Abstract

With the remarkable ability of hepatitis C virus (HCV) to establish persistent infections that can lead to progressive liver pathology and the poor response of prevalent HCV genotypes to the current treatment, HCV represents a significant global health problem. Studies of HCV replication in cell culture were virtually impossible until the development of subgenomic replicons that replicate autonomously in the human hepatoma cell line Huh-7. Many improvements to the replicon system have been made allowing the establishment of transient replication assays for HCV genotypes 1a, 1b, and 2a. Specifically, the identification of adaptive mutations that drastically enhance HCV genotype 1 replication and the isolation of highly permissive Huh-7 sublines led to the development of replication-competent full-length genomes in addition to a collection of robustly replicating subgenomes derived from genotype 1 sequences. More recently, the cell tropism of HCV subgenomic replicons has been expanded to non-hepatoma cell lines and mouse hepatocytes. The HCV replicon system has opened new avenues for detailed molecular studies of RNA replication and HCV-host interactions as well as the development of active inhibitors of HCV replication. Finally, the identification of genotype 2a-derived replicons that efficiently replicate in cell culture without adaptive mutations has facilitated the development of systems supporting the complete virus life cycle.

Introduction

Persistent infection with HCV has emerged as one of the primary causes of chronic liver disease, with an estimated 170 million carriers throughout the world (WHO, 2000). Viral persistence develops in ~80% of infected individuals and although the acute phase of infection is frequently asymptomatic or associated with mild and non-specific symptoms, these patients are at risk for developing chronic liver disease (Alter and Seeff, 2000). Approximately 20% of chronic carriers will develop cirrhosis, and some of these cases will progress to hepatocellular carcinoma. Consequently, HCV-induced chronic liver disease is now recognized as the leading indication for orthotopic liver transplantation in the United States (Fishman et al., 1996).

Hepatitis C viruses have a high level of genetic heterogeneity and thus have been grouped by their degree of sequence identity into six separate genotypes and further divided into numerous subtypes (Simmonds et al., 1993; Robertson et al., 1998). Geographic distribution and responses to current therapy differ between genotypes. Genotypes 1a and 1b are the most prevalent in the United States and Western Europe, followed by infections with genotype 2 and 3 strains. The only licensed therapy for chronic hepatitis C infection is polyethylene glycol (PEG)-conjugated interferon (IFN)-α given in combination with the guanosine analog ribavirin; while ~90% of patients persistently infected with HCV genotypes 2 and 3 clear the virus, only 50% of patients infected with HCV genotypes 1, 4, 5, and 6 mount a sustained response (Poynard et al., 2003). Clearly, there is a need for the development of more effective therapeutic strategies to improve the clinical treatment of HCV-associated hepatitis.

HCV has been classified as the sole member of the genus Hepacivirus within the Flaviviridae family, which also includes the classical flaviviruses, such as West Nile and yellow fever viruses, and the animal pestiviruses, such as bovine viral diarrhea virus (BVDV). Like these related viruses, HCV is enveloped with a positive-sense, single-stranded RNA genome. The HCV genome is ~9.6 kb in length and consists of a 5′ non-translated region (NTR) and a long open reading frame (ORF) encoding all the virus-specific proteins followed by a 3′ NTR, comprised of a short variable sequence, a poly(U)/polypyrimidine [poly(U/UC)] tract, and a highly conserved terminal sequence (Fig. 1A). Translation of the genomic RNA is mediated by an internal ribosome entry site (IRES) located within the 5′ NTR (reviewed in Rijnbrand and Lemon, 2000). The resulting polyprotein precursor of about 3000 amino acids is co- and post-translationally cleaved into at least 10 different products by a combination of host cell signal peptidases and two viral proteases. At the N-terminus are the structural proteins C (capsid), E1, and E2 (envelope glycoproteins) followed by the short hydrophobic peptide, p7. The C-terminal two-thirds of the polyprotein comprise the non-structural proteins (NS) 2, 3, 4A, 4B, 5A, and 5B (Fig. 1A). In addition, the frameshift (F) or alternative reading frame protein (ARFP), encoded in an overlapping reading frame within the N-terminus of the HCV polyprotein, is synthesized by ribosomal frameshift (Walewski et al., 2001; Xu et al., 2001; Varaklioti et al., 2002).

Fig. 1

Structure of the HCV genome and Con1-derived bicistronic replicon, location of adaptive mutations in the HCV polyprotein of subgenomic replicons, and the positions of these mutated residues in the crystal structures of the NS3 protease, the NS3 helicase, (more...)

As discussed later, the non-structural proteins NS3-5B are sufficient for subgenomic replicon replication in cell culture (Lohmann et al., 1999). These proteins are presumed to function as structural and enzymatic components of the HCV replication complex or replicase, and the enzymatic properties of the non-structural proteins are fairly well defined (Table 1 and reviewed in Reed and Rice, 1999; Blight et al., 2002a). The N-terminus of NS3 is a serine protease that forms a stable complex with its cofactor NS4A to mediate cleavage of the HCV polyprotein at the NS3/4A, 4A/4B, 4B/5A, and 5A/5B junctions. The C-terminal two-thirds of NS3 harbors an RNA nucleoside triphosphatase (NTPase)/helicase activity capable of unwinding nucleic acid duplexes. How the NS3 helicase/NTPase contributes to the RNA replication process is currently unknown, although it is thought to unwind regions of extensive secondary structure in the template or double-stranded RNAs resulting from synthesis of the complementary negative-sense RNA intermediate (see below). NS5B, the C-terminal cleavage product of the polyprotein, is the RNA-dependent RNA polymerase (RdRp). The roles of NS4B and NS5A in RNA replication, however, are less clear. NS4B is an integral membrane protein that induces a distinct membrane alteration, designated the membranous web (Egger et al., 2002). NS5A is phosphorylated predominantly on serine residues by one or more unidentified cellular kinases producing two NS5A phosphoprotein variants; basal- (p56) and hyper- (p58) phosphorylated forms of NS5A (Kaneko et al., 1994; Tanji et al., 1995; Reed et al., 1997). Surprisingly, NS2 was not required for subgenomic replication in cell culture (Lohmann et al., 1999) and thus the role of NS2 in the viral life cycle is not completely clear, although it has been shown to form a distinct autoprotease with the serine protease domain of NS3 (NS2-3; Table 1) responsible for cleavage at its own C-terminus (Grakoui et al., 1993; Hijikata et al., 1993).

