Hussein H. Aly, Department of Microbiology and Immunology, Graduate School of Medicine, Hokkaido University, Kita-ku, Sapporo 060-8638, Japan. Tel: +81 11 706 5073; fax: +81 11 706 5056; email: firstname.lastname@example.org
Chronic hepatitis C virus (HCV) infection affects approximately 170 million people worldwide. HCV infection is a major global health problem as it can be complicated with liver cirrhosis and hepatocellular carcinoma. So far, there is no vaccine available and the non-specific, interferon (IFN)-based treatments now in use have significant side-effects and are frequently ineffective, as only approximately 50% of treated patients with genotypes 1 and 4 demonstrate HCV clearance. The lack of suitable in vitro and in vivo models for the analysis of HCV infection has hampered elucidation of the HCV life cycle and the development of both protective and therapeutic strategies against HCV infection. The present review focuses on the progress made towards the establishment of such models.
Chronic HCV infection is a major cause of mortality and morbidity throughout the world, infecting approximately 3.1% of the world's population (1). Only a fraction of acutely infected individuals are able to clear the infection spontaneously, whereas approximately 80% of infected individuals develop a chronic infection (2, 3). Patients with chronic HCV are at increased risk for developing liver fibrosis, cirrhosis, and/or hepatocellular carcinoma. Currently, these long-term complications of chronic HCV infection are the leading indication for liver transplantation (4, 5). Because of the high incidence of new infections by blood transfusions in the 1980s before the discovery of the virus, and because morbidity associated with chronic HCV infection generally takes decades to develop, it is expected that the burden of disease in the near future will rise dramatically.
HCV is an enveloped flavivirus, with a positive-stranded RNA genome of approximately 9600 nucleotides. The coding region is flanked by 5′ and 3′ non-coding regions, which are important for the initiation of translation and regulation of genomic duplication, respectively. The coding region itself is composed of a single open reading frame, which encodes a polyprotein precursor of approximately 3000 amino acids. This polyprotein is cleaved by host and viral proteases into structural and NS proteins (Fig. 1). Replication of the HCV genome involves the synthesis of a full-length negative-stranded RNA intermediate, which in turn provides a template for the de novo production of positive-stranded RNA. Both these synthesis steps are mediated by the viral RNA-dependent RNA polymerase NS5B (6–8). NS5B lacks proofreading abilities, and this leads to a high mutation rate and the generation of numerous quasispecies. HCV isolates can be classified into seven major genotypes, which vary in sequence by more than 30%. In addition to the distinct prevalence and global spread of the virus, the genotype is an important factor determining disease progression and responses to antiviral therapy (9).
Currently, the only licensed treatment for HCV is the combination of (pegylated)-interferon-alpha (IFN-α) and ribavirin. Although the success rate of treatment has improved substantially, standard therapy is not effective in all patients. Moreover, severe adverse effects and high costs limit the compliance and global application of this treatment. The development of prophylaxis and novel therapeutics to treat HCV infection has been hampered by the lack of suitable in vitro and in vivo culture systems. In this review, we describe the development of in vitro culture systems for HCV.
Dr Bartenschlager's group was the first to establish a convenient reproducible in vitro cell culture system for the study of HCV replication (10). They created antibiotic-resistant HCV genomes to select replication-competent viral clones by conveying antibiotic resistance to cells. This was achieved by replacing the structural protein-coding sequences, as well as p7 of the consensus genome Con1, by the neomycin resistance gene. In addition, a second IRES was introduced to promote translation of the non-structural protein-coding sequences important for viral replication (Fig. 2). Upon transfection of these so-called subgenomic replicons in specific cell lines, drug-resistant cell colonies were isolated in which high levels of viral replication occurred. Subsequent analysis confirmed that these HCV replicons indeed were capable of self-amplification through synthesis of a negative-strand replication intermediate, and could be stably propagated in cell culture for many years (10, 11).
HCV replication was supported by several cell types such as HuH6 (12), HepG2 (13), Li23 (14), and 293 cells (15), with the human hepatoma cell line HuH-7 being the most permissive (16). Interestingly, removal of replicon RNA from these cell clones by treatment with type 1 IFN rendered the cells more permissive to reintroduction of replicons, resulting in higher replication rates. Examples of these highly permissive cells are HuH-7.5 and HuH-7-Lunet cells (16, 17). The efficient replication in the replicon systems was found to depend on tissue-culture-adaptive mutations. Introduction of these specific mutations in the wild-type consensus sequence significantly enhanced viral replication in vitro (18–22). Mutational hot spots were found clustered primarily in the NS3, NS4B, and NS5A regions. The mechanisms behind the enhanced replication caused by these tissue-culture-adaptive mutations are still largely unknown, and the interesting fact that these mutations are not commonly found in patients suggests that these may have a toll on the viral fitness.
