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Tan SL, editor. Hepatitis C Viruses: Genomes and Molecular Biology. Norfolk (UK): Horizon Bioscience; 2006.

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Hepatitis C Viruses: Genomes and Molecular Biology.

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Chapter 11HCV Replicon Systems

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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, and the NS5B RdRp.

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.

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.

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 1b

For many years, the only HCV replicons able to autonomously replicate in cultured Huh-7 cells were derived from the Con1 strain. This restriction has now been overcome by the development of replication-competent subgenomic RNAs derived from independent genotype 1b isolates. Unlike the Con1 replicons, cell culture adaptation does not appear to be required for efficient replication of subgenomes derived from the HCV-N isolate, nor for the G418 selection of Huh-7 clones (Guo et al., 2001; Ikeda et al., 2002). Instead, a unique four amino acid insertion naturally present within the ISDR of the HCV-N NS5A protein was sufficient for persistent replication in Huh-7 cells. Removal of these four amino acids from the HCV-N replicon drastically reduced its capacity to confer resistance to G418, but replication could be restored by incorporation of the highly adaptive Con1 mutation, S2204I in NS5A (Ikeda et al., 2002). Thus, this natural four amino acid insertion in HCV-N NS5A behaves like a cell culture-acquired adaptive mutation. For the HCV-BK strain, systematic mutagenesis of the NS3 coding region showed that efficient replicon replication required a mutation in the helicase domain of NS3 (R1496M) in addition to the S2204I substitution in NS5A (Grobler et al., 2003). NS5A adaptive mutations S2197P, S2204I, or ΔS2201 (Fig. 1B) were necessary for productive replication of chimeric HC-J4 replicons containing the 5′ NTR and first 75 amino acids of NS3 from the Con1 strain (Maekawa et al., 2004). While the above genotype 1b replicons were derived from cDNA clones, subgenomic replicons have also been constructed from HCV genome RNA replicating at low levels in cultured cells infected with human serum containing genotype 1b HCV. A very low number of G418-resistant colonies was obtained after transfection of Huh-7 cells, or more permissive Huh-7 sublines, with subgenomic replicons derived from the human T-cell line MT-2C infected with HCV isolate M1LE (Kishine et al., 2002) or from human non-neoplastic hepatocytes (PH5CH8) infected with HCV isolate HCV-O (Kato et al., 2003a). Subgenomes replicating in G418-selected cell clones harbored mutations in NS3 as well as NS4B or NS5A. Although the adaptive advantage of these mutations for M1LE and HCV-O replication has not been determined, substitutions at these positions in the Con1 polyprotein have been shown to enhance Con1 subgenomic replication (Lohmann et al., 2001; Lohmann et al., 2003; Lanford et al., 2003).

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.

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.

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...)

Genotype 2a

Subgenomic replicons derived from the genotype 2a JFH-1 clone, initially isolated from a patient with fulminant hepatitis, represent the only non-genotype 1 sequence currently capable of efficient replication in cell culture (Kato et al., 2003b). Interestingly, JFH-1 subgenomes replicate with high efficiency in Huh-7 cells in the absence of adaptive mutations; the G418-resistant colony-forming ability of the unmodified bicistronic replicon is 60-fold higher than a Con1 subgenomic RNA harboring highly adaptive mutations (Kato et al., 2003b). Although adaptive mutations are not a prerequisite for efficient JFH-1 replication, amino acid changes in the replicase proteins were identified in the majority of G418-selected replicon-containing Huh-7 clones. Of those tested, one mutation, H2476L in NS5B, enhanced the G418 transduction efficiency by only 3-fold, which is well below the level of enhancement seen for single highly adaptive Con1 mutations (Blight et al., 2000; Guo et al., 2001; Lohmann et al., 2003; Lanford et al., 2003). Nonetheless, the colonies derived from JFH-1 replicons containing this NS5B mutation were significantly larger than those obtained after transfection of unmodified JFH-1 subgenomes (Kato et al., 2003b), suggesting that this mutation confers a higher replication phenotype in Huh-7 cells. Furthermore, the high replication efficiency of unmodified JFH-1 replicons allowed HCV RNA and proteins to be monitored in transient replication assays (Fig. 4). Based on the data presented by Kato and coworkers (Kato et al., 2003b), the JFH-1 subgenomic RNA is the most efficient replicon tested so far. Additionally, it appears that adaptive mutations may not always be necessary for efficient replication in cell culture. Instead, the requirement for adaptive mutations is dependent on the individual HCV isolate. Identification of the JFH-1 determinants that promote this high level of RNA replication could provide insights into the mechanisms of HCV RNA replication. Replication of a genotype that diverges from genotype 1 isolates by ~30% not only represents an important advance for replication studies, but also for drug discovery efforts.

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).

Drug Resistance Genes

Engineering alternative drug resistance genes may permit faster selection of replication-positive cells as well as allow different replicon sequences to be sequentially or simultaneously selected within the same cell. Neo-encoding Con1 replicons have been successfully selected in the same cell with Con1 bicistronic replicons encoding either blasticidin S deaminase (Evans et al., 2004a) or hygromycin phosphotransferase (Appel et al., 2005a) genes in place of the Neo cassette (Fig. 3A). This approach found: (i) no evidence of recombination between different subgenomes (Evans et al., 2004a; Appel et al., 2005a); (ii) a high level of competition between subgenomic RNAs, suggesting that host cell machinery required for HCV replication is limiting (Evans et al., 2004a) and; (iii) replicons harboring lethal mutations in NS3, NS4B, or NS5B or mutations that disrupt the structure of the N-terminal membrane anchor of NS5A could not be rescued by co-expression of functional subgenomic RNAs (Evans et al., 2004a; Appel et al., 2005a). Thus, it appears that HCV replication complexes are relatively closed structures preventing or limiting the exchange of viral proteins. However, deletions in NS5A or multiple mutations affecting potential phosphorylation sites in the central region of NS5A could be complemented in trans, suggesting that NS5A is loosely associated with the intracellular membranes that provide the scaffold of the HCV replication complex (Appel et al., 2005a). Alternatively, a monocistronic replicon encoding the hygromycin phosphotransferase gene establishes productive replication in Huh-7 cells and confers resistance to hygromycin (Frese et al., 2002). In this replicon, ubiquitin was inserted in-frame between hygromycin phosphotransferase and the NS3-5B coding region so that translation of the entire ORF is directed by the HCV 5′ NTR and NS3 is released from the polyprotein by a cellular ubiquitin carboxyl-terminal hydrolase-mediated cleavage event at the ubiquitin/NS3 junction (Fig. 3A).

Reporter Genes

Assays for colony formation are time consuming and assume that the frequency of drug-resistant colonies observed with a given replicon is directly proportional to its intrinsic replication activity. With the identification of adaptive mutations that facilitate efficient HCV replication, transient RNA replication assays that allow a more rapid and direct analysis of relative replication efficiencies have been developed. Reporters such as luciferase and ß-lactamase, as well as a transactivator inducing secreted alkaline phosphatase (SEAP), have been used to monitor replication at early times after transfection of Huh-7 cells.

Firefly luciferase has successfully replaced the Neo gene in Con1 (Krieger et al., 2001) and HCV-O (Ikeda et al., 2005) bicistronic replicons (Fig. 3B), thus enabling replication to be monitored at various times following transfection by measuring the luciferase activity relative to a polymerase-defective replicon. After 48–72 hours, the luciferase activities seen with an adapted Con1 replicon are about 100-fold higher than the negative control (Krieger et al., 2001). The luciferase activity directly correlates with the levels of HCV RNA synthesis, demonstrating that luciferase is a reliable marker of replication (Krieger et al., 2001). Enhanced replication levels, and thus higher luciferase activities (~5-fold), as well as more reproducible results were achieved by placing luciferase under the translational control of the IRES from poliovirus instead of the HCV IRES (Fig. 3B; Lohmann et al., 2003). Although luciferase activity allows the rapid determination of relative replication levels in the population of transfected cells, it does not provide a direct measure of the number of cells supporting replication. This restriction has been overcome by the development of bicistronic Con1 replicons containing a ß-lactamase reporter (Fig. 3B; Murray et al., 2003). In this strategy, Huh-7 cells supporting active HCV replication are identified using a cell-permeable fluorescent substrate that is cleaved by ß-lactamase expressed in the cell, leading to blue fluorescence. More recently, it has been reported that the C-terminal domain of NS5A (between residues 2370 and 2412) is dispensable for replicon function (Appel et al., 2005b) and heterologous sequences can be inserted within this region with only a moderate reduction in replication (Moradpour et al., 2004b; Appel et al., 2005b). Thus, viable Con1 replicons carrying an in-frame insertion of enhanced green fluorescent protein (GFP) at position 2356 or 2390 in NS5A were created (Fig. 3B); however, the G418 transduction efficiency of GFP-expressing replicons was ≥25-fold lower than the parental replicon constructs (Moradpour et al., 2004b). Although the GFP signal is readily visualized in G418-selected cell clones by fluorescence microscopy allowing active replication complexes to be tracked in real time (Moradpour et al., 2004b and see below), it is not clear if the replication competence of these GFP-expressing replicons is sufficient for the detection and quantification of GFP-positive cells in transient replication assays. In an independent report a firefly luciferase-expressing Con1 subgenomic RNA (Fig. 3B) carrying a GFP insertion between positions 2370 and 2412 of NS5A replicated to levels about 100-fold below the parental replicon that lacked GFP and this lower replication efficiency prevented the direct visualization of the NS5A-GFP fusion protein at 72 hr post-transfection (Appel et al., 2005b).