Table 1

Summary of the enzymatic functions of the HCV non-structural proteins.

The mechanisms of viral attachment and cellular entry and the precise intracellular steps in HCV RNA replication, virus assembly, and virion release are largely unknown due to the previous lack of suitable cell culture systems for HCV. Some details have begun to emerge since the development of subgenomic replicons that recapitulate the intracellular steps of RNA replication (Fig. 4). Briefly, input positive-sense HCV RNA is translated and the resultant polyprotein is processed into the individual HCV proteins. The non-structural proteins assemble in association with intracellular membranes into the replication complex that transcribes the input RNA molecule to generate a complementary negative-sense RNA intermediate that presumably remains base-paired with its template. This double-stranded replicative form is transcribed asymmetrically, leading to the preferential accumulation of positive-sense RNAs that are then available for further translation or synthesis of the negative-sense RNA intermediate.

Fig. 4

The intracellular steps involved in HCV RNA replication and the various methods used to monitor these steps in stable antibiotic-resistant cell clones or in transient replication assays. After transfection of Huh-7 cells with in vitro transcribed RNAs (more...)

Since the molecular cloning of the HCV genome 16 years ago, there has been a great deal of progress in defining HCV genome structure and protein function. However, the lack of a reliable and robust cell culture system has presented a major obstacle to studies on the viral life cycle and for developing effective antiviral drugs. These hurdles have been overcome by the development of subgenomic and genomic replicons for HCV. In this chapter, we will describe the development of the HCV replicon system along with the most recent advances and applications of this system.

Development of HCV Replicons

Numerous attempts have been made to propagate HCV in cell culture by infection with virus-containing inoculum. Replication has been detected in hepatoma, B- and T-cell lines, primary cultures of human or chimpanzee hepatocytes, and peripheral blood mononuclear cells; however, replication levels were frequently transient and always so low that HCV RNA synthesis could only be monitored by highly sensitive reverse transcription (RT)-PCR assays and thus were not amenable to detailed studies of HCV replication (reviewed in Bartenschlager and Lohmann, 2001). For many positive-sense RNA viruses, including the closely related flaviand pestiviruses, productive replication can be efficiently launched by transfecting permissive cells with genomic RNAs transcribed in vitro from cloned virus genomes. With this approach, the viral entry and uncoating steps are bypassed; however, transfection of HCV RNAs transcribed from cDNA clones with proven infectivity never reproducibly established HCV replication in the many cell lines tested (reviewed in Bartenschlager and Lohmann, 2001). This was most likely due to the generally low levels of replicated RNA and was further complicated by the difficulty of distinguishing input RNA from plus strands produced by RNA replication (Fig. 4).

Although HCV is notoriously difficult to grow in cell culture, HCV subgenomic replicons that efficiently replicate in a human hepatoma cell line, Huh-7, have been developed (Lohmann et al., 1999). The development of this system was inspired by the observation that the structural proteins were not required for replication of several positive-sense RNA viruses including flavi- and pestiviruses, and by the successful design of self-replicating replicons for the flavivirus Kunjin (Khromykh and Westaway, 1997) and the pestivirus BVDV (Behrens et al., 1998). The first generation functional HCV replicons were derived from the consensus Con1 cDNA that was isolated from the liver of a patient chronically infected with a genotype 1b strain and comprised: (i) the HCV 5′ NTR and the first 12 codons of the capsid protein fused in-frame with the selectable marker gene, neomycin phosphotransferase (Neo), which upon expression confers resistance to the cytotoxic drug G418; (ii) the IRES element from encephalomyocarditis virus (EMCV), which directs translation of the HCV non-structural proteins; and (iii) the HCV 3′ NTR. Fig. 1A depicts the structure of the bicistronic replicon encoding the HCV polyprotein NS3-5B. Transfection of Huh-7 cells with transcripts synthesized in vitro and selection with G418 resulted in a low number of surviving cell colonies. Independent G418-resistant cell colonies harbored replicons that replicated RNA to high levels (1,000–5,000 copies of positive-sense HCV RNA per cell), while the negative-sense replicative intermediate was 5–10-fold lower, consistent with asymmetric RNA synthesis. Autonomous HCV replication was verified by the efficient labeling of HCV RNA with [³H] uridine in the presence of actinomycin D, an inhibitor of DNA- but not RNA- dependent RNA polymerases (Lohmann et al., 1999). HCV non-structural proteins in G418- selected cell clones localized exclusively to the cytoplasm in close association with membranes of the endoplasmic reticulum (ER) (Pietschmann et al., 2001; Mottola et al., 2002), a predicted site of HCV RNA replication (discussed below).

Identification of Adaptive Mutations

Despite the high levels of subgenomic RNA replication within a selected cell clone, G418 resistance arose in a very low frequency of transfected cells (~1 colony per 10⁶ transfected cells; Lohmann et al., 1999; Blight et al., 2000). This was due to two restrictions: first, replicon RNAs had to acquire adaptive mutations to efficiently replicate in the Huh-7 cell line; and second, only a low number of cells in the culture support efficient HCV replication (discussed in detail below). Sequence analysis of Con1-derived HCV RNAs replicating in cell clones after G418 selection identified in most cases at least one mutation in the non-structural coding region, but not in the highly conserved 5′ or 3′ NTRs (Blight et al., 2000; Krieger et al., 2001; Lohmann et al., 2001; Guo et al., 2001; Lohmann et al., 2003; Lanford et al., 2003). The impact of individual mutations on HCV RNA replication was tested by engineering mutations into the parental replicon and determining the number of G418-resistant colonies after transfection of a defined amount of in vitro transcribed RNA, or by transient replication assays. As discussed below, most mutations were found to enhance RNA replication in Huh-7 cells; however, the degree of adaptation was highly dependent on the particular substitution.