HCV replicons have proven to be extremely valuable for studies on the process of HCV replication, as well as for testing novel antiviral compounds that specifically target the protease activity of NS3 or the polymerase activity of NS5 (23).
Cell culture-derived infectious HCV
Studies using HCV replicons have provided detailed knowledge on the mechanisms of replication of HCV. However, an apparent shortcoming of these models was that stable cell clones containing self-replicating replicons and expressing all viral proteins remained unable to release infectious HCV particles. The inability to secrete viral particles may be the consequence of adaptive mutations, which are needed to enhance viral replication rates, but at the same time may block viral assembly. Indeed, replicons without adaptive mutations show very low replication rates (16, 24). A different situation emerged when the first genotype 2a consensus genome was established (25, 26).
A subgenomic replicon constructed from a clone called JFH-1, isolated from a Japanese patient with fulminant hepatitis C, replicated up to 20-fold higher in HuH-7 cells as compared to Con1 replicons, and did not require adaptive mutations for efficient replication in vitro (26). Transfection of HuH-7 and HuH-7.5.1 cells with the in vitro-transcribed full-length JFH-1 genome or a recombinant chimeric genome with another genotype 2a isolate, J6, resulted in the secretion of viral particles that were infectious in cultured cells (Fig. 3), in chimeric mice, and in chimpanzees (27–29).
The infectivity of cells could be neutralized with antibodies against the HCV entry receptor CD81, antibodies against E2, or immunoglobulins from chronically infected patients. Importantly, the replication of cell-cultured HCV in this system was inhibited by IFN-α as well as by several HCV-specific antiviral compounds (29). Since 2005, chimeric JFH-1-based genomes have been constructed of all seven known HCV genotypes. Similar to the J6-JFH-1 chimera, in these so-called intergenotypic recombinants, the structural genes (core, E1, and E2), p7, and NS2 of JFH-1 were replaced by genotype-specific sequences which often resulted in lower infectious virion production than wild-type JFH-1 (30–32). Most NS proteins of intergenotypic chimeras originate from JFH-1, and therefore these genomes are unlikely to reflect genotype-specific characteristics of replication. However, these intergenotypic chimeras may become critically important in the study of differences in HCV entry or to assess the efficacy of HCV entry inhibitors. Interestingly, production of infectious genotype 1a HCV in cells transfected with synthetic RNA (H77-S) derived from a prototype virus (H77-C) was also reported (33). H77-S carries adaptive mutations that promote efficient viral RNA replication in HuH-7.5 cells. These mutations are located within the NS3/4A protease complex, and the NS5A protein (34) H77-S showed similar replication efficiency to JFH-1 isolate; however, it showed lower expression of HCV core protein, and lower production of infectious HCV particles (33).
Serum-derived HCV infection
The previously mentioned models used to study HCV infection are based on subclones of HuH-7 cells infected with JFH1 recombinant virus or its derivatives (27). HuH-7 cells and its subclones, however, do not support the entire life cycle of the bbHCV present in the blood of patients (35). Moreover, HCV has considerable diversity and variability. It is generally classified into six major genotypes and more than 100 subtypes (36). JFH1, however, is a single isolate of HCV genotype 2a that was originally derived from a patient with rare fulminant hepatitis (27). Thus, usage of HCV particles isolated from patient serum could be more useful to study authentic HCV infection.
Many researchers have attempted to develop an in vitro system for bbHCV (37–39). These current systems, however, are still insufficient due to their low efficiency for infectivity and replication of bbHCV. Normal human hepatocytes are the ideal system in which to study HCV infectivity. When cultured in vitro, however, they proliferate poorly and divide only a few times (40). Continuous proliferation could be achieved by introducing oncogenes, the HPV/E6E7 immortalized multiple cell types that were phenotypically and functionally similar to the parental cells (41–45). We established a human primary non-neoplastic hepatocyte cell line transduced with the HPV18/E6E7 that retained primary hepatocyte characteristics even after prolonged culture (35). We further improved the susceptibility of HPV18/E6E7-immortalized hepatocytes (HuS-E/2 cells) to bbHCV infectivity by impairing the innate immune response of these cells through suppression of interferon regulatory factor-7 (IRF-7) expression. These cells were useful to assay infectivity of HCV strains other than JFH-1, HCV replication, innate immune system engagement of HCV, and screening of anti-HCV agents. This infection system using non-neoplastic cells also suggested that IRF-7 plays an important role in eliminating HCV infection. Using this system, the suppressive effect of tamoxifen and mir199 on HCV replication was reported (46, 47).
