Assoc. Professor SA Locarnini, Research and Molecular Development, Victorian Infectious Diseases Reference Laboratory, 10 Wreckyn Street, North Melbourne, Vic. 3051, Australia. Email: firstname.lastname@example.org
Abstract Hepatitis C virus (HCV) is now the major cause of transfusion-associated and parenterally transmitted viral hepatitis and accounts for a significant proportion of hepatitis cases worldwide. The majority of infections become persistent and approximately 20% of chronically infected individuals develop cirrhosis, which is strongly associated with progression to hepatocellular carcinoma. Molecular biological investigations into the structure and function of HCV and its genes has led to the identification of a number of potential targets for selective antiviral intervention. The present review summarizes current research activity into these novel drug targets and addresses the basis for clinical non-response in the current interferon-α-based therapies. Future therapeutic strategies that utilize HCV-specific antiviral agents should prove effective in controlling active viral replication, but the risk of emergence of drug-resistance will need to be addressed due to the quasispecies feature of HCV replication.
Hepatitis C virus (HCV) infects an estimated 170 million persons worldwide and thus represents a very significant viral pandemic. The virus establishes persistent infection in the majority of infections.1,2 Chronic hepatitis C (CH-C) is a fibrotic liver disease that slowly progresses over several decades, resulting in cirrhosis in approximately 20–25% of patients.1,2 However, progression to chronic disease occurs in the majority of HCV-infected persons.3 Unfortunately, advances in HCV research have been hampered by the inability to grow the virus easily in cell culture. However, there have been some recent important advances into the pathogenesis of the infection and also improvements in therapeutic options and outcomes.
Virology and pathogenesis
The HCV is an RNA-containing virus that belongs to the family Flaviviridae, with the most closely related human viruses being the hepatitis G virus (or GBV-C), yellow-fever virus and dengue virus.4 The natural targets of HCV are hepatocytes and possibly B lymphocytes.5,6 Viral replication is extremely vigorous and an enormous yield phenotype is typical of active replication. It has been estimated that more than 10 trillion virion particles are produced every day in an infected individual.7 Replication occurs through an RNA-dependent RNA polymerase mechanism and the viral replicase lacks a ‘proof reading’ function, resulting in the rapid evolution of diverse but related swarms of quasispecies within an infected person.
The HCV is a plus-sense single-stranded RNA virus of 9.5 kb. The genome consists of a single open reading frame and two untranslated regions (UTR) at each end. It encodes a polyprotein of approximately 3000 amino acids, which is cleaved into single proteins by a host signal peptidase in the structural region and the HCV-encoded protease in the non-structural (NS) region (Fig. 1). The structural region contains the core protein and two envelope proteins E1 and E2. There are two regions in E2, called hypervariable regions 1 and 2 (HVR1 and HVR2), which show extreme sequence variability due to high rates of mutation that are thought to be the result of selective pressure generated by the host's immune response through the production of virus-specific antibodies. The E2 protein also contains the binding site for CD81, a tetraspanin expressed on hepatocytes and B lymphocytes, and this has been claimed to be the putative HCV receptor or coreceptor.8 The low-density lipoprotein receptor has also been proposed as a potential receptor for HCV.9,10
The NS proteins of HCV have been assigned functions as proteases in the case of NS2, NS3 (the N-terminal part) and NS4A, helicase (for the C-terminal part of NS3) and replicase (or RNA-dependent RNA polymerase or RDRP) for the NS5B. The crystal structure of NS3 and NS5 has been recently resolved11,12 but the function and properties of the other HCV NS proteins, for example p7, are less well defined. A region in the NS5A gene has been identified as the interferon-sensitivity-determining region (ISDR) and has been linked to the clinical response during interferon therapy.13 More recently, Taylor et al. demonstrated that the E2 protein can also interfere with the antiviral action of interferon (IFN)-α.14
In most persons who become infected with HCV, viremia persists, accompanied by variable degrees of hepatic inflammation and fibrosis. Early studies of chronic HCV infection indicated that only a small percentage of hepatocytes were actually infected, but more recent estimates have suggested that over 50% or more of hepatocytes are infected with virus.15
The presence of lymphocytes within the hepatic parenchyma has been interpreted as evidence of immune-mediated damage, as in the case of HBV infection.16 However, recent studies of acute HCV infection in both chimpanzees and humans has suggested that immune-mediated control of viral replication may be possible. Viral clearance of transient infections was associated with the development and persistence of strong, virus-specific responses by cytotoxic T lymphocytes17–19 and T-helper (Th) cells.18 The responses of Th cells appear to be critical because the loss of these cells has been linked to the re-emergence of viremia.20 The recent observation that viral diversity is reduced in those persons in whom the infection is cleared (i.e. fewer quasispecies being observed) is also consistent with the occurrence of greater immune-mediated control of the virus.21