Highly adaptive mutations lie within the NS4B, NS5A, and NS5B coding regions, with the majority clustering in NS5A, just upstream of the sequence termed the IFN sensitivity-determining region (ISDR; Fig. 1B), a region that has been implicated in the effectiveness of IFN treatment (Enomoto et al., 1995; Enomoto et al., 1996). Highly adaptive amino acid substitutions have been identified at nine positions in Con1 NS5A (Fig. 1B; Blight et al., 2000; Krieger et al., 2001; Guo et al., 2001; Lohmann et al., 2003; Lanford et al., 2003). The most efficient adapted replicon contains a single serine to isoleucine substitution at position 2204 in NS5A (S2204I; Fig. 1B) and establishes replication in ~10% of transfected Huh-7 cells (Blight et al., 2000). The >10,000-fold improvement in colony-forming efficiency compared to the parental Con1 replicon was sufficient for the detection of HCV RNA and proteins shortly after RNA transfection. By 96 hours, HCV RNA levels were almost 500-fold higher than a replicon carrying a lethal mutation in the NS5B RdRp (Blight et al., 2000). In addition to amino acid substitutions, an in-frame deletion of 47 amino acids encompassing the putative ISDR (Δ2207–2254; Fig. 1B) (Blight et al., 2000) and a deletion of the serine residue at position 2201 (ΔS2201; Fig. 1B) (Guo et al., 2001) also enhance replicon replication in Huh-7 cells.

Based on the predicted topology of the integral membrane protein NS4B, amino acid substitutions reside in distinct cytoplasmic domains of this protein (Guo et al., 2001; Lohmann et al., 2003). Mutations at these sites have a strong impact on Con1 replication with a K1846T substitution enhancing replication to a greater extent than V1897A (Lohmann et al., 2003). In contrast to the highly adaptive mutations in NS4B and NS5A, amino acid substitutions within NS5B are only moderately enhancing. For instance, a single amino acid substitution at position 2884 (R2884G; Fig. 1B and E) increases G418-colony formation by ~500-fold compared to the parental sequence (Lohmann et al., 2001).

Another cluster of adaptive mutations (Fig. 1B) mapped to the solvent-accessible surface of the NS3 crystal structure and at a distance from the active sites of the protease and helicase (Fig. 1C and D). Interestingly, the mutations lying within NS3 were always found in conjunction with highly adaptive substitutions (Blight et al., 2000; Krieger et al., 2001; Lohmann et al., 2001; Guo et al., 2001; Lohmann et al., 2003; Lanford et al., 2003). By themselves, these NS3 mutations have minimal or no impact on replicon replication (Krieger et al., 2001; Lohmann et al., 2001; Lohmann et al., 2003; Lanford et al., 2003), but can enhance replication synergistically when combined with each other or with highly adaptive mutations in NS4B, NS5A, or NS5B (Krieger et al., 2001; Lohmann et al., 2001; Lohmann et al., 2003; Lanford et al., 2003). To illustrate this point, two amino acid substitutions in NS3 (E1202G and T1280I) together with either K1846T in NS4B (Lohmann et al., 2003) or S2197P in NS5A (Krieger et al., 2001) enhance Con1 replication to levels above those observed for the replicons harboring the single NS4B or NS5A adaptive mutations. In contrast, combinations of highly adaptive mutations in NS4B, NS5A, and NS5B are antagonistic, albeit to different extents. Combining highly adaptive mutations in NS5A with each other (Blight et al., 2002b; Lohmann et al., 2003) or with the NS5B R2884G substitution (Lohmann et al., 2003) severely impair or completely abolish replication. Thus, it appears that the mechanism(s) of cell culture adaptation achieved by mutations in NS3 is different from the one exerted by substitutions in NS4B, NS5A, and NS5B.

At this stage, we can only speculate about the mechanism(s) for adaptive mutation-enhanced replication and the synergy between mutations in NS3 and those in NS4B, NS5A, or NS5B. Most mutations conferring cell culture adaptation have not been found in natural isolates of HCV and almost invariably target amino acid residues that are conserved between different HCV genotypes, suggesting that mutations represent a specific adaptation to the Huh-7 cell environment. Given that adaptive mutations reside on the surface of the available crystal structures for NS3 and NS5B (Fig. 1C–E) and do not affect the active sites of these enzymes, it is assumed that mutations modulate interactions among viral proteins and/or between viral and cellular components of the HCV replication complex. There is accumulating evidence that the suppression of NS5A hyperphosphorylation may represent a mechanism of replicon adaptation. In support of this, Con1 subgenomic RNA no longer requires adaptive mutations to efficiently replicate when Huh-7 cells are treated with inhibitors that block NS5A hyperphosphorylation (Neddermann et al., 2004). Additionally, site-directed mutagenesis of the serine residues involved in NS5A hyperphosphorylation led to a decrease in p58 formation with a corresponding increase in HCV replication (Appel et al., 2005b). Similarly, highly adaptive mutations targeting serine residues in NS5A either ablate (eg. S2204I; Blight et al., 2000) or impair NS5A hyperphosphorylation (eg. S2197P/C; Blight et al., 2000). In addition, the replication-enhancing mutations in NS4B also reduce the level of NS5A hyperphosphorylation (Evans et al., 2004b; Appel et al., 2005b). Thus, impaired NS5A hyperphosphorylation is a critical requirement for efficient Con1 RNA replication in Huh-7 cells. Perhaps hyperphosphorylation of NS5A performs a regulatory role in the HCV life cycle and adaptive mutations that suppress NS5A hyperphosphorylation prevent the dissociation of the replication complex, thereby allowing the establishment of ongoing efficient replication (Evans et al., 2004b; Appel et al., 2005b). Alternatively, it has been shown that antiviral pressures of the host cell triggered by HCV RNA replication contribute to the acquisition of adaptive mutations. Specifically, when a Huh-7 cell clone supporting replication of an IFN-sensitive Con1 subgenomic RNA was maintained long term in culture, mutations accumulated throughout the HCV-coding region. The fitness of this replicon variant was significantly enhanced and was resistant to the host defenses triggered by productive replication and by IFN-α treatment (Sumpter Jr. et al., 2004).