The first cistron of the bicistronic Con1, HCV-N, (Yi et al., 2002) and H77 (Yi and Lemon, 2004) replicons have been modified to include the human immunodeficiency virus (HIV) tat protein, a potent transcriptional transactivator of the HIV long terminal repeat (LTR) promoter (Yi et al., 2002). Briefly, the tat-coding sequence was fused to a picornaviral 2A protease sequence followed by the Neo selectable marker (Fig. 3B), such that upon translation, the autocatalytic protease activity of 2A mediates cleavage at its C-terminus, liberating Neo. Replication in transfected cells leads to the intracellular accumulation of tat, which in turn activates the LTR-SEAP cassette, which is stably integrated into the genome of the transfected Huh-7 cell. SEAP is subsequently expressed and secreted from replication-positive cells and five days after transfection of adapted replicons, extracellular SEAP can reach levels 100-fold above the amount secreted from cells transfected with replication-defective mutants (Yi et al., 2002).

In addition to the use of these reporter gene replicon systems for rapid determination of replicative ability, these systems are amenable to high throughput tests including screening large compound libraries for anti-HCV activity. For instance, the ß-lactamase replicon system has been successfully adapted to a high-throughput screening assay to identify inhibitors of HCV replication (Zuck et al., 2004). Furthermore, replicons carrying firefly or renilla luciferase fused to the neomycin phosphotransferase gene (Fig. 3B) have been used to assess the effect of human IFN-α and ribavirin on HCV replication (Tanabe et al., 2004; Ikeda et al., 2005). Another group determined the level of biologically active IFN-α in sera taken from HCV carriers undergoing IFN treatment by using cells harboring a bicistronic replicon where the first cistron comprises a firefly luciferase-ubiquitin-Neo cassette (Fig. 3B; Vrolijk et al., 2003).

Monocistronic Subgenomic Replicons

The ability to monitor HCV replication without selection eliminates the requirement for bicistronic replicons. In fact, heterologous sequences such as the Neo gene and the EMCV IRES reduce the replicative capacity of Con1- and H77-derived subgenomic RNAs (Blight et al., 2002b; Blight et al., 2003). Robust replication is observed with a monocistronic replicon composed of the 5′ NTR followed by the entire capsid sequence fused to the NS2-NS5B coding region, such that cleavage between capsid and NS2 is mediated by the host cell signal peptidase and translation is under the control of the HCV IRES (Fig. 3C; Blight et al., 2003). The replication-competence of this subgenomic RNA, as well as the monocistronic hygromycin phosphotransferase-encoding replicon described above (Fig. 3A; Frese et al., 2002), demonstrates that the EMCV IRES is not a requirement for efficient expression of the HCV replicase region and subgenomic replication in Huh-7 cells. Since this monocistronic replicon lacks antibiotic resistance or reporter genes (Fig. 3C), replication in transfected cells can only be monitored by the detection of HCV-specific RNA and proteins. Quantitative real-time RT-PCR (Blight et al., 2000; Blight et al., 2002b; Blight et al., 2003) and Northern blot hybridization (Krieger et al., 2001; Pietschmann et al., 2002; Lohmann et al., 2003; Kato et al., 2003b; Yi and Lemon, 2004) have been used to analyze HCV RNA synthesis (Fig. 4), and HCV protein expression has been successfully detected by a collection of assays including Western blot, fluorescent activated cell sorting (FACS), metabolic labeling and immunoprecipitation with HCV-specific antisera, and immunostaining of cell monolayers (Fig. 4; Pietschmann et al., 2002; Blight et al., 2002b; Blight et al., 2003; Kato et al., 2003b). HCV subgenomic replication can now be studied in the absence of heterologous sequences, alleviating concerns that the experimental phenotype is related to the expressed heterologous gene or foreign IRES element.

Full-Length Replicons

Full-length or genomic HCV replicons derived from Con1, H77, HCV-O, and HCV-N productively replicate in Huh-7 cells or in highly permissive Huh-7 sublines (Ikeda et al., 2002; Pietschmann et al., 2002; Blight et al., 2002b; Blight et al., 2003; Yi and Lemon, 2004; Ikeda et al., 2005). Full-length HCV RNAs carry the complete HCV open reading frame (C-NS5B) and replication is dependent on cell culture-adaptive mutations. Bicistronic derivatives encoding Neo (Ikeda et al., 2002; Pietschmann et al., 2002; Blight et al., 2002b; Ikeda et al., 2005) or a renilla luciferase-Neo fusion (Ikeda et al., 2005) have been generated (Fig. 3D), facilitating the selection of stable G418-resistant cell lines supporting full-length HCV replication. The number of Huh-7 cells able to support full-length replication is much lower than that seen for the subgenomic derivative carrying the same adaptive mutations and the average level of full-length RNA replication is about 5-fold lower than subgenomic replication (Pietschmann et al., 2002; Blight et al., 2002b; Blight et al., 2003). So far, there is no evidence of HCV particle assembly and release from Huh-7 cells supporting replication of Con1 (Pietschmann et al., 2002; Blight et al., 2002b) or H77 (Blight et al., 2003) full-length RNAs containing cell culture-adaptive mutations. Although unmodified full-length HCV RNAs generated from these HCV strains produce infectious virus in the chimpanzee model (Kolykhalov et al., 1997; Yanagi et al., 1997; Bukh et al., 2002), Con1 genomes harboring cell culture-adaptive mutations are severely attenuated in vivo (Bukh et al., 2002), suggesting that adaptive mutations inhibit virus particle assembly. In support of this, Huh-7 cells supporting replication of a full-length RNA containing the non-structural proteins from the genotype 2a JFH-1 strain that lacks adaptive mutations assemble and release infectious virus particles (Bartenschlager et al., 2004; Heller et al., 2005). Additionally, virus production has also been observed in Huh-7 cells transfected with plasmid DNA carrying an unmodified infectious full-length genotype 1b HCV genome flanked by self-cleaving hammerhead ribozymes to generate the exact 5′ and 3′ ends of intracellular transcribed RNA (Heller et al., 2005). The development of cell culture systems supporting the complete virus life cycle now allows studies directed towards defining the mechanisms of viral particle assembly (see Chapter 16).

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.

Huh-7 Cell Line

Although cell culture-adaptive mutations are essential for genotype 1 replication in Huh-7 cells, there is convincing evidence that the environment within the Huh-7 cell also governs the ability of HCV RNAs to replicate. First, the relative replication efficiencies of subgenomic RNAs in transient replication assays can vary by as much as 100-fold between different passages of Huh-7 cells. These differences are independent of the adaptive mutation(s) introduced into the replicon and are not due to differences in HCV RNA translation or stability (Lohmann et al., 2003), suggesting that efficient replication depends on host cell conditions or specific cellular factors. Secondly, replication of subgenomic RNAs, despite the inclusion of highly adaptive mutations, can only be detected in a subset of transfected Huh-7 cells (~10% for the highly adaptive mutation S2204I in NS5A; Blight et al., 2000; Blight et al., 2002b), suggesting that many Huh-7 cells do not provide an optimal environment for HCV replication. Additionally, the number of G418-resistant colonies obtained after transfection of selectable replicons was significantly higher in cell clones from which the replicon had been eliminated by extended treatment with IFN-α ("curing") than that observed for naïve Huh-7 cells (Blight et al., 2002b; Murray et al., 2003; Ikeda et al., 2005). Furthermore, rare G418-selected Huh-7 clones "cured" of Con1 replicons that had not acquired adaptive mutations during the selection process were more permissive for HCV replication than "cured" cells originally supporting adapted Con1 subgenomic RNA replication (Blight et al., 2002b). These results demonstrate that cellular environments more conducive to HCV replication are ultimately selected and, in Huh-7 cells that harbor conditions most supportive for viral replication, transfected Con1 replicon RNA does not need to adapt to efficiently replicate.