The relatively weak responses of cytotoxic T lymphocytes in persons with chronic HCV infection seems to be insufficient to contain viremia and genetic evolution of the virus, but unfortunately sufficient to cause collateral damage through the elaboration of inflammatory cytokines in the liver.22,23
Viral replication: life cycle and antiviral targets
As well as encoding the viral structural and non-structural proteins, the HCV-RNA genome also contains two UTR at the 5′-and 3′ terminus of the viral genome that are important for the control of translation and the transcription of viral RNA. The 5′-UTR of the genomic RNA encodes an internal ribosome entry site (IRES), which is responsible for the initiation of translation.24 The 3′-UTR of the genomic RNA contains a stretch of poly-U-ribonucleotides followed by a region of 98–100 nucleotides (n.t.).25 This 98–100-n.t. region is highly conserved throughout all of the HCV genotypes and isolates. This part of the 3′-UTR contains three unique stem and loop structures as well as important regulatory signals for the initiation of RNA replication. It has been speculated that this structure may also have a role in RNA stability or packaging of the RNA into viral core particles. The 3′-UTR of the negative-sense RNA must contain the RNA initiation signals for the synthesis of genomic sense RNA while the 5′-UTR of the negative-sense RNA would contain the termination and release signal(s) for those newly synthesized progeny RNA strands.
Hepatitis C virus life cycle
The HCV replication cycle can be summarized as follows (Fig. 2): (i) penetration of the host cell and liberation of the genomic RNA from the virus particle into the cytoplasm; (ii) translation of the input RNA, processing of the viral polyprotein and formation of a replicase complex associated with intracellular membranes; (iii) utilization of the input plus-strand for synthesis of a minus-strand RNA intermediate (RI) and possibly the formation of double-stranded (d.s.) RNA, which has been called the replicative form (RF) RNA; (iv) production of new plus-strand RNA molecules, which in turn can be used for synthesis of new minus strands, RI and even RF; (v) translation of the new plus-strand RNA molecules for polyprotein expression or packaging into progeny virions; and (vi) release of virus from the infected cell.26
Attachment and entry
The first step in a virus life cycle is the attachment of the infectious particle to the host cell, for which a specific interaction between a receptor on the cell surface and a viral attachment protein on the viral surface is required. Recently, the ligand CD81 was identified as a putative HCV receptor based on its strong interaction with E2 as well as with virus particles in vitro.8 However, whether virus binding to CD81 is followed by internalization of the virus particle is not yet known because this step of receptor-mediated endocytosis is critical to the initiation of active viral replication.
The HCV, as well as other members of the Flaviviridae family, may also enter the cell by binding to low-density lipoprotein (LDL) receptors. Agnello et al. have demonstrated a direct correlation between the level of cell surface-expressed LDL receptor and the number of positive cells endocytosing HCV.10 This result strongly suggests that HCV particles associated with LDL bind to the LDL receptor. Whether interaction with the LDL receptor9 or CD81 leads to a productive infection still remains to be determined.
Polyprotein translation and processing
Once inside the cytoplasm the genomic RNA is directly translated, and this is a hallmark feature of plus-stranded RNA viruses. Translation of the viral RNA is mediated by an IRES element.27,28 This RNA element resides approximately between nucleotides 40 and 355 of the 5′-UTR and forms four highly structured domains (I–IV).24
Activity of the HCV IRES is influenced by several factors. First, the X-tail at the 3′UTR end of the HCV genome appears to enhance IRES-dependent translation.29 Second, several cellular factors have been demonstrated to bind to the HCV IRES and, in most cases, stimulate translation. These include poly-pyrimidine-tract-binding protein (PTB),30,31 the La antigen,32 heterogeneous nuclear ribonucleoprotein L33 and as yet unidentified proteins with apparent molecular masses of 120, 87 and 25 kDa.34,35 The requirements for cellular factors for IRES activity may also explain the dependence on the cell cycle. Using cell lines stably expressing bicistronic reporter constructs with a cap-dependently expressed upstream reporter and a downstream reporter translated from the HCV IRES, it was found that IRES-dependent translation was greatest in mitotic and lowest in quiescent (G0) cells.36 One possible explanation would be that HCV translation is regulated by cellular proteins that vary in abundance or activity during the cell cycle.