As observed for many other viruses, especially those with high mutation rates, passages in cell culture for prolonged periods of time can result in the accumulation of mutations that often improve virus replication in vitro but frequently lead to attenuation in vivo. Similarly, cell culture-adaptive mutations that facilitate efficient HCV replication in Huh-7 cells give rise to highly attenuated phenotypes in vivo. Intrahepatic inoculation of chimpanzees, the only recognized animal model for HCV infection, with full-length Con1 genomes harboring three cell culture-adaptive mutations (E1202G and T1280I in NS3 and S2197P in NS5A; Fig. 1B) failed to develop a productive infection (Bukh et al., 2002). A Con1 genome with the single S2197P substitution in NS5A replicated poorly, and one week after inoculation circulating HCV genomes were detectable but had reverted to the original wild-type Con1 sequence (Bukh et al., 2002). Thus, the attenuation in vivo of Con1 genomes carrying cell culture-acquired mutations may explain why Huh-7 cells supporting full-length Con1 replication do not produce infectious virus particles (Pietschmann et al., 2002; Blight et al., 2002b and discussed below).

Generation of Replicons from Other HCV Isolates

Slight variations in HCV sequence can dramatically alter the replicative ability of engineered replicons and as a result creating functional subgenomes for different HCV isolates has not been straightforward. Of the six HCV genotypes, viable replicons have only been reported for genotype 1 and 2 strains. Six genotype 1b isolates - Con1 (discussed above), HCV-N (Guo et al., 2001; Ikeda et al., 2002), HCV-BK (Grobler et al., 2003), HC-J4 (Maekawa et al., 2004), 1B-2/HCV-O (Kato et al., 2003a), and 1B-1/M1LE (Kishine et al., 2002) - productively replicate in Huh-7 cells. Replication-competent genotype 1a replicons are derived from the Hutchinson strain (H77 or HCV-H; Blight et al., 2003; Gu et al., 2003; Grobler et al., 2003; Yi and Lemon, 2004; Liang et al., 2005) and efficient replication of JFH-1, classified as a genotype 2a virus, has been also demonstrated in cell culture (Kato et al., 2003b).

Genotype 1a

The identification of efficiently replicating replicons corresponding to genotype 1a strains has proven even more challenging than generating functional genotype 1b subgenomic RNAs. Attempts to construct a replication-competent replicon from the HCV-1 infectious clone have been unsuccessful, despite the inclusion of adaptive mutations identified in the genotype 1b Con1 replicon (Lanford et al., 2003). Similar negative results were obtained for the H77 strain until highly permissive cell lines were isolated (Blight et al., 2003; Grobler et al., 2003) or H77-Con1 chimeric replicons were constructed (Gu et al., 2003; Yi and Lemon, 2004). Although intrahepatic inoculation of H77 RNA is associated with high viremia during the acute phase of infection in the chimpanzee (Kolykhalov et al., 1997; Yanagi et al., 1997), replicons derived from this infectious H77 molecular clone require at least two adaptive mutations to productively replicate in cell culture. Interestingly, the single adaptive mutation S2204I in NS5A that was identified in the Con1 replicon was a crucial prerequisite for obtaining G418-resistant colonies supporting H77 replication (Blight et al., 2003; Gu et al., 2003; Grobler et al., 2003; Yi and Lemon, 2004), indicating that at least one of the Con1 adaptive mutations is also effective in a genotype 1a sequence.

Transfection of a highly permissive Huh-7 subline, Huh-7.5 (Blight et al., 2002b and see below), with S2204I-containing H77 replicons allowed the establishment of the first G418-resistant colonies supporting H77 replication (Blight et al., 2003). Analysis of H77 RNAs replicating in these G418-selected cell clones identified a second amino acid substitution in the helicase domain of NS3 (A1226D or P1496L; Fig. 2A and C). Both of these NS3 mutations, when combined individually with S2204I in NS5A, increased the replicative capacities of subgenomic H77 RNA with the greatest enhancement seen with the P1496L substitution. Similarly, Grobler et al. (Grobler et al., 2003) independently found that P1496L (or S1222T) together with S2204I is sufficient for productive replication of H77 RNA in a hyper-permissive Huh-7 subline, MR2. Like the synergistic NS3 mutations identified in the Con1 strain, H77 residues S1222, A1226, and P1496 map to the surface of the NS3 helicase and are not located in the nucleotide, metal, or nucleic acid binding sites (Fig. 2C). Additionally, replacement of proline with leucine at position 1496 has no effect on the in vitro unwinding activity of purified NS3 helicase (Grobler et al., 2003), suggesting that these mutations are involved in mediating interactions with viral or cellular factors rather than increasing the enzymatic capacity.

Fig. 2

Location of adaptive mutations that enhance H77 replicon replication in Huh-7 cells. (A) The NS3 to NS5B polyprotein with the positions of individual mutations proven to facilitate efficient replication in combination with S2204I in NS5A (bold) shown (more...)