A major limitation of the immortalized hepatocytes infection system was the failure to produce infectious HCV particles. Because the 3-D cell culture condition more closely reproduces the in vivo environment of hepatocytes (48), culturing these cells in this manner may support the entire HCV life cycle. Similarly, a previous report showed the production of HCV particles from the FLC4 hepatocyte line transfected with HCV-RNA and cultured in a 3-D radial-flow bioreactor (RFB). The RFB system is composed of a dedicated device containing 1 × 109 FLC4 cells with a culture area of 2.7 m2. A more convenient, smaller and easy to use 3-D culture system is required for the study of the several aspects of bbHCV infection. (49). A hybrid artificial liver support system was developed using animal hepatocytes cultured in a 3-D/HF. This bioartificial liver showed several characteristic features of liver tissue for more than 4 months (50–52).
By growing our HuSE/2 cells in a similar 3-D culture (53) the gene expression profile was improved to more closely match that of human primary hepatocytes. We used this small 3-D culture system and showed it to be ideal for culturing HuS-E/2 cells for the study of bbHCV infection (Fig. 4) (54). Using this system we observed not only the enhancement of HCV replication, but also the production of infectious HCV particles in the medium using the 3-D/HF system. The cell mass formed by the 3-D culture system, most likely the polar character, was essential for the life cycle of bbHCV. Using microarray comparison of gene expression between 2-D and 3-D cultured cells, we found a higher activation of the PPAR-α signaling pathway which was shown to be important for the improvement of HCV replication in 3-D culture. Suppression of the PPAR-α signaling pathway using its antagonist MK886 markedly suppressed HCV replication in two different cell lines (53). A recent study showed that the induction of PPAR-α or PPAR-γ led to the suppression or enhancement of HCV replication, respectively, in HuH-7 cells (55). Using HuH-7-derived clones, three different independent studies confirmed our data, showing the suppression of HCV replication by PPAR-α blockers such as (MK886) (56, 57) or 2-chloro-5-nitro-N-(pyridyl) benzamide (BA) (58). Furthermore, no effect of PPAR-γ was observed on HCV replication (58).
Delayed production of infectious particles was also observed in cells infected with some HCV strains after prolonged culture (54). It is likely that mutation of the HCV genome and/or selection of clones during prolonged culture improved the productivity of infectious particles. This lack of production of infectious particles soon after infection may serve to avoid an early strong response from the host immune system, and demonstrates a novel mechanism of latent infection by HCV. Similarly, fluctuation in HCV proliferation was observed during the prolonged culture of 3-D-HuS-E/2 cells infected with bbHCV (54); this fluctuation was associated with a change in viral quasispecies, suggesting that an HCV strain having a growth advantage proliferates selectively and dominantly in these culture conditions. Because the progressive emergence of each dominant strain was only temporary, it is highly likely that the infection and proliferation of such an HCV strain is suppressed by cellular mechanism(s). Our results showed two cellular mechanisms functioning to do this. The first is the involvement of the innate immune system, as evidenced by the secretion of IFN-α during the first week of infection. The second mechanism is HCV-induced apoptosis. Although HCV-induced apoptosis was not found when HCV-1b was used for infection, it was found in all cases where HCV-2a was used, suggesting a higher cytopathic tendency of the HCV-2a genotype.
Mouse cells permissible to HCV infection
The development of prophylaxis and novel therapeutics to treat HCV infection has been hampered by the lack of suitable animal models, a deficit resulting from the limited species tropism of HCV. Chimpanzees are the only available immunocompetent in vivo experimental system, but their use is limited by ethical concerns, restricted availability and prohibitively high costs (59).