The viral polyprotein is translated at the rough endoplasmic reticulum (ER) and cleaved co- and post-translationally by host cell signalases and two viral proteases (Fig. 1). As deduced from hydrophobic sequences preceding the cleavage sites and the dependence on microsomal membranes, the C-NS2 region is processed by host signal peptidases cleaving at the C/E1, E1/E2, E2/p7, p7/NS2 junctions (Figs 1,2). Processing between NS2 and NS3 is a rapid intramolecular reaction and is accomplished by the NS2-3 proteinase.37–39
Processing of the NS3–5B region is mediated by the NS3 proteinase with the following preferred but not obligatory order of cleavages: NS3/4A→NS5A/B→NS4A/B→NS4B/5A.40–43 Processing at the NS3/4A site is a co-translational intramolecular reaction whereas cleavage at the other sites can be mediated intermolecularly and thus these represent attractive antiviral targets.
The proteolytic activity of NS3 is greatly stimulated by NS4A both in transfected cells and in various in vitro assay systems. A feature conserved in many plus-strand RNA virus families is that proteinase and NTPase/helicase activities reside in a single polypeptide, and the same is found with NS3 of HCV.26
A feature of HCV-infected cells is the proliferation of smooth membranes44 and most or all of the HCV polyprotein cleavage products, in particular NS3–5B, form a replicase complex associated with intracellular membranes that most likely contains abundant cellular proteins.45 The formation of such a complex is a feature typical of plus-strand RNA viruses such as poliovirus or flaviviruses,46,47 and it allows the production of viral proteins and RNA in a distinct compartment. In addition, this complex formation permits the tight coupling of functions residing in different polypeptide chains.26
The individual steps underlying RNA replication are largely unknown. It is obvious that the NS5B RDRP is the main enzyme catalyzing the synthesis of minus- and plus-strand RNA. In vitro the enzyme prefers a primer-dependent initiation of RNA synthesis, either by elongation of a primer hybridized to an RNA homopolymer or via a ‘copy back’ mechanism when using heteropolymeric templates.48–53 In the case of a ‘copy back’ mechanism, sequences at the 3′ end would have to fold back intramolecularly and hybridize, thereby generating a 3′ end that can be used for elongation, resulting in a product approximately twice the length of the input template.26 However, at least under certain experimental conditions, HCV NS5B, as well as RDRP of the closely related pestivirus bovine viral diarrhoea virus (BVDV), can initiate RNA synthesis de novo and it is quite plausible and more likely that this mechanism also operates in vivo.54–56
Cellular proteins and components are also involved in HCV-RNA synthesis. One example is PTB, which is found to specifically interact with viral sequences at the 3′-NTR.57–59 Another candidate is glyceraldehyde-3-phosphate de-hydrogenase which binds to the poly(U)-sequence in the 3′-NTR.60 Cellular proteins provisionally called p87 and p130 have been identified by UV cross-linking experiments with the X-tail sequence, but the nature of these proteins remains to be determined.61
Virion assembly and release
Particle formation most probably is initiated by core protein interacting with the RNA genome. Although in vitro core protein binds to RNA without detectable specificity, recent evidence indicates a preferential intracellular binding to RNA sequences in the 5′ half of the HCV genome.62 Such binding may not only accomplish a selective packaging of the plus-stranded genome but would also repress translation from the IRES, providing a potential mechanism to switch from translation/replication to assembly.62
A feature typical of the HCV envelope proteins E1 and E2 is their retention in the ER compartment when expressed in various heterologous systems in cell culture.63 This retention is achieved by signals in the transmembrane domains of E1 and E2, suggesting that viral nucleocapsids acquire their envelope by budding through ER membranes. In this process the virus would be exported via the constitutive secretory pathway. In agreement with this assumption, complex N-linked glycans were found on the surface of partially purified virus particles, suggesting virus transit through the Golgi.64
Despite the lack of convenient cell culture and animal model systems to study HCV in detail, a number of advances in the molecular virology of the virus have been made. In particular (i) definition of molecular clones that are infections in the chimpanzee animal model of HCV infection;65–67 (ii) development of a subgenomic replicon system in Huh7 cells;68 and (iii) construction of a transgenic mouse model for HCV infection.69 More dramatically, recent progress in the structural biology of the virus has led to the determination of high-resolution 3-D structures of a number of the key virally encoded enzymes, including the NS3 protease,11,70,71 NS3 helicase72–74 and NS5B RDRP.12,75 In some cases these structures have been determined as a complex with specific substrates, cofactors (NS4A) and inhibitors. Specific in vitro assays have been developed to examine viral enzymatic activities for identification testing and subsequent development of potential antiviral agents.