Alternatively, the requirements for productive H77 replication in the parental Huh-7 cell line were defined through the construction of chimeric replicons between Con1 and H77 sequences harboring S2204I in NS5A (Gu et al., 2003; Yi and Lemon, 2004). One study (Yi and Lemon, 2004) identified adaptive mutations within NS3, NS4A, and NS5A that act cooperatively to enhance H77 replication. Maximal non-chimeric H77 replication was achieved with a combination of mutations Q1067R and V1655I in NS3, K1691R in NS4A, and K2040R and S2204I in NS5A (Fig. 2A). In contrast to the NS3 mutations found in the helicase domain (Fig. 2C; Blight et al., 2003; Grobler et al., 2003), the substitutions identified in NS3 by Yi and Lemon (Yi and Lemon, 2004; Fig. 2A) are located in close proximity to the protease active site in the NS3/4A crystal structure (Q1067 and G1188; Fig. 2B) or in the P3 position of the NS3/4A cleavage site potentially influencing substrate recognition during cis-cleavage at this junction (V1655; Fig. 2A). Furthermore, K1691 in NS4A is located immediately downstream of the sequence involved in complex formation with NS3. Thus, these mutations (Yi and Lemon, 2004) may facilitate HCV replication via a mechanism different from those amino acid substitutions previously identified in the helicase domain of NS3 (Blight et al., 2003; Grobler et al., 2003). Similarly, another laboratory (Gu et al., 2003) achieved efficient replication of a replicon that was predominantly H77 derived, except the 5′ NTR and N-terminal 75 amino acids of NS3 were from the Con1 genotype 1b strain. In the single G418-resistant cell clone analyzed, four amino acid changes were identified across NS3, NS5A, and NS5B; however, it is unclear which mutations or combination of mutations was responsible for augmenting chimeric or non-hybrid replication (Gu et al., 2003).

In complete contrast, an H77-derived replicon encoding the puromycin N-acetyltransferase (PAC) gene instead of the Neo cassette (Fig. 3A) does not require adaptive mutations to autonomously replicate and confer resistance to puromycin in Huh-7 cells, although S2204I in NS5A did evolve in a minority of cell clones (Liang et al., 2005). Additionally, unlike the studies discussed above (Blight et al., 2003; Gu et al., 2003; Grobler et al., 2003; Yi and Lemon, 2004), inclusion of S2204I in NS5A did not significantly improve the colony-forming efficiency of PAC-expressing replicons (~3-fold improvement; Liang et al., 2005). The ability of PAC-expressing H77 replicons to establish replication in Huh-7 cells without adaptive mutations is puzzling and may reflect the different selective pressure. Perhaps cellular environments that are conducive to non-adapted H77 replication are more efficiently selected by puromycin than G418. In support of this, transfection of total cellular RNA extracted from a replicon-containing cell clone led to a 50- fold increase in puromycin-resistant colonies, suggesting that translation of cellular mRNAs initially introduced with replicon RNA created an intracellular milieu in naïve Huh-7 cells favorable for the establishment of HCV replication.

Fig. 3

Organization of replication-competent HCV replicons reported so far. (A) Antibiotic selectable replicons. (Top) Biscistonic HCV replicons are composed of the 5′ NTR, a small portion of the capsid-coding region (open box), an antibiotic resistance (more...)

Development of Alternative Replicon Derivatives

The identification of adaptive mutations that dramatically enhance genotype 1 HCV replication in cell culture has made it possible to explore alternative drug resistance genes (Fig. 3A; Frese et al., 2002; Evans et al., 2004a; Liang et al., 2005; Appel et al., 2005a) and develop transient replication assays utilizing replicon derivatives expressing or activating the expression of easily quantifiable reporter enzymes (Fig. 3B; Krieger et al., 2001; Yi et al., 2002; Lohmann et al., 2003; Murray et al., 2003; Ikeda et al., 2005). Furthermore, robustly replicating monocistronic replicons, containing one translation module and eliminating non-HCV sequences (Fig. 3C; Blight et al., 2003), have also been generated, as well as replication-competent full-length RNAs that stably or transiently replicate in Huh-7 cells (Fig. 3D; Ikeda et al., 2002; Pietschmann et al., 2002; Blight et al., 2002b; Blight et al., 2003; Yi and Lemon, 2004; Ikeda et al., 2005).

Cell Lines Permissive for HCV Replication

In vivo, hepatocytes are believed to be the major site of HCV replication; however, some evidence suggests that extrahepatic cells, including lymphocytes, monocytes, and dendritic cells, may also harbor the virus (Blight and Gowans, 1995; Laskus et al., 2000; Goutagny et al., 2003). Productive replication of HCV replicons in vitro appears to be extremely cell-type specific, with human hepatoma Huh-7 cells being the most permissive cell line identified so far, although, as described below, the environment within these cells affects the efficiency of HCV replication. Extensive effort has been devoted to identifying other permissive cell lines and recently, the cell repertoire was expanded to include additional continuous human hepatoma cell lines and a murine hepatoma cell line, as well as non-hepatic cell lines such as the human cervical cancer-derived HeLa cell line and 293 cells established from human embryonic kidney.

Applications of the HCV Replicon System

Since the introduction of HCV replicons in 1999 and the subsequent identification of adaptive mutations a year later, numerous researchers have utilized the replicon system to probe the mechanisms of HCV replication, define the roles of individual proteins, identify the viral and cellular determinants of HCV replication, and examine the interplay between HCV and the Huh-7 cell. This section provides specific examples to highlight the utility of this cell culture model to address these fundamental questions about HCV biology.

The availability of stable cell lines that harbor autonomously replicating subgenomic RNAs has facilitated the study of viral protein expression, subcellular localization of HCV replication, and the structure, function, and biochemical properties of the replication complex. Additionally, the mechanisms by which HCV counteracts the host antiviral response, the effects of HCV replication on host cell function and the importance of host factors for efficient replication have begun to be unraveled. The HCV polyprotein is proteolytically processed in a preferential order with rapid cleavages at the NS3/4A and NS5A/5B sites, while the NS4A-4B-5A precursor is processed at a slower rate (Pietschmann et al., 2001), confirming previous studies using heterologous expression systems (Lin et al., 1994; Bartenschlager et al., 1994; Tanji et al., 1994). The mature HCV proteins have half-lives ranging from 10 to 16 hours, except the hyperphosphorylated form of NS5A, which appears less stable (Pietschmann et al., 2001). Similar to all positive-sense RNA viruses investigated so far (reviewed in Ahlquist et al., 2003; Salonen et al., 2005), HCV reorganizes intracellular membranes to form a membranous web. This altered membrane represents a site of HCV replication in Huh-7 cells (Gosert et al., 2003), and by utilizing the replicon encoding the NS5A-GFP fusion (Fig. 3B), active HCV replication complexes have been directly visualized in living cells by fluorescence microscopy (Moradpour et al., 2004b). Interestingly, fluorescence is strongest in subconfluent cells, consistent with earlier studies showing that subgenomic RNA replication is strongly influenced by the proliferation status of the cells with replication stimulated in the S phase of the cell cycle, but rapidly declining in confluent or serum-starved cells (Pietschmann et al., 2001; Scholle et al., 2004). The ability to track functional replication complexes should aid in defining the steps involved in membranous web formation and in the assembly and turnover of replication complexes. Although the origin of the membranous web has not been defined, its close proximity to the ER and the ER localization of the non-structural proteins in replicon-containing cells (Pietschmann et al., 2001; Mottola et al., 2002; El-Hage and Luo, 2003; Miyanari et al., 2003) suggest the web is derived from membranes of the ER. In contrast, the association of HCV RNA and non-structural proteins with NP40-insoluble membranes in replicon-containing cells and their cofractionation with caveolin-2 suggest that active replication complexes reside on lipid rafts recruited from intracellular sites (Shi et al., 2003; Gao et al., 2004; Aizaki et al., 2004). Clearly more experimentation is required to define the origin of the membranes on which the HCV replication complex assembles.

