlate with HCV clearance and response to therapy.155e158 The drug development pipeline contains several compounds that hold promise to achieve the goal of a shorter and more tolerable therapy, and are also like to drastically improve treatment response rates. As outlined in this review, multiple additional targets have emerged or are beginning to emerge that may add to the arsenal of therapeutic intervention. Future standard of care therapy will hopefully address the needs of all patients and will lead to a cure in most if not all chronic HCV carriers. However, it remains unclear how highly potent antiviral drug cocktails will affect the global HCV disease burden. High costs, lack of adequate infrastructure for distribution and medical supervision may lessen the impact of future therapies in resource-poor environments. Thus, development of potent, pan-genotypic costeffective therapeutic and prophylactic vaccines remains a priority to conquer the global HCV health problem.
Acknowledgements We thank Sophana Ung (CIIL) for his help in preparing some of the illustrations. Contributors Alexander Ploss and Jean Dubuisson contributed equally. Funding This work was supported in part by award number RC1DK087193 (AP) from the National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Diseases, Infectious Disease Society of America Astella Young Investigator Award (AP) and the French Agence Nationale de Recherche sur le Sida et les hepatites virales (ANRS) (JD). Competing interests None. Provenance and peer review Commissioned; externally peer reviewed.

HCV ASSEMBLY AS A THERAPEUTIC TARGET

The cell-culture system for HCV has allowed the optimisation of drug screening assays which might indicate novel molecules acting at different steps in the viral life cycle, including virus assembly.78 79 146 As for the other steps, blocking virus assembly can be achieved by targeting viral components or cellular factors (gure 2). Approaches to develop antiviral molecules targeting viral proteins are only starting to emerge. A screening test for molecules inhibiting core dimerisation has indeed recently been developed.147 The p7 polypeptide is another potential target, however the currently available compounds against this protein have a modest antiviral effect (reviewed in Steinmann and Pietschmann148). Other viral proteins could also be interesting viral targets. Due to its major role in HCV assembly, the C-terminal domain of NS5A is of particular interest. HCV envelope proteins are also potentially interesting targets since they play several interconnected functions. Besides viral components, cellular factors involved in HCV morphogenesis can also be interesting candidates. Due to their role in glycoprotein folding, inhibitors of a-glucosidases have been developed as inhibitors of HCV assembly.149 150 Two a-glucosidase inhibitors, UT-231B (an imminosugar) and celgosivir (MX-3253da castanospermine prodrug), made it to phase II clinical trials, but both have since been stopped.151 152 The recent identication of DGAT1 as a cellular factor essential for core protein localisation around LDs suggests that this cellular protein could also be an interesting target for therapeutic intervention.127 Indeed DGAT1 inhibitors, currently in early clinical trials for obesity-associated diseases, could be tested for their potential activity against HCV. It is worth noting that another diacylglycerol acyltransferase (DGAT2) is also involved in LD biogenesis,153 but HCV only targets DGAT1. Furthermore, DGAT2generated LDs form normally in DGAT1 inhibitortreated cells, suggesting limited effects of DGAT1 inhibitors on the cellular functions.127 Since HCV assembly is closely linked to VLDL biogenesis, targeting this lipoprotein pathway could offer another possibility for therapeutic intervention. The grapefruit avonoid naringenin has been shown to inhibit VLDL secretion in vitro and in vivo and it can inhibit HCV secretion in cell culture.154 In the same line, MTP inhibitors could also be interesting to test against HCV.142

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Gut 2012;61(Suppl 1):i25ei35. doi:10.1136/gutjnl-2012-302048

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New advances in the molecular biology of hepatitis C virus infection: towards the identification of new treatment targets
Alexander Ploss and Jean Dubuisson Gut 2012 61: i25-i35

doi: 10.1136/gutjnl-2012-302048

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