The most permissive "cured" subline identified so far (Huh-7.5; Blight et al., 2002b) has the capacity to support high levels of subgenomic HCV replication in >75% of transfected cells. Furthermore, Huh-7.5 cells more readily support RNAs with lower replicative abilities, such as full-length Con1 replicons (Blight et al., 2002b) and H77-derived RNAs (Blight et al., 2003). Increased permissiveness in Huh-7.5 cells is due to mutational inactivation of the retinoic acid inducible gene-I (RIG-I), a cytoplasmic protein that recognizes structured RNA to induce type I IFN production via activation of transcription factors interferon regulatory factor (IRF)-3 and NF-κB. Complementation with functional RIG-I restores IRF-3 signaling in Huh-7.5 cells and converts this hyper-permissive cell line to a relatively non-permissive phenotype (Sumpter Jr. et al., 2005). Thus, RIG-I-mediated activation of IRF-3 is a critical determinant of cellular permissiveness for HCV replication.

Additional Cell Lines

Attempts by many investigators to propagate HCV RNAs in other cell lines were unsuccessful until 2003, when Zhu and coworkers described replication of Con1 subgenomic RNAs in HeLa cells and in the murine hepatoma cell line Hepa1–6, albeit with low efficiency (Zhu et al., 2003). Due to the high error rate of the NS5B RdRp, replicon RNAs prepared from Huh-7 cell lines in which persistent replication had been established were expected to have greater genetic variance than HCV RNAs generated by in vitro transcription from cloned cDNA templates. Indeed, transfection of HeLa cells with total RNA from Con1 replicon-harboring Huh-7 cells gave rise to a low number of G418-resistant cell colonies. Replicons in G418-resistant HeLa cells maintained the Huh-7 cell adaptive mutations, but acquired several additional mutations in the non-structural proteins. Interestingly, the colony-forming efficiency of replicon RNAs isolated from HeLa cells was significantly higher in naïve HeLa cells than Huh-7 cells. In addition, HeLa cell-derived replicon RNAs were more efficient at establishing G418-resistance in HeLa cells than replicons obtained from Huh-7 cells. These results indicate that replicon variants have been selected that can replicate more efficiently in HeLa cells. However, in vitro transcribed subgenomic RNAs carrying both the Huh-7 and HeLa cell-specific mutations only conferred G418 resistance in a few HeLa cells, and thus the relative contribution of these mutations to productive replication in HeLa cells has not been determined. Replicon RNAs derived from HeLa cells, but not from Huh-7 cells, were also able to replicate and confer resistance to G418 in a few Hepa1-6 cells. Sequence analysis revealed that replicons from these mouse cells had preserved the majority of the mutations found in HeLa cells, and only a few additional mutations were identified. Moreover, total RNA isolated from one of the Hepa1–6 replicon-containing cell lines did not increase the colony-forming efficiency compared to replicon RNA from HeLa cells, suggesting that HeLa-derived subgenomes were already adapted for replication in the mouse cells (Zhu et al., 2003).

Con1 replicon replication has also been established in human embryonic kidney 293 cells. By co-culturing replicon-containing Huh-7 cells with 293 cells, rare hybrids closely resembling parental 293 cells were selected that supported subgenomic RNA replication (Ali et al., 2004). Nucleotide sequence analysis of replicating HCV RNA in hybrid 293 cells identified a large number of mutations that appear to facilitate replication in 293 cells; transfection of total cellular RNA isolated from one of these replicon-expressing hybrid clones was able to establish replication in naïve 293 cells. As observed by Zhu and coworkers (Zhu et al., 2003), in vitro transcribed replicon RNA, containing all the mutations identified in this hybrid 293 clone, failed to confer resistance to G418 in naïve 293 cells (Ali et al., 2004). The differences in colony-forming capacity between in vitro transcribed Con1 subgenomic RNA and replicon RNA isolated from stable cell clones remains a mystery, although it is possible that RNA molecules co-purifying with the replicating replicon RNA facilitates the establishment of Con1 replication in these less permissive cell lines.

Although in vitro transcripts derived from genotype 1b replicons have not successfully established replication in non-Huh-7 cells (Zhu et al., 2003; Ali et al., 2004), G418 resistance in human hepatocyte- (HepG2 and IMY-N9; Date et al., 2004) and non-hepatocyte- (HeLa and 293; Kato et al., 2005) derived cell lines has been achieved by the genotype 2a replicon, JFH-1. The efficiency of colony formation was 10- to 1000-fold lower compared to Huh-7 cells (Date et al., 2004; Kato et al., 2005), and the colony-forming efficiency and colony size in HepG2 and IMY-N9 cells was increased following transfection of JFH-1 replicons harboring the Huh-7 specific adaptive mutation in NS5B (H2496L; Date et al., 2004). Many G418-selected cell clones did not contain mutations in the HCV coding region and, when coding changes were found, they were not shared between independent clones and had not been previously identified in JFH-1 replicons replicating in Huh-7 cells (Date et al., 2004; Kato et al., 2005). Interestingly, in eight of the nine 293-derived cell clones analyzed, amino acid substitutions were not identified (Kato et al., 2005). These findings suggest that adaptive mutations are not essential for JFH-1 replication in either hepatocyte- or non-hepatocyte-derived cell lines.

Collectively, the cell tropism for HCV genotypes 1b and 2a has not only been expanded to include additional human hepatoma cell lines, but also non-liver derived human cells and murine hepatocytes, disproving the previous hypothesis that HCV replication is governed by hepatocyte- and primate-specific factors. Moreover, the ability of HCV replicons to replicate in a murine hepatoma cell line offers some hope that a mouse model for HCV infection may be developed in the future.

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.