The important virus-specific targets for antiviral drug development include (i) processing of the viral protein by NS2-3 and NS3 (+NS4A); (ii) viral RNA replication that uses the NS3 helicase and NS5B RDRP; and (iii) viral regulatory elements, such as the 5′ UTR encoding the IRES for translation and 3′ UTR used in the generation of plus- and minus-strand syntheses. The importance of combination therapies to multiple enzyme targets has been demonstrated in the clinical setting with HIV/AIDS due to the selection of resistant virus. In the future treatment of chronic HCV infection with these types of agents, a number of antiviral drugs directed at multiple targets will be required in order to reduce or eliminate HCV quasispecies that could be selected out as drug resistant.
Strategies for inhibition of hepatitis C virus viral processing by NS2/3 and NS3/4A
As outlined earlier, the processing of HCV non-structural proteins is mediated by two viral-encoded enzymes, namely a metalloprotease and a serine protease.37,44,76 The NS2/3 protease is responsible for the cis zinc-dependent cleavage between NS2/3. The region of NS3 involved in the NS2/3 protease activity overlaps with the serine protease domain. However, the NS2/3 proteolytic activity is distinct from the serine protease activity. Mutagenesis studies have shown that residues His952 and Cys992 are essential for autocatalytic activity.37,44 A number of groups have developed assays for the characterization of this enzyme and for the development of new antiviral agents, but no specific inhibitor of this process has reached clinical phase 1 testing.
The serine protease activity of NS3 requires NS4A as a cofactor for the cleavage of NS3/4A, while NS4B/5A increases the efficiency of cleavage between NS5A/5B and NS4A/4B. Direct comparison of the NS3 serine protease crystal structure with and without NS4A has shown that NS4A is required to improve the anchoring and orientation of the catalytic triad within the serine protease.11 Zinc is also an essential structural component for this serine protease.11
A number of pharmaceutical companies have developed high-throughput assays to screen for potential HCV protease inhibitors. The X-ray crystal structure of the NS3 protease domain with and without NS4A has aided in the development and modification of inhibitors that can specifically inhibit HCV protease activity.70 The first lead compounds directed at the protease were small molecule inhibitors based on the substrate-cleavage site.77 These molecules have not progressed into clinical studies due to the rather featureless substrate-binding groove of the catalytic component of the enzyme.70 Thus, the interaction between NS3 and NS4A and the zinc-binding domain may serve as better targets for protease in the future, while it has been proposed that because the helicase function of NS3 is also closely associated with this interaction, that small RNA molecules (RNA aptamers) could inhibit both protease and helicase activity.78
Strategies for inhibition of hepatitis C virus viral RNA replication
Helicase activity of NS3 The HCV helicase is unusual because it can unwind DNA–RNA and DNA–DNA substrates as well as RNA–RNA hybrids. It uses 3′/3′ substrates but not 5′/5′ substrates because it can unwind only in a 3′ to 5′ direction.79 The crystal structure of NS3 may aid in the future design and modification of antiviral agents targeted to various sites of this unusual enzyme, which would successfully inhibit helicase activity. These targets include the NTP binding site, the binding sites for the single- and double-stranded RNA or DNA and the interaction between the various domains. In studies to investigate potential antiviral agents, Kumar et al. tested RNA molecules containing random RNA (120 random bases) for specific binding to NS3.80 The RNA aptamers inhibited both helicase and protease activity. These agents may provide potential lead compounds for molecules that inhibit this important target.