HCV subgenomic replicon-containing Huh-7 cells also provide a source of membrane fractions containing crude replication complexes for biochemical studies (Ali et al., 2002; Hardy et al., 2003; Lai et al., 2003). These complexes retain enzymatic activity as evidenced by the synthesis of replicon-length RNA from the endogenous (co-fractionating) template RNA. Furthermore, partially single-stranded and double-stranded replicative forms are synthesized, transcription of single- and double-stranded RNAs are differentially effected by Mg²⁺ and Mn²⁺, RNA synthesis is actinomycin D-resistant, and de novo initiation of RNA transcription occurs in these isolated membrane fractions. Thus, the use of enzymatically active HCV replication complexes rather than employing NS5B alone offers an in vitro system to probe the structure and function of the replicase and to evaluate potential inhibitors targeting RNA replication. Interestingly, these crude replication complexes have failed to utilize exogenously added template RNA (Ali et al., 2002; Hardy et al., 2003; Lai et al., 2003) and most of the viral RNA in these membrane-bound complexes is nuclease resistant (El-Hage and Luo, 2003; Miyanari et al., 2003; Yang et al., 2004), consistent with the notion that HCV replication complexes are relatively closed structures. Furthermore, treatment of these complexes with proteinase K degrades more than 90% of the viral proteins with no effect on RNA transcription (Miyanari et al., 2003), thus only a minor fraction of HCV proteins are engaged in RNA synthesis and this fraction is protected within the replication complex.

Like many viral infections, HCV triggers the host cell antiviral response in part through the accumulation of replication intermediates or the presence of double-stranded RNA structures within the HCV genome (Pflugheber et al., 2002; Wang et al., 2003; Sumpter Jr. et al., 2005). Persistent HCV infections frequently develop (Alter and Seeff, 2000), suggesting that HCV has evolved efficient mechanisms to counteract the intracellular antiviral response. These mechanisms are beginning to be elucidated using stable cell lines supporting persistent subgenomic HCV replication. The protease action of the HCV NS3/4A complex has been shown to disrupt two independent signaling pathways, toll-like receptor 3 (TLR3) and RIG-I, that both induce type I IFN production (Li et al., 2005; Sumpter Jr. et al., 2005; Breiman et al., 2005). While the protease target in the RIG-I signaling pathway has yet to be identified (Sumpter Jr. et al., 2005; Breiman et al., 2005), the adaptor protein (toll-IL-1 receptor domain-containing adaptor inducing IFN-β; TRIF) linking TLR3 to the kinases responsible for activating the latent transcription factors, IRF-3 and NF-κB, is proteolytically cleaved (Li et al., 2005). Additionally, NS5A has been implicated in the ability of HCV to block the host response to double-stranded RNA. Direct binding of NS5A with protein kinase R (PKR) disrupts signaling events that activate IRF-1 (Pflugheber et al., 2002) or limit RNA translation (Wang et al., 2003).

Inhibition of host factors either through RNA interference or expression of dominant-negative mutants of the protein has facilitated the identification of host cell factors important for productive HCV replication. For example, by these strategies polypyrimidine tract binding protein (PTB; Zhang et al., 2004; Domitrovich et al., 2005), La autoantigen (Zhang et al., 2004; Domitrovich et al., 2005), and human vesicle-associated membrane transport protein A (hVAP-A; Gao et al., 2004; Zhang et al., 2004) have been found to be important for HCV replication. Furthermore, hVAP-A interacts with NS5B (Gao et al., 2004) and NS5A (Evans et al., 2004b; Gao et al., 2004) and recently it has been suggested that NS5A hyperphosphorylation disrupts interactions between hVAP-A and NS5A to negatively regulate HCV replication (Evans et al., 2004b).

Reverse genetics in the replicon system has become an important tool to define HCV RNA sequences and protein determinants critical for productive RNA replication in Huh-7 cells. For instance, the first 125 nucleotides of the 5′ NTR are sufficient for RNA replication, demonstrating that the regions required for translation and replication overlap (Friebe et al., 2001; Kim et al., 2002; Reusken et al., 2003; Luo et al., 2003). Mapping studies have been conducted on the 3′ NTR and confirm the observations made in chimpanzees experimentally inoculated with RNA transcripts carrying similar mutations in the 3′ NTR (Kolykhalov et al., 2000; Yanagi et al., 1999). The terminal 98 nucleotides and poly(U/UC) tract are indispensable for replication, although the poly(U/UC) region can be shortened without affecting HCV replication (Friebe and Bartenschlager, 2002; Yi and Lemon, 2003). In contrast, the upstream variable region can be deleted, resulting in a 100-fold reduction in G418-resistant colonies (Friebe and Bartenschlager, 2002; Yi and Lemon, 2003). More recently, critical cis-acting replication sequences in addition to the 5′ and 3′ NTRs have been identified. Mutational disruption of a computer-predicted highly conserved stem-loop structure located within the 3′ terminal coding region of NS5B, designated 5BSL3.2, blocks subgenomic replication (Lee et al., 2004a; You et al., 2004; Friebe et al., 2005) and can be restored when an intact copy of this RNA element is inserted into the 3′ NTR (Friebe et al., 2005). Furthermore, 5BSL3.2 and the terminal 98 nucleotides form a pseudoknot structure that is indispensable for HCV replication (Friebe et al., 2005).