References

  1. Ahlquist P, Noueiry AO, Lee W, Kushner DB, Dye BT. Host factors in positive-strand RNA virus genome replication. J. Virol. 2003;77:8181–8186. [PMC free article: PMC165243] [PubMed: 12857886]
  2. Aizaki H, Lee KJ, Sung VM, Ishiko H, Lai MM. Characterization of the hepatitis C virus RNA replication complex associated with lipid rafts. Virology. 2004;324:450–461. [PubMed: 15207630]
  3. Ali N, Tardif KD, Siddiqui A. Cell-free replication of the hepatitis C virus subgenomic replicon. J. Virol. 2002;76:12001–12007. [PMC free article: PMC136887] [PubMed: 12414942]
  4. Ali S, Pellerin C, Lamarre D, Kukolj G. Hepaitis C virus subgenomic replicons in the human embryonic kidney 293 cell line. J. Virol. 2004;78:491–501. [PMC free article: PMC303421] [PubMed: 14671129]
  5. Alter HJ, Seeff LB. Recovery, persistence and sequelae in hepatitis C virus infection: a perspective on the long-term outcome. Semin. Liver Dis. 2000;20:17–25. [PubMed: 10895429]
  6. Appel N, Herian U, Bartenschlager R. Efficient rescue of hepatitis C virus RNA replication by trans-complementation with nonstructural protein 5A. J. Virol. 2005a;79:896–909. [PMC free article: PMC538567] [PubMed: 15613318]
  7. Appel N, Pietschmann T, Bartenschlager R. Mutational analysis of hepatitis C virus nonstructural protein 5A: potential role of differential phosphorylation in RNA replication and identification of a genetically flexible domain. J. Virol. 2005b;79:3187–3194. [PMC free article: PMC548472] [PubMed: 15709040]
  8. Bartenschlager R, Ahlborn-Laake L, Mous J, Jacobsen H. Kinetic and structural analyses of hepatitis C virus polyprotein processing. J. Virol. 1994;68:5045–5055. [PMC free article: PMC236447] [PubMed: 8035505]
  9. Bartenschlager R, Frese M, Pietschmann T. Novel insights into hepatitis C virus replication and persistence. Adv. Virus Res. 2004;63:71–180. [PubMed: 15530561]
  10. Bartenschlager R, Lohmann V. Novel cell culture systems for the hepatitis C virus. Antiviral Res. 2001;52:1–17. [PubMed: 11530183]
  11. Beaulieu PL, Bousquet Y, Gauthier J, Gillard J, Marquis M, McKercher G, Pellerin C, Valois S, Kukolj G. Non-nucleoside benzimidazole-based allosteric inhibitors of the hepatitis C virus NS5B polymerase: inhibition of subgenomic hepatitis C virus RNA replicons in Huh-7 cells. J. Med. Chem. 2004;47:6884–6892. [PubMed: 15615537]
  12. Behrens S-E, Grassmann CW, Thiel H-J, Meyers G, Tautz N. Characterization of an autonomous subgenomic pestivirus RNA replicon. J. Virol. 1998;72:2364–2372. [PMC free article: PMC109536] [PubMed: 9499097]
  13. Blight KJ, Gowans EJ. In situ hybridization and immunohistochemical staining of hepatitis C virus products. Viral Hepatitis Rev. 1995;1:143–155.
  14. Blight KJ, Grakoui A, Hanson HL, Rice CM. Ou J-HJ, editor. The molecular biology of hepatitis C virus. Boston: Kluwer Academic Publishers; Hepatitis viruses. 2002a:81–108.
  15. Blight KJ, Kolykhalov AA, Rice CM. Efficient initiation of HCV RNA replication in cell culture. Science. 2000;290:1972–1974. [PubMed: 11110665]
  16. Blight KJ, McKeating JA, Marcotrigiano J, Rice CM. Efficient replication of hepatitis C virus genotype 1a RNAs in cell culture. J. Virol. 2003;77:3181–3190. [PMC free article: PMC149761] [PubMed: 12584342]
  17. Blight KJ, McKeating JA, Rice CM. Highly permissive cell lines for subgenomic and genomic hepatitis C virus RNA replication. J. Virol. 2002b;76:13001–13014. [PMC free article: PMC136668] [PubMed: 12438626]
  18. Breiman A, Grandvaux N, Lin R, Ottone C, Akira S, Yoneyama M, Fujita T, Hiscott J, Meurs EF. Inhibition of RIG-I-dependent signaling to the interferon pathway during hepatitis C virus expression and restoration of signaling by IKKε J. Virol. 2005;79:3969–3978. [PMC free article: PMC1061556] [PubMed: 15767399]
  19. Bukh J, Pietschmann T, Lohmann V, Krieger N, Faulk K, Engle RE, Govindarajan S, Shapiro M, St Claire M, Bartenschlager R. Mutations that permit efficient replication of hepatitis C virus RNA in Huh-7 cells prevent productive replication in chimpanzees. Proc. Natl. Acad. Sci. USA. 2002;99:14416–14421. [PMC free article: PMC137898] [PubMed: 12391335]
  20. Carroll SS, Tomassini JE, Bosserman M, Getty K, Stahlhut MW, Eldrup AB, Bhat B, Hall D, Simcoe A, LaFemina R, et al. Inhibition of hepatitis C virus RNA replication by 2′-modified nucleoside analogs. J. Biol. Chem. 2003;278:11979–11984. [PubMed: 12554735]
  21. Date T, Kato T, Miyamoto M, Zhao Z, Yasui K, Mizokami M, Wakita T. Genotype 2a hepatitis C virus subgenomic replicon can replicate in HepG2 and IMY-N9 cells. J. Biol. Chem. 2004;279:22371–22376. [PubMed: 14990575]
  22. Di Bisceglie AM, Hoofnagle JH. Optimal therapy of hepatitis C. Hepatology. 2002;36:S121–127. [PubMed: 12407585]
  23. Domitrovich AM, Diebel KW, Ali N, Sarker S, Siddiqui A. Role of La autoantigen and polypyrimidine tract-binding protein in HCV replication. Virology. 2005;335:72–86. [PubMed: 15823607]
  24. Dubuisson J, Penin F, Moradpour D. Interaction of hepatitis C virus proteins with host cell membranes and lipids. Trends in Cell Biology. 2002;12:517–523. [PubMed: 12446113]
  25. Egger D, Wolk B, Gosert R, Bianchi L, Blum HE, Moradpour D, Bienz K. Expression of hepatitis C virus proteins induces distinct membrane alterations including a candidate viral replication complex. J. Virol. 2002;76:5974–5984. [PMC free article: PMC136238] [PubMed: 12021330]
  26. Einav S, Elazar M, Danieli T, Glenn JS. A nucleotide binding motif in hepatitis C virus (HCV) NS4B mediates HCV RNA replication. J. Virol. 2004;78:11288–11295. [PMC free article: PMC521822] [PubMed: 15452248]
  27. El-Hage N, Luo G. Replication of hepatitis C virus RNA occurs in a membrane-bound replication complex containing nonstructural viral proteins and RNA. J. Gen. Virol. 2003;84:2761–2769. [PubMed: 13679611]
  28. Elazar M, Cheong KH, Liu P, Greenberg HB, Rice CM, Glenn JS. Amphipathic helix-dependent localization of NS5A mediates hepatitis C virus RNA replication. J. Virol. 2003;77:6055–6061. [PMC free article: PMC154017] [PubMed: 12719597]
  29. Elazar M, Liu P, Rice CM, Glenn JS. An N-terminal amphipathic helix in hepatitis C virus (HCV) NS4B mediates membrane association, correct localization of replication complex proteins, and HCV RNA replication. J. Virol. 2004;78:11393–11400. [PMC free article: PMC521809] [PubMed: 15452261]
  30. Enomoto N, Sakuma I, Asahina Y, Kurosaki M, Murakami T, Yamamoto C, Izumi N, Marumo F, Sato C. Comparison of full-length sequences of interferon-sensitive and resistant hepatitis C virus 1b. J. Clin. Invest. 1995;96:224–230. [PMC free article: PMC185192] [PubMed: 7542279]
  31. Enomoto N, Sakuma I, Asahina Y, Kurosaki M, Murakami T, Yamamoto C, Ogura Y, Izumi N, Marumo F, Sato C. Mutations in the nonstructural protein 5A gene and response to interferon in patients with chronic hepatitis C virus 1b infection. N. Engl. J. Med. 1996;334:77–81. [PubMed: 8531962]
  32. Evans MJ, Rice CM, Goff SP. Genetic interactions between hepatitis C virus replicons. J. Virol. 2004a;78:12085–12089. [PMC free article: PMC523274] [PubMed: 15479852]
  33. Evans MJ, Rice CM, Goff SP. Phosphorylation of hepatitis C virus nonstructural protein 5A modulates its protein interactions and viral RNA replication. Proc. Natl. Acad. Sci. USA. 2004b;101:13038–13043. [PMC free article: PMC516513] [PubMed: 15326295]