Polymerase activity of NS5B Several groups have developed HCV polymerase assays containing recombinant NS5B or NS2–5B for which elongation activity has been demonstrated.51,81 Recently Zhong et al. measured the template requirements for NS5B and found that HCV RDRP uses di- or trinucleotides efficiently to initiate intramolecular copy-back RNA replication.81 The initiation complex consisted of the polymerase, template and primer assembled at the 3′ terminus of the template RNA. The initial docking of the RNA on NS5B polymerase required a single-stranded template RNA molecule of at least 5 bp to occupy the template groove near the fingers subdomain. A unique β-hairpin loop in the thumb subdomain of NS5B may play an important role in properly positioning the single-stranded template for initiation of RNA synthesis. The original crystal structure of the HCV RDRP identified a unique catalytic domain in its enzymatic function and found that the RDRP actually fully encircled the active site very much like a ‘donut structure’.12
In addition to polymerase assays containing only the NS5B protein, RNA polymerase activity can now be analyzed in the context of the HCV replicon. Recently Moradpour et al. have developed a monoclonal antibody that specifically inhibits the RNA polymerase activity of NS5B in the context of the HCV replicon.82 This provides a promising new approach for the design of other compounds that may inhibit HCV replication. Interferon-α can also directly inhibit HCV replication in cell-based assay systems that contain the full-length HCV genome,83 and a number of pharmaceutical companies are developing small molecular inhibitors of the HCV NS5B enzyme that are now beginning to enter into phase 1 clinical evaluation.
Strategies for the inhibition of cis-acting RNA sequences
The IRES encoded by the 5′UTR and the conserved 98-bp RNA structure at the 3′ UTR of the positive sense RNA provide ideal targets that will block the translation and the transcription of the HCV genome. Antisense oligonucleotides directed to these regions of the HCV genome have been effective in preventing HCV replication.84–86 Antisense oligonucleotides consist of DNA or RNA sequences specifically designed to bind an RNA target in order to form RNA–RNA (antisense RNA) or RNA–DNA (antisense DNA) hybrids. The formation of such hybrids can inhibit translation of HCV–RNA and/or RNA replication. The inhibitory effects of antisense oligonucleotides may be mediated, in part, by the intracellular degradation of RNA in RNA–DNA hybrids; this degradation is mediated by RNase H. Cellular deaminases may also be involved in mediating the effects of antisense oligonucleotides; these intracellular catalyze the modification of RNA–RNA hybrids. Phosphorothioate, methylphosphonate, and phosphotriester analogs of antisense oligonucleotides have been effective in increasing the resistance of these antisense oligonucleotides to nuclease attack and also in improving cellular uptake, intracellular stability and subsequent hybridization.
Ribozymes have been developed to inhibit HCV replication by cleaving the target HCV genomic RNA.87,88 Ribozymes are naturally occurring short RNA molecules with endoribonuclease activity that are capable of catalyzing sequence-specific cleavage of RNA. The specificity of such catalytic RNA is determined by flanking sequences that are complementary to the target RNA. Cleavage occurs 3′ to the nucleotide triplet ‘NUX’, where N is any base and X any base except G. Hammerhead and hairpin ribozymes are two families of ribozymes that have in vitro antiviral activity against a variety of HCV-RNA targets. A number of groups have developed nuclease-resistant ribozymes for the treatment of HCV infection; these ribozymes are administered intravenously or subcutaneously and they have favorable pharmacokinetics and tissue distribution in mice.88 The HCV ribozyme described in this report was found to be retained in liver cells, providing strong support for their potential use as therapeutic agents for the treatment of chronic hepatitis C.88 The DNA analogs of ribozymes have been developed; they are also effective inhibitors against HCV.89
Until recently, structure-based drug design has been limited largely to protein targets because of the relative lack of RNA structural information. The availability of high-resolution 3-D RNA structures has undergone a dramatic increase over recent years (Ramos et al. 1997;90 Batey et al. 199991). An analysis of these coordinates has revealed that several structural motifs are repeated in different contexts throughout several structure databases.92 For example, tetra-loops, U-turns, dinucleotide platforms and the alpha-sarcin/ricin loop (SRL) motif. This recently available repertoire of RNA motifs has provided considerable insight into the 3-D structure of RNAs.92 Klinck et al. have used motif prediction in conjunction with experimental evidence, as a tool for structure prediction as applied to viral RNA for potential drug target development.92 These workers identified a 27-n.t. fragment in the 5′-UTR IRES of HCV, located in the subdomain IIId which included a SRL motif in its internal loop. Thus, it can now be possible to use this substrate for the purposes of novel drug design.92