As mentioned above, HCV replication occurs in conjunction with rearranged membranes, and thus it is not surprising that most of the HCV non-structural proteins contain membrane-anchoring segments (NS4A, 4B, 5A, and 5B; reviewed in Dubuisson et al., 2002; Moradpour et al., 2003). Membrane association of NS5A and NS5B, mediated by an N-terminal amphipathic helix in NS5A (Elazar et al., 2003) and the C-terminal 21 amino acid residues of NS5B (Moradpour et al., 2004a; Lee et al., 2004b), is essential for productive HCV replication in Huh-7 cells. Although the determinants for membrane association have been defined and their importance in HCV replication is beginning to be recognized, the composition and interactions required to assemble functional replication complexes are poorly understood. Nonetheless, sequences that may mediate interactions essential for replicase assembly are being identified. For instance, solvent-exposed residues in the N-terminal helix of NS5A (Penin et al., 2004) and sequences within the transmembrane region of NS5B (Ivashkina et al., 2002; Moradpour et al., 2004a; Lee et al., 2004b) appear to play critical, but as yet undefined, roles in subgenomic replication beyond those of membrane insertion. Two regions of NS5A previously reported to mediate interactions with NS5B in vitro (Shirota et al., 2002) have been shown to be important for productive HCV replication (Shimakami et al., 2004).

Finally, by applying reverse genetics in the replicon system, a GTP-binding motif (Einav et al., 2004) and an amphipathic helix (Elazar et al., 2004) in NS4B as well as a conserved zinc-binding motif in the N-terminal domain of NS5A (Tellinghuisen et al., 2004) have been found to be important for efficient HCV replication, although at this stage the exact role of these motifs in replication has not been determined. As discussed above, hyperphosphorylated NS5A is detrimental to HCV replication, while basal phosphorylation of NS5A has recently been shown to be nonessential for productive HCV replication in Huh-7 cells (Appel et al., 2005b). Thus, further experimentation is warranted to determine whether phosphorylation of NS5A is required at all for RNA replication or whether it serves a regulatory purpose, such as signaling the switch between RNA replication and virus particle assembly. As illustrated above, subgenomic replicons provide an excellent model system for examining HCV-host interactions and for molecular studies of HCV replication.

Inhibition of Replicon Replication

With the development of HCV replicons it became possible to analyze the role of cytokines in the cellular defense against HCV. Genotype 1 replication in cell culture is sensitive to the antiviral programs induced by IFN-α (Blight et al., 2000; Frese et al., 2001; Guo et al., 2001; Gu et al., 2003; Kato et al., 2003a; Lanford et al., 2003; Tanabe et al., 2004; Kanda et al., 2004; Ikeda et al., 2005), IFN-β (Kato et al., 2003a; Larkin et al., 2003), IFN-γ (Frese et al., 2002; Kato et al., 2003a; Lanford et al., 2003; Larkin et al., 2003), IFN-λ (Robek et al., 2005), and interleukin-1 (IL-1; Zhu and Liu, 2003), but not tumor necrosis factor-α (Lanford et al., 2003). In all cases examined so far, the 50% inhibitory concentrations (IC₅₀) for IFN-αin Huh-7 cells are very low (0.5–3 IU/ml; Gu et al., 2003; Tanabe et al., 2004; Ikeda et al., 2005) and are independent of the ISDR (Blight et al., 2000). Type I IFNs induce the transcription of a large number of genes that encode effector proteins with antiviral activities, including PKR, 2′–5′ oligoadenylate synthase (OAS), IFN-stimulated gene 56 (ISG56), and MxA guanosine triphosphatase. Although MxA inhibits the replication of a broad variety of RNA viruses (Haller and Kochs, 2002), it appears that IFN activity in Huh-7 cells proceeds via a MxA-independent pathway since expression of MxA does not inhibit HCV replication and expression of a dominant-negative MxA does not interfere with the antiviral effects of IFN (Frese et al., 2001). Other studies have suggested that IFN-αblocks HCV replication through translational control involving PKR (Wang et al., 2003) and ISG56 (Sumpter Jr. et al., 2004). Thus, the underlying mechanisms of antiviral activity of type I IFNs as well as IFN-γ, IFN-λ, and IL-1 in the replicon system remain unresolved.

Ribavirin significantly improves the rate of sustained viral clearance compared to monotherapy with IFN-α (McHutchison et al., 1998; Di Bisceglie and Hoofnagle, 2002). Similarly, ribavirin and IFN-α in combination elicit strong synergistic inhibitory effects on subgenomic HCV replication (Tanabe et al., 2004; Kanda et al., 2004). The underlying therapeutic mechanism of ribavirin is unknown, but may involve induction of lethal mutagenesis, inhibition of RdRp activity, depletion of intracellular nucleotide pools via inhibition of the host enzyme inosine monophosphate dehydrogenase (IMPDH), or stimulation of the cellular immune response. In Huh-7 cells, ribavirin exhibits antiviral activity by acting as an RNA mutagen inducing error-prone HCV replication (Lanford et al., 2003; Zhou et al., 2003; Tanabe et al., 2004; Kanda et al., 2004). On the other hand, exogenous guanosine can suppress the mutagenic effect of ribavirin and potent IMPDH inhibitors enhance the antiviral capacity of ribavirin, suggesting that ribavirin can inhibit HCV replication by depleting GTP pools (Zhou et al., 2003). The HCV replicon system is being utilized to generate ribavirin-resistant variants. Interestingly, two conserved mutations in the C-terminal region of NS5A independently confer a low level of ribavirin resistance, while ribavirin-resistant HCV replication could also be attributed to defects in ribavirin import rather than mutations in the replicon RNA (Pfeiffer and Kirkegaard, 2005). Thus, HCV replicons will continue to provide a valuable model system for elucidating the mechanisms of ribavirin action, synergism with IFN, and ribavirin resistance in cultured cells.