  34. Fishman JA, Rubin RH, Koziel MJ, Periera BJ. Hepatitis C virus and organ transplantation. Transplantation. 1996;62:147–154. [PubMed: 8755808]
  35. Frese M, Pietschmann T, Moradpour D, Haller O, Bartenschlager R. Interferon-α inhibits hepatitis C virus subgenomic RNA replication by an MxA-independent pathway. J. Gen. Virol. 2001;82:723–733. [PubMed: 11257176]
  36. Frese M, Schwarzle V, Barth K, Krieger N, Lohmann V, Mihm S, Haller O, Bartenschlager R. Interferon-γ inhibits replication of subgenomic and genomic hepatitis C virus RNAs. Hepatology. 2002;35:694–703. [PubMed: 11870386]
  37. Friebe P, Bartenschlager R. Genetic analysis of sequences in the 3′ nontranslated region of hepatitis C virus that are important for RNA replication. J. Virol. 2002;76:5326–5338. [PMC free article: PMC137049] [PubMed: 11991961]
  38. Friebe P, Boudet J, Simorre JP, Bartenschlager R. Kissing-loop interaction in the 3′ end of the hepatitis C virus genome essential for RNA replication. J. Virol. 2005;79:380–392. [PMC free article: PMC538730] [PubMed: 15596831]
  39. Friebe P, Lohmann V, Krieger N, Bartenschlager R. Sequences in the 5′ nontranslated region of hepatitis C virus required for RNA replication. J. Virol. 2001;75:12047–12057. [PMC free article: PMC116100] [PubMed: 11711595]
  40. Gao L, Aizaki H, He J-W, Lai MM. Interactions between viral nonstructural proteins and host protein hVAP-33 mediate the formation of hepatitis C virus RNA replication complex on lipid raft. J. Virol. 2004;78:3480–3488. [PMC free article: PMC371042] [PubMed: 15016871]
  41. Gosert R, Egger D, Lohmann V, Bartenschlager R, Blum HE, Bienz K, Moradpour D. Identification of the hepatitis C virus RNA replication complex in Huh-7 cells harboring subgenomic replicons. J. Virol. 2003;77:5487–5492. [PMC free article: PMC153965] [PubMed: 12692249]
  42. Goutagny N, Fatmi A, De Ledinghen V, Penin F, Couzigou P, Inchauspe G, Bain C. Evidence of viral replication in circulating dendritic cells during hepatitis C virus infection. J. Infect. Dis. 2003;187:1951–1958. [PubMed: 12792872]
  43. Grakoui A, McCourt DW, Wychowski C, Feinstone SM, Rice CM. A second hepatitis C virus-encoded proteinase. Proc. Natl. Acad. Sci. USA. 1993;90:10583–10587. [PMC free article: PMC47821] [PubMed: 8248148]
  44. Grobler JA, Markel EJ, Fay JF, Graham DJ, Simcoe AL, Ludmerer SW, Murray EM, Migliaccio G, Flores OA. Identification of a key determinant of hepatitis C virus cell culture adaptation in domain II of NS3 helicase. J Biol Chem. 2003;278:16741–16746. [PubMed: 12615931]
  45. Gu B, Gates AT, Isken O, Behrens SE, Sarisky RT. Replication studies using genotype 1a subgenomic hepatitis C virus replicons. J Virol. 2003;77:5352–5359. [PMC free article: PMC153987] [PubMed: 12692237]
  46. Guo JT, Bichko VV, Seeger C. Effect of alpha interferon on the hepatitis C virus replicon. J Virol. 2001;75:8516–8523. [PMC free article: PMC115097] [PubMed: 11507197]
  47. Haller O, Kochs G. Interferon-induced Mx proteins: dynamin-like GTPases with antiviral activity. Traffic. 2002;3:710–717. [PubMed: 12230469]
  48. Hardy R, Marcotrigiano J, Blight KJ, Majors JE, Rice CM. Hepatitis C virus RNA synthesis in a cell-free system isolated from replicon-containing hepatoma cells. J Virol. 2003;77:2029–2037. [PMC free article: PMC140877] [PubMed: 12525637]
  49. Heller T, Saito S, Auerbach J, Williams T, Moreen TR, Jazwinski A, Cruz B, Jeurkar N, Sapp R, Luo G, Liang TJ. An in vitro model of hepatitis C virion production. Proc. Natl. Acad. Sci. USA. 2005;102:2579–2583. [PMC free article: PMC549006] [PubMed: 15701697]
  50. Hijikata M, Mizushima H, Akagi T, Mori S, Kakiuchi N, Kato N, Tanaka T, Kimura K, Shimotohno K. Two distinct proteinase activities required for the processing of a putative nonstructural precursor protein of hepatitis C virus. J Virol. 1993;67:4665–4675. [PMC free article: PMC237852] [PubMed: 8392606]
  51. Hinrichsen H, Benhamou Y, Wedemeyer H, Reiser M, Sentjens RE, Calleja JL, Forns X, Erhardt A, Cronlein J, Chaves RL, et al. Short-term antiviral efficacy of BILN 2061, a hepatitis C virus serine protease inhibitor, in hepatitis C genotype 1 patients. Gastroenterology. 2004;127:1347–1355. [PubMed: 15521004]
  52. Ikeda M, Abe K, Dansako H, Nakamura T, Naka K, Kato N. Efficient replication of a full-length hepatitis C virus genome, strain O, in cell culture, and development of a luciferase reporter system. Biochem Biophys Res Comm. 2005;329:1350–1359. [PubMed: 15766575]
  53. Ikeda M, Yi M, Li K, Lemon SM. Selectable subgenomic and genome-length dicistronic RNAs derived from an infectious molecular clone of the HCV-N strain of hepatitis C virus replicate efficiently in cultured Huh7 cells. J Virol. 2002;76:2997–3006. [PMC free article: PMC135991] [PubMed: 11861865]
  54. Ivashkina N, Wolk B, Lohmann V, Bartenschlager R, Blum HE, Penin F, Moradpour D. The hepatitis C virus RNA-dependent RNA polymerase membrane insertion sequence is a transmembrane segment. J Virol. 2002;76:13088–13093. [PMC free article: PMC136709] [PubMed: 12438637]
  55. Kanda T, Yokosuka O, Imazeki F, Tanaka M, Shino Y, Shimada H, Tomonaga T, Nomura F, Nagao K, Ochiai T, Saisho H. Inhibition of subgenomic hepatitis C virus RNA in Huh-7 cells: ribavirin induces mutagenesis in HCV RNA. J Viral Hepat. 2004;11:479–487. [PubMed: 15500548]
  56. Kaneko T, Tanji Y, Satoh S, Hijikata M, Asabe S, Kimura K, Shimotohno K. Production of two phosphoproteins from the NS5A region of the hepatitis C viral genome. Biochem Biophys Res Commun. 1994;205:320–326. [PubMed: 7999043]
  57. Kapadia SB, Brideau-Anderson A, Chisari FV. Interference of hepatitis C virus RNA replication by short interfering RNAs. Proc. Natl. Acad. Sci. USA. 2004;100:2014–2018. [PMC free article: PMC149950] [PubMed: 12566571]
  58. Kato N, Sugiyama K, Namba K, Dansako H, Nakamura T, Takami M, Naka K, Nozaki A, Shimotohno K. Establishment of a hepatitis C virus subgenomic replicon derived from human hepatocytes infected in vitro. Biochem. Biophys. Res. Comm. 2003a;306:756–766. [PubMed: 12810084]
  59. Kato T, Date T, Miyamoto M, Furusaka A, Tokushige K, Mizokami M, Wakita T. Efficient replication of the genotype 2a hepatitis C virus subgenomic replicon. Gastroenterology. 2003b;125:1808–1817. [PubMed: 14724833]
  60. Kato T, Date T, Miyamoto M, Zhao Z, Mizokami M, Wakita T. Nonhepatic cell lines HeLa and 293 support efficient replication of the hepatitis C virus genotype 2a subgenomic replicon. J Virol. 2005;79:592–596. [PMC free article: PMC538706] [PubMed: 15596851]
  61. Khromykh AA, Westaway EG. Subgenomic replicons of the flavivirus Kunjin: construction and applications. J Virol. 1997;71:1497–1505. [PMC free article: PMC191206] [PubMed: 8995675]
  62. Kim JL, Morgenstern KA, Griffith JP, Dwyer MD, Thomson JA, Murcko MA, Lin C, Caron PR. Hepatitis C virus NS3 RNA helicase domain with a bound oligonucleotide: the crystal structure provides insights into the mode of unwinding. Structure. 1998;6:89–100. [PubMed: 9493270]
  63. Kim JL, Morgenstern KA, Lin C, Fox T, Dwyer MD, Landro JA, Chambers SP, Markland W, Lepre CA, O'Malley ET, et al. Crystal structure of the hepatitis C virus NS3 protease domain complexed with a synthetic NS4A cofactor peptide. Cell. 1996;87:343–355. [PubMed: 8861917]