Antiviral drug resistance
To date, no example of classical antiviral drug resistance has been documented. The two agents approved for use in treating patients with chronic HCV infections, IFN-α and ribavirin, appear to have more of an immune modulating role rather than a direct antiviral effect on HCV infection and replication. As outlined earlier, a number of small molecule inhibitors of HCV replication are progressing into the clinical evaluation phase and, as with other similar agents that are active against specific viruses, classical drug resistance will almost certainly emerge.93
In the case of IFN-α, viruses have evolved a number of mechanisms to circumvent its antiviral effect. Interferon-α is produced by most cells in response to viral infection. The binding of IFN-α to its receptor mediates a signal cascade that results in the transcriptional induction of IFN-induced genes.94 Although most of these genes are of unknown function, several have demonstrable antiviral activity. For instance, 2′, 5′ oligoadenylate synthetase (2,5 OAS), upon activation by double-stranded RNA, synthesizes 2′-5′ polyadenylate chains, which in turn, activate RNase L. This latest RNase degrades both viral and cellular RNA not associated with polyribosomes. MxA is a guanosine triphosphatase and inhibits the replication of RNA viruses. Major histocompatibility complex class II antigens are also induced by IFN-α and mediate presentation of viral antigen to the immune system. The best-studied IFN-α-induced antiviral gene is the double-stranded RNA-activated protein kinase, PKR. It has many cellular roles including proapoptotic functions, growth control, and differentiation activity and, of course, inhibition of translation in response to viral infection.
All of these IFN-α-induced genes have been examined for their roles in HCV resistance to IFN-α, most of them at the level of protein expression.95 The proteins that are expressed during the IFN response are enzymes that require further activation, such as 2,5 OAS or PKR. Therefore, enzyme activity, and not protein expression, is the key to dissecting the effects of IFN-α on viruses or the effects of viral genes on the IFN-α response.96
Two HCV proteins, NS5A and E2, have been implicated in the ability of the virus to regulate the host response to double-stranded RNA. Both proteins have been shown to bind and inhibit PKR.14,95,96 However, several molecular epidemiological studies have correlated the sequence of the PKR-interacting region of E2 or NS5A with HCV persistence also, and the widespread ‘resistance’ to the current IFN-based therapy for HCV infection.97,98 These studies have suggested that E2 and NS5A may contribute to viral persistence by inhibiting the intracellular antiviral response that is triggered by double-stranded RNA and signaled by PKR.96,99 Enomoto et al. originally identified the ISDR of NS5A and showed that ≥4 amino acid mutations between codons 2209 and 2248 of the carboxyterminal part of the NS5A protein (mutant type), as compared to the HCV-1b prototype (wild-type), could be correlated with sustained virological responses to IFN-α.100 Taylor et al. demonstrated that the HCV E2 protein contains a 12-amino-acid sequence that is similar to the PKR autophosphorylation site and the translation initiation factor eIF2a phosphorylation site, the latter of which is a target of PKR.14 This has been termed the PKR-e-IF2a phosphorylation homology domain (PePHD). These investigators showed that PePHD of genotype 1 isolates but not type 2 and 3 isolates, inhibited the kinase activity of PKR and thus abolished its inhibitory effect on protein synthesis and cell growth.14 Taken together, HCV can thus interfere with the IFN-α-induced signal transduction pathways, thereby circumventing its antiviral effect.
As more data are collected, there is growing evidence that HCV has evolved a number of mechanisms to alter the host intracellular environment sufficiently to favor persistent virus replication. The challenge for future drug development will be to counter these viral replicative processes and design therapeutic strategies that will completely inhibit HCV replication.
The author would like to thank Angeline Bartholomeusz for her assistance with this manuscript.