Subgenomic replicons encode all the known cis-acting RNA sequences and viral enzymes (Table 1) required for replication that are now prime targets for antiviral drug design. Thus, the replicon system provides an excellent screening platform to identify compounds that effectively block enzymatic activities of HCV-encoded proteins and to evaluate the inhibitory effect of nucleic-acid based approaches including antisense oligonucleotides, ribozymes, and small interfering RNAs (siRNAs). Small-molecule inhibitors of the HCV NS3/4A serine protease that are effective in the nanomolar range have been identified (Lamarre et al., 2003; Pause et al., 2003) and nucleoside analogues and non-nucleoside small molecules have been explored as RdRp inhibitors (Carroll et al., 2003; Tomei et al., 2004; Beaulieu et al., 2004; Ludmerer et al., 2005; Tomassini et al., 2005). A peptidomimetic inhibitor of the protease, BILN 2061, provided the first proof-of-principle for preclinical evaluation of new antiviral drugs using the replicon system. BILN 2061 was very active at inhibiting genotype 1 subgenomic replication (IC₅₀ 3–4 nmol/L; Lamarre et al., 2003) and in a phase 1 clinical trial, BILN 2061 administered orally to HCV genotype 1-infected patients led to a 100–1000-fold drop in circulating virus within two days of treatment (Lamarre et al., 2003; Hinrichsen et al., 2004). In contrast, only half of the patients persistently infected with HCV genotype 2 or 3 who received BILN 2061 for 48 hours responded with a reduction in viral RNA greater than 1 log₁₀ (Reiser et al., 2005), underscoring the need to develop replicon-based screening assays for the remaining HCV genotypes (genotypes 3, 4, 5 and 6). Another major factor limiting the efficacy of therapies to combat HCV infection will be the ability of HCV to develop resistance to specific antiviral drugs, but the HCV replicon system will allow potential drug-resistant variants to be rapidly identified and characterized. For example, drug-resistant substitutions in the NS3 protease domain emerge when replicon-containing cells are cultured in the presence of active inhibitors of the HCV protease (Trozzi et al., 2003; Lin et al., 2004; Lu et al., 2004) and a single mutation within the NS5B polymerase conferred resistance to a nucleoside analog, although replicons carrying this mutation were attenuated (Migliaccio et al., 2003). Replicon-containing cells are also being used to test the efficacy of RNA interference against HCV RNAs. Synthetic or stably expressed siRNAs and small hairpin RNAs targeting various regions of the HCV non-structural coding sequence (NS3, NS4B, and NS5B) as well as the 5′ NTR efficiently suppress HCV replication, albeit with different efficiencies (Randall et al., 2003; Yokota et al., 2003; Seo et al., 2003; Wilson et al., 2003; Kapadia et al., 2004; Kronke et al., 2004; Takigawa et al., 2004). Interestingly, siRNAs are more effective at reducing HCV RNA levels than high doses (100 IU/ml) of IFN-α (Kapadia et al., 2004). Furthermore, replicating RNA can be cleared from >98% of siRNA-transfected cells (Randall et al., 2003), and siRNAs, introduced into Huh-7 cells prior to HCV replicons, effectively prevent the establishment of HCV replication (Wilson et al., 2003). Thus, these studies support the principle of siRNA-based HCV antiviral therapy; however, the challenges that have hindered nucleic acid therapies in the past still need to be resolved. As we have already begun to witness, the HCV replicon system will be fundamental for determining the antiviral potency of HCV inhibitors, optimizing drug regimens, monitoring for drug-resistance, and assessing the efficacy of nucleic-acid based antiviral strategies.

Concluding Remarks

The development of subgenomic replicons capable of autonomous replication in the human hepatoma cell line Huh-7 marked an important turning point for HCV research. The identification of cell culture-adaptive mutations and highly permissive Huh-7 sublines has enabled the development of transient replication assays as well as replication-competent monocistronic subgenomes, replicons that encode reporter genes, and full-length HCV genomes. Replicons derived from isolates belonging to genotype 1a, 1b, and 2a are now available and the cell tropism for HCV genotypes 1b and 2a has been expanded to other hepatoma cell lines, non-liver-derived cells, and murine hepatocytes. For the first time, HCV replication can be studied at the molecular level in cell culture. Many investigators have capitalized on this system to investigate important questions related to HCV biology that have plagued the field since the molecular cloning of the HCV genome 16 years ago. While significant advances have been made, there are many questions that remain which undoubtedly will be answered by future research. For instance, what are the mechanisms underlying cell culture adaptation of genotype 1 isolates? Why does the JFH-1 genotype 2a sequence not require adaptive mutations to replicate in cell culture? What are the factors that facilitate more efficient HCV replication in Huh-7 cells than in the other cell lines tested? Why do adaptive mutations prevent virus particle assembly? The recent discovery that full-length RNAs encoding the unmodified non-structural proteins from genotype 2a JFH-1 produce infectious virus particles overcomes one of the remaining barriers and now allows the complete viral life cycle to be studied in cell culture. Finally, subgenomic RNAs are already proving valuable for the development and evaluation of antiviral drugs as well as screening for the emergence of drug resistance. The replicon system should accelerate the development of effective drugs to cure individuals chronically infected with HCV.

Acknowledgments

We are grateful to Dan Ader, John Majors, Sondra Schlesinger, and Milton Schlesinger for critical reading of the manuscript. Supported in part by the Ellison Medical Foundation New Scholars in Global Infectious Disease Research Program to K.J.B. (ID-NS-0119-03). E.A.N. is supported by the Monticello College Foundation Olin Fellowship for Women. We apologize to colleagues for the omission of other key literature citations due to space limitations.

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