  64. Kim YK, Kim CS, Lee SH, Jang SK. Domains I and II in the 5′ nontranslated region of the HCV genome are required for RNA replication. Biochem Biophys Res Commun. 2002;290:105–112. [PubMed: 11779140]
  65. Kishine H, Sugiyama K, Hijikata M, Kato N, Takahashi H, Noshi T, Nio Y, Hosaka M, Miyanari Y, Shimotohno K. Subgenomic replicon derived from a cell line infected with the hepatitis C virus. Biochem Biophys Res Comm. 2002;293:993–999. [PubMed: 12051758]
  66. Kolykhalov AA, Agapov EV, Blight KJ, Mihalik K, Feinstone SM, Rice CM. Transmission of hepatitis C by intrahepatic inoculation with transcribed RNA. Science. 1997;277:570–574. [PubMed: 9228008]
  67. Kolykhalov AA, Mihalik K, Feinstone SM, Rice CM. Hepatitis C virus encoded enzymatic activities and conserved RNA elements in the 3′ nontranslated region are essential for virus replication in vivo. J. Virol. 2000;74:2046–2051. [PMC free article: PMC111684] [PubMed: 10644379]
  68. Krieger N, Lohmann V, Bartenschlager R. Enhancement of hepatitis C virus RNA replication by cell culture-adaptive mutations. J Virol. 2001;75:4614–4624. [PMC free article: PMC114214] [PubMed: 11312331]
  69. Kronke J, Kittler R, Buchholz F, Windisch MP, Pietschmann T, Bartenschlager R, Frese M. Alternative approaches for efficient inhibition of hepatitis C virus RNA replication by small interfering RNAs. J Virol. 2004;78:3436–3446. [PMC free article: PMC371081] [PubMed: 15016866]
  70. Lai VC, Dempsey S, Lau JY, Hong Z, Zhong W. In vitro RNA replication directed by replicase complexes isolated from the subgenomic replicon cells of hepatitis C virus. J Virol. 2003;77:2295–2300. [PMC free article: PMC140981] [PubMed: 12525668]
  71. Lamarre D, Anderson PC, Bailey M, Beaulieu P, Bolger G, Bonneau P, Bos M, Cameron DR, Cartier M, Cordingley MG, et al. An NS3 protease inhibitor with antiviral effects in humans infected with hepatitis C virus. Nature. 2003;426:186–189. [PubMed: 14578911]
  72. Lanford RE, Guerra B, Lee H, Averett DR, Pfeiffer B, Chavez D, Notvall L, Bigger C. Antiviral effect and virus-host interactions in response to alpha interferon, gamma interferon, poly(I)-poly(C), tumor necrosis factor alpha, and ribavirin in hepatitis C virus subgenomic replicons. J Virol. 2003;77:1092–1104. [PMC free article: PMC140845] [PubMed: 12502825]
  73. Larkin J, Jin L, Farmen M, Venable D, Huang Y, Tan SL, Glass JI. Synergistic antiviral activity of human interferon combinations in the hepatitis C virus replicon system. J Interferon Cytokine Res. 2003;23:247–257. [PubMed: 12804067]
  74. Laskus T, Radkowski M, Piasek A, Nowicki M, Horban A, Cianciara J, Rakela J. Hepatitis C virus in lymphoid cells of patients coinfected with human immunodeficiency virus type 1: evidence of active replication in monocytes/macrophages and lymphocytes. J Infect Dis. 2000;181:442–448. [PubMed: 10669324]
  75. Lee HK, Shin H, Wimmer E, Paul A. cis-acting RNA signals in the NS5B C-terminal coding sequence of the hepatitis C virus genome. J. Virol. 2004a;78:10865–10877. [PMC free article: PMC521798] [PubMed: 15452207]
  76. Lee KJ, Choi J, Ou JH, Lai MM. The C-terminal transmembrane domain of hepatitis C virus (HCV) RNA polymerase is essential for HCV replication in vivo. J. Virol. 2004b;78:3797–3802. [PMC free article: PMC371049] [PubMed: 15016899]
  77. Lesburg CA, Cable MB, Ferrari E, Hong Z, Mannarino AF, Weber PC. Crystal structure of the RNA-dependent RNA polymerase from hepatitis C virus reveals a fully encircled active site. Nat Struct Biol. 1999;6:937–943. [PubMed: 10504728]
  78. Li K, Foy E, Ferreon JC, Nakamura M, Ferreon ACM, Ikeda M, Ray SC, Gale M Jr, Lemon SM. Immune evasion by hepatitis C virus NS3/4A protease-mediated cleavage of the Toll-like receptor 3 adaptor protein TRIF. Proc. Natl. Acad. Sci. USA. 2005;102:2992–2997. [PMC free article: PMC548795] [PubMed: 15710891]
  79. Liang C, Rieder E, Hahm B, Jang SK, Paul A, Wimmer E. Replication of a novel subgenomic HCV genotype 1a replicon expressing a puromycin resistance gene in Huh7 cells. Virology. 2005;333:41–53. [PubMed: 15708591]
  80. Lin C, Lin K, Luong YP, Rao BG, Wei YY, Brennan DL, Fulghum JR, Hsiao HM, Ma S, Maxwell JP, et al. In vitro resistance studies of hepatitis C virus serine protease inhibitiors, VX-950 and BILN 2061. J Biol Chem. 2004;279:17508–17514. [PubMed: 14766754]
  81. Lin C, Prágai B, Grakoui A, Xu J, Rice CM. Hepatitis C virus NS3 serine proteinase: trans-cleavage requirements and processing kinetics. J. Virol. 1994;68:8147–8157. [PMC free article: PMC237280] [PubMed: 7966606]
  82. Lohmann V, Hoffmann S, Herian U, Penin F, Bartenschlager R. Viral and cellular determinants of hepatitis C virus RNA replication in cell culture. J Virol. 2003;77:1–13. [PMC free article: PMC149776] [PubMed: 12584326]
  83. Lohmann V, Korner F, Dobierzewska A, Bartenschlager R. Mutations in hepatitis C virus RNAs conferring cell culture adaptation. J Virol. 2001;75:1437–1449. [PMC free article: PMC114050] [PubMed: 11152517]
  84. Lohmann V, Korner F, Koch JO, Herian U, Theilmann L, Bartenschlager R. Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science. 1999;285:110–113. [PubMed: 10390360]
  85. Lu L, Pilot-Matias TJ, Stewart KD, Randolph JT, Pithawalla R, He W, Huang PP, Klein LL, Mo H, Molla A. Mutations conferring resistance to a potent hepatitis C virus serine protease inhibitor in vitro. Antimicrob. Agents Chemother. 2004;48:2260–2266. [PMC free article: PMC415624] [PubMed: 15155230]
  86. Ludmerer SW, Graham DJ, Boots E, Murray EM, Simcoe A, Markel EJ, Grobler JA, Flores OA, Olsen DB, Hazuda DJ, Lafemina RL. Replication fitness and NS5B drug sensitivity of diverse hepatitis C virus isolates characterized by using a transient replication assay. Antimicrob Agents Chemother. 2005;49:2059–2069. [PMC free article: PMC1087645] [PubMed: 15855532]
  87. Luo G, Xin S, Cai Z. Role of the 5′-proximal stem-loop structure of the 5′ untranslated region in replication and translation of hepatitis C virus RNA. J Virol. 2003;77:3312–3318. [PMC free article: PMC149781] [PubMed: 12584356]
  88. Maekawa S, Enomoto N, Sakamoto N, Kurosaki M, Ueda E, Kohashi T, Watanabe H, Chen CH, Yamashiro T, Tanabe Y, et al. Introduction of NS5A mutations enables subgenomic HCV replicon derived from chimpanzee-infectious HC–J4 isolate to replicate efficiently in Huh-7 cells. J Viral Hepat. 2004;11:394–403. [PubMed: 15357644]
  89. McHutchison JG, Gordon SC, Schiff ER, Shiffman ML, Lee WM, Rustgi VK, Goodman ZD, Ling MH, Cort S, Albrecht JK. Interferon alfa-2b alone or in combination with ribavirin as initial treatment for chronic hepatitis C. Hepatitis Interventional Therapy Group. N Engl J Med. 1998;339:1485–1492. [PubMed: 9819446]
  90. Migliaccio G, Tomassini JE, Carroll SS, Tomei L, Altamura S, Bhat B, Bartholomew L, Bosserman MR, Ceccacci A, Colwell LF, et al. Characterization of resistance to non-obligate chain-terminating ribonucleoside analogs that inhibit hepatitis C virus replication in vitro. J. Biol. Chem. 2003;278:49164–49170. [PubMed: 12966103]
  91. Miyanari Y, Hijikata M, Yamaji M, Hosaka M, Takahashi H, Shimotohno K. Hepatitis C virus non-structural proteins in the probable membranous compartment function in viral genome replication. J Biol Chem. 2003;278:50301–50308. [PubMed: 12963739]
  92. Moradpour D, Brass V, Bieck E, Friebe P, Gosert R, Blum HE, Bartenschlager R, Penin F, Lohmann V. Membrane association of the RNA-dependent RNA polymerase is essential for hepatitis C virus RNA replication. J. Virol. 2004a;78 [PMC free article: PMC524999] [PubMed: 15542678]
  93. Moradpour D, Evans MJ, Gosert R, Yuan Z, Blum HE, Goff SP, Lindenbach BD, Rice CM. Insertion of green fluorescent protein into nonstructural protein 5A allows direct visualization of functional hepatitis C virus replication complexes. J Virol. 2004b;78:7400–7409. [PMC free article: PMC434129] [PubMed: 15220413]
  94. Moradpour D, Gosert R, Egger D, Penin F, Blum HE, Bienz K. Membrane association of hepatitis C virus nonstructural proteins and identification of the membrane alteration that harbors the viral replication complex. Antiviral Res. 2003;60:103–109. [PubMed: 14638405]
  95. Mottola G, Cardinali G, Ceccacci A, Trozzi C, Bartholomew L, Torrisi MR, Pedrazzini E, Bonatti S, Migliaccio G. Hepatitis C virus nonstructural proteins are localized in a modified endoplasmic reticulum of cells expressing viral subgenomic replicons. Virology. 2002;293:31–43. [PubMed: 11853397]
  96. Murray EM, Grobler JA, Markel EJ, Pagnoni MF, Paonessa G, Simon AJ, Flores OA. Persistent replication of hepatitis C virus replicons expressing β-lactamase reporter in subpopulations of highly permissive Huh7 cells. J Virol. 2003;77:2928–2935. [PMC free article: PMC149762] [PubMed: 12584317]
  97. Neddermann P, Quintavalle M, Di Pietro C, Clementi A, Cerretani M, Altamura S, Bartholomew L, De Francesco R. Reduction of hepatitis C virus NS5A hyperphosphorylation by selective inhibition of cellular kinases activates viral RNA replication in cell culture. J Virol. 2004;78:13306–13314. [PMC free article: PMC524975] [PubMed: 15542681]
  98. Pause A, Kukolj G, Bailey M, Brault M, Do F, Halmos T, Lagace L, Maurice R, Marquis M, McKercher G, et al. An NS3 serine protease inhibitor abrogates replication of subgenomic hepatitis C virus RNA. J Biol Chem. 2003;278:20374–20380. [PubMed: 12646587]
  99. Penin F, Brass V, Appel N, Ramboarina S, Montserret R, Ficheux D, Blum HE, Bartenschlager R, Moradpour D. Structure and function of the membrane anchor domain of hepatitis C virus nonstructural protein 5A. J Biol Chem. 2004;279:40835–40843. [PubMed: 15247283]
  100. Pfeiffer JK, Kirkegaard K. Ribavirin resistance in hepatitis C virus replicon-containing cell lines conferred by changes in the cell line or mutations in the replicon RNA. J Virol. 2005;79:2346–2355. [PMC free article: PMC546591] [PubMed: 15681435]
  101. Pflugheber J, Fredericksen B, Sumpter R Jr, Wang C, Ware F, Sodora DL, Gale M Jr. Regulation of PKR and IRF-1 during hepatitis C virus RNA replication. Proc. Natl. Acad. Sci. USA. 2002;99:4650–4655. [PMC free article: PMC123702] [PubMed: 11904369]
  102. Pietschmann T, Lohmann V, Kaul A, Krieger N, Rinck G, Rutter G, Strand D, Bartenschlager R. Persistent and transient replication of full-length hepatitis C virus genomes in cell culture. J Virol. 2002;76:4008–4021. [PMC free article: PMC136109] [PubMed: 11907240]
  103. Pietschmann T, Lohmann V, Rutter G, Kurpanek K, Bartenschlager R. Characterization of cell lines carrying self-replicating hepatitis C virus RNAs. J Virol. 2001;75:1252–1264. [PMC free article: PMC114031] [PubMed: 11152498]
  104. Poynard T, Yuen MF, Ratziu V, Lai CL. Viral hepatitis C. Lancet. 2003;362:2095–2100. [PubMed: 14697814]
  105. Randall G, Grakoui A, Rice CM. Clearance of replicating hepatitis C virus replicon RNAs in cell culture by small interfering RNAs. Proc. Natl. Acad. Sci. USA. 2003;100:235–240. [PMC free article: PMC140937] [PubMed: 12518066]
  106. Reed KE, Rice CM. Overview of hepatitis C virus genome structure, polyprotein processing, and protein properties. In: Hagedorn C, Rice CM, editors. Hepatitis, C virus. Berlin: Springer-Verlag; 1999. pp. 55–84. [PubMed: 10592656]
  107. Reed KE, Xu J, Rice CM. Phosphorylation of the hepatitis C virus NS5A protein in vitro and in vivo: properties of the NS5A-associated kinase. J. Virol. 1997;71:7187–7197. [PMC free article: PMC192058] [PubMed: 9311791]
  108. Reiser M, Hinrichsen H, Benhamou Y, Reesink HW, Wedemeyer H, Avendano C, Riba N, Yong CL, Nehmiz G, Steinmann G. Antiviral efficacy of NS3-serine protease inhibitor BILN-2061 in patients with chronic genotype 2 and 3 hepatitis C. Hepatology. 2005;41:832–835. [PubMed: 15732092]
  109. Reusken CB, Dalebout TJ, Eerligh P, Bredenbeek PJ, Spaan WJ. Analysis of hepatitis C virus/classical swine fever virus chimeric 5′NTRs: sequences within the hepatitis C virus IRES are required for viral RNA replication. J Gen Virol. 2003;84:1761–1769. [PubMed: 12810870]
  110. Rijnbrand RCA, Lemon SM. Internal ribosome entry site-mediated translation in hepatitis C virus replication. In: Hagedorn C, Rice CM, editors. Hepatitis C virus. Berlin: Springer-Verlag; 2000. pp. 85–116. [PubMed: 10592657]
  111. Robek MD, Boyd BS, Chisari FV. Lambda interferon inhibits hepatitis B and C virus replication. J Virol. 2005;79:3851–3854. [PMC free article: PMC1075734] [PubMed: 15731279]
  112. Robertson B, Myers G, Howard C, Brettin T, Bukh J, Gaschen B, Gojobori T, Maertens G, Mizokami M, Nainan O, et al. Classification, nomenclature, and database development for hepatitis C virus (HCV) and related viruses: proposals for standardization. International Committee on Virus Taxonomy [news] Arch Virol. 1998;143:2493–2503. [PubMed: 9930205]
  113. Salonen A, Ahola T, Kaariainen L. Viral RNA replication in association with cellular membranes. Curr Top Microbiol Immunol. 2005;285:139–173. [PubMed: 15609503]
  114. Scholle F, Li K, Bodola F, Ikeda M, Luxon BA, Lemon SM. Virus-host cell interactions during hepatitis C virus RNA replication: impact of polyprotein expression on the cellular transcriptome and cell cycle association with viral RNA synthesis. J Virol. 2004;78:1513–1524. [PMC free article: PMC321413] [PubMed: 14722306]
  115. Seo MY, Abrignani S, Houghton M, Han JH. Small interfering RNA-mediated inhibition of hepatitis C virus replication in the human hepatoma cell line Huh-7. J. Virol. 2003;77 [PMC free article: PMC140638] [PubMed: 12477890]
  116. Shi ST, Lee KJ, Aizaki H, Hwang SB, Lai MMC. Hepatitis C virus RNA replication occurs on a detergent-resistant membrane that cofractionates with caveolin-2. J Virol. 2003;77:4160–4168. [PMC free article: PMC150636] [PubMed: 12634374]
  117. Shimakami T, Hijikata M, Luo H, Ma Y, Kaneko S, Shimotohno K, Murakami S. Effect of interaction between hepatitis C virus NS5A and NS5B on hepatitis C virus RNA replication with the hepatitis C virus replicon. J Virol. 2004;78:2738–2748. [PMC free article: PMC353754] [PubMed: 14990694]
  118. Shirota Y, Luo H, Qin W, Kaneko S, Yamashita T, Kobayashi K, Murakami S. Hepatitis C virus (HCV) NS5A binds RNA-dependent RNA polymerase (RdRP) NS5B and modulates RNA-dependent RNA polymerase activity. J Biol Chem. 2002;277:11149–11155. [PubMed: 11801599]
  119. Simmonds P, Holmes EC, Cha T-A, Chan S-W, McOmish F, Irvine B, Beall E, Yap PL, Kolberg J, Urdea MS. Classification of hepatitis C virus into six major genotypes and a series of subtypes by phylogenetic analysis of the NS5 region. J Gen Virol. 1993;74:2391–2399. [PubMed: 8245854]
  120. Sumpter R Jr, Loo YM, Foy E, Li K, Yoneyama M, Fujita T, Lemon SM, Gale M Jr. Regulating intracellular antiviral defense and permissiveness to hepatitis C virus RNA replication through a cellular RNA helicase, RIG-I. J Virol. 2005;79:2689–2699. [PMC free article: PMC548482] [PubMed: 15708988]
  121. Sumpter R Jr, Wang C, Foy E, Loo YM, Gale M Jr. Viral evolution and interferon resistance of hepatitis C virus RNA replication in a cell culture model. J Virol. 2004;78:11591–11604. [PMC free article: PMC523295] [PubMed: 15479800]
  122. Takigawa Y, Nagano-Fujii M, Deng L, Hidajat R, Tanaka M, Mizuta H, Hotta H. Suppression of hepatitis C virus replicon by RNA interference directed against the NS3 and NS5B regions of the viral genome. Microbiol Immunol. 2004;48:591–598. [PubMed: 15322339]
  123. Tanabe Y, Sakamoto N, Enomoto N, Kurosaki M, Ueda E, Maekawa S, Yamashiro T, Nakagawa M, Chen CH, Kanazawa N, et al. Synergistic inhibition of intracellular hepatitis C virus replication by combination of ribavirin and interferon-α J Infect Dis. 2004;189:1129–1139. [PubMed: 15031779]
  124. Tanji Y, Hijikata M, Hirowatari Y, Shimotohno K. Hepatitis C virus polyprotein processing: kinetics and mutagenic analysis of serine proteinase-dependent cleavage. J Virol. 1994;68:8418–8422. [PMC free article: PMC237315] [PubMed: 7966638]
  125. Tanji Y, Kaneko T, Satoh S, Shimotohno K. Phosphorylation of hepatitis C virus-encoded nonstructural protein NS5A. J Virol. 1995;69:3980–3986. [PMC free article: PMC189129] [PubMed: 7769656]
  126. Tellinghuisen TL, Marcotrigiano J, Gorbalenya AE, Rice CM. The NS5A protein of hepatitis C virus is a zinc metalloprotein. J Biol Chem. 2004;279:48576–485 87. [PubMed: 15339921]
  127. Tomassini JE, Getty K, Stahlhut MW, Shim S, Bhat B, Eldrup AB, Prakash TP, Carroll SS, Flores O, Maccoss M, et al. Inhibitory effect of 2′-substituted nucleosides on hepatitis C virus replication correlates with metabolic properties in replicon cells. Antimicrob Agents Chemother. 2005;49:2050–2058. [PMC free article: PMC1087620] [PubMed: 15855531]
  128. Tomei L, Altamura S, Bartholomew L, Bisbocci M, Bailey C, Bosserman M, Cellucci A, Forte E, Incitti I, Orsatti L, et al. Characterization of the inhibition of hepatitis C virus RNA replication by nonnucleosides. J Virol. 2004;78:938–946. [PMC free article: PMC368780] [PubMed: 14694125]
  129. Trozzi C, Bartholomew L, Ceccacci A, Biasiol G, Pacini L, Altamura S, Narjes F, Muraglia E, Paonessa G, Koch U, et al. In vitro selection and characterization of hepatitis C virus serine protease variants resistant to an active-site peptide inhibitor. J Virol. 2003;77:3669–3679. [PMC free article: PMC149541] [PubMed: 12610142]
  130. Varaklioti A, Vassilaki N, Georgopoulou U, Mavromara P. Alternate translation occurs within the core coding region of the hepatitis C viral genome. J Biol Chem. 2002;277:17713–17721. [PubMed: 11884417]
  131. Vrolijk JM, Kaul A, Hansen BE, Lohmann V, Haagmans BL, Schalm SW, Bartenschlager R. A replicon-based bioassay for the measurement of interferons in patients with chronic hepatitis C. J. Virol. Methods. 2003;110:201–209. [PubMed: 12798249]
  132. Walewski JL, Keller TR, Stump DD, Branch AD. Evidence for a new hepatitis C virus antigen encoded in an overlapping reading frame. RNA. 2001;7:710–721. [PMC free article: PMC1370123] [PubMed: 11350035]
  133. Wang C, Pflugheber J, Sumpter R Jr, Sodora DL, Hui D, Sen GC, Gale M Jr. Alpha interferon induces distinct translational control programs to suppress hepatitis C virus RNA replication. J Virol. 2003;77:3898–3912. [PMC free article: PMC150642] [PubMed: 12634350]
  134. WHO. Hepatitis C - global prevalence (update) Wkly. Epidemiol. Rec. 2000;75:18–19. [PubMed: 10686829]
  135. Wilson JA, Jayasena S, Khvorova A, Sabatinos S, Rodrigue-Gervais IG, Arya S, Sarangi F, Harris-Brandts M, Beaulieu S, Richardson CD. RNA interference blocks gene expression and RNA synthesis from hepatitis C replicons propagated in human liver cells. Proc. Natl. Acad. Sci. USA. 2003;100:2783–2788. [PMC free article: PMC151418] [PubMed: 12594341]
  136. Xu Z, Choi J, Yen TS, Lu W, Strohecke RA, Govindarajan S, Chien D, Selby MJ, Ou J. Synthesis of a novel hepatitis C virus protein by ribosomal frameshift. EMBO J. 2001;20:3840–3848. [PMC free article: PMC125543] [PubMed: 11447125]
  137. Yanagi M, Purcell RH, Emerson SU, Bukh J. Transcripts from a single full-length cDNA clone of hepatitis C virus are infectious when directly transfected into the liver of a chimpanzee. Proc. Natl. Acad. Sci. USA. 1997;94:8738–8743. [PMC free article: PMC23104] [PubMed: 9238047]
  138. Yanagi M, St Claire M, Emerson SU, Purcell RH, Bukh J. In vivo analysis of the 3′ untranslated region of the hepatitis C virus after in vitro mutagenesis of an infectious cDNA clone. Proc Natl Acad Sci USA. 1999;96:2291–2295. [PMC free article: PMC26776] [PubMed: 10051634]
  139. Yang G, Pevear DC, Collett MS, Chunduru S, Young DC, Benetatos CA, Jordan R. Newly synthesized hepatitis C virus replicon RNA is protected from nuclease activity by a protease-sensitive factor(s) J Virol. 2004;78:10202–10205. [PMC free article: PMC514995] [PubMed: 15331754]
  140. Yi M, Bodola F, Lemon SM. Subgenomic hepatitis C virus replicons inducing expression of a secreted enzymatic reporter protein. Virology. 2002;304:197–210. [PubMed: 12504562]
  141. Yi M, Lemon SM. 3′ nontranslated RNA signals required for replication of hepatitis C virus RNA. J Virol. 2003;77:3557–3568. [PMC free article: PMC149512] [PubMed: 12610131]
  142. Yi M, Lemon SM. Adaptive mutations producing efficient replication of genotype 1a hepatitis C virus RNA in normal Huh7 cells. J Virol. 2004;78:7904–7915. [PMC free article: PMC446091] [PubMed: 15254163]
  143. Yokota T, Sakamoto N, Enomoto N, Tanabe Y, Miyagishi M, Maekawa S, Yi L, Kurosaki M, Taira K, Watanabe M, Mizusawa H. Inhibition of intracellular hepatitis C virus replication by synthetic and vector-derived small interfering RNAs. EMBO Reports. 2003;4:602–608. [PMC free article: PMC1319196] [PubMed: 12740604]
  144. You S, Stump DD, Branch AD, Rice CM. A cis-acting replication element in the sequence encoding the NS5B RNA-dependent RNA polymerase is required for hepatitis C virus RNA replication. J Virol. 2004;78:1352–1366. [PMC free article: PMC321395] [PubMed: 14722290]
  145. Zhang J, Yamada O, Sakamoto T, Yoshida H, Iwai T, Matsushita Y, Shimamura H, Araki H, Shimotohno K. Down-regulation of viral replication by adenoviral-mediated expression of siRNA against cellular cofactors for hepatitis C virus. Virology. 2004;320:135–143. [PubMed: 15003869]
  146. Zhou S, Liu R, Baroudy BM, Malcolm BA, Reyes GR. The effect of ribavirin and IMPDH inhibitors on hepatitis C virus subgenomic replicon RNA. Virology. 2003;310:333–342. [PubMed: 12781720]
  147. Zhu H, Liu C. Interleukin-1 inhibits hepatitis C virus subgenomic RNA replication by activation of extracellular regulated kinase pathway. J Virol. 2003;77:5493–5498. [PMC free article: PMC153991] [PubMed: 12692250]
  148. Zhu Q, Guo JT, Seeger C. Replication of hepatitis C virus subgenomes in nonhepatic epithelial and mouse hepatoma cells. J Virol. 2003;77:9204–9210. [PMC free article: PMC187424] [PubMed: 12915536]
  149. Zuck P, Murray EM, Stec E, Grobler JA, Simon AJ, Strulovici B, Inglese J, Flores OA, Ferrer M. A cell-based B-lactamase reporter gene assay for the identification of inhibitors of hepatitis C virus replication. Anal Biochem. 2004;334:344–355. [PubMed: 15494142]
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