A convenient small-animal model supporting the HCV life cycle could significantly accelerate the preclinical testing of vaccine and drug candidates, as well as facilitate in vivo studies of HCV pathogenesis. A murine model was described in which overexpression of a uPA transgene resulted not only in neonatal bleeding disorders, but also in severe liver toxicity (60). Importantly, the diseased liver could be replaced by donor hepatocytes of murine origin, as well as by hepatocytes from rats, woodchucks, and humans once the uPA transgenic mice were backcrossed on an immunodeficient background. Mice with chimeric human livers that were inoculated with serum from HCV-positive donors developed prolonged HCV infections with high viral titers and evidence for active replication of the virus in chimeric human livers (61). At present, the chimeric human liver uPA/SCID mouse model is physiologically closest to a natural human infection and therefore represents the most successful small-animal model for HCV infection. Several shortcomings, however, limit its widespread use and application. Most importantly, the immunodeficiency required to allow successful xenotransplantation precludes studies on the adaptive immune response, immunopathology, and active immunization strategies (vaccine development). Second, only a few laboratories have reported successful generation of these chimeras, because this model requires high-quality human donor hepatocytes and the actual transplantation is difficult to carry out in small animals with a tendency to bleed. Finally, the efficacy of human hepatocyte engraftment is highly variable in these animals, ranging from approximately 2% to 92% after additional treatment with an antibody to asialo- GM-1 (62).
The successful establishment of the HCV life cycle in mouse hepatocytes is another tempting alternative to overcome these problems. In addition to missing or incompatible positive regulators of HCV replication, dominant-negative restriction factors might be present in mouse hepatocytes. Altered or exacerbated innate antiviral responses, the inability of HCV proteins to overcome murine defenses, or mouse-specific restriction factors similar to those that control retroviral infection, such as Fv1, TRIM5α or APOBEC3 cytidine deaminases, could impair HCV replication in mouse cells.
In mammalian cells, the host detects and responds to infection by RNA-viruses, including HCV, by primarily recognizing viral RNA through several distinct pathogen recognition receptors (PRR), including the cell surface and endosomal RNA sensors TLR3 and TLR7, and the cytoplasmic RNA sensors RIG-I and MDA5 (Fig. 5) (63). The detection of virus infection by these receptors leads to the induction of IFN and their downstream IFN-inducible anti-viral genes through distinct signaling pathways (64). Type I IFN is an important regulator of viral infections in the innate immune system (65). Another type of IFN, IFN-lambda, affects the prognosis of HCV infection, and its response to antiviral therapy (66,67). Variations in the type or intensity of the antiviral response between hosts are known to restrict the tropism of certain viruses, such as myxoma virus, which is only permissive in mouse cells that have impaired IFN responses. Similarly, we previously reported that the impairment of IRF-7, and suppression of the interferon response improved HCV replication in immortalized primary human hepatocytes. (35)
Mutations impairing the function of the RIG-I gene and the induction of IFN were essential in establishing HCV infectivity in human HuH-7.5 cells (68). Similarly, the HCV-NS3/4a protease is known to cleave the IPS-1 adaptor molecule, inducing further downstream blocking of the IFN-inducing signaling pathway (69). These data clearly demonstrate that the host RIG-I pathway is crucial for suppressing HCV proliferation in human hepatocytes. Using a similar strategy, we investigated whether suppressing the antiviral host innate immune system conferred any advantage on HCV proliferation in mouse hepatocytes (70). We examined the possibility of HCV replication in mice lacking the expression of key factors that modulate the type I IFN-inducing pathways (Fig. 6). Only gene silencing of IFNAR or IPS-1 was sufficient to establish spontaneous HCV replication in mouse hepatocytes.
To establish a cell line permissive for HCV replication, which is required for further in vitro studies of the HCV life cycle in mouse hepatocytes, we immortalized IFNAR- and IPS-1-ko mice hepatocytes with SV40 T antigen. Upon expression of the human (h)CD81 gene, these newly established cell lines were able to support HCV infection and replication for the first time in mouse hepatocytes. Using these cell lines, we demonstrated that the suppression of IPS-1 enhances HCV infection and replication in mouse hepatocytes through the suppression of both IFN induction and an IFN-independent J6JFH1-induced cytopathic effect. We also showed for the first time the importance of the HCV structural region for viral replication, as JFH1 chimera containing the J6 structure region showed a privilege for spontaneous replication over full-length JFH1 or the subgenomic JFH1 replicon. IRF-3-ko MEF were previously shown to support HCV replication more efficiently than wild MEF (71). As the knockout of IPS-1 mainly suppresses signaling in response to virus RNA detection, and maintains an intact IFN response and induction to other stimulants, it may result in minimum interference to adaptive immune responses as compared to IRF-3 or IFNAR-ko.
We have established an in vitro culture system that can support the entire life cycle of a variety of HCV isolates and genotypes. Although this in vitro model system may not completely reproduce the in vivo situation, we believe it is the first in vitro system showing HCV strain-dependent virus/cell interaction including induction of cellular apoptosis and/or evasion from the cellular innate immune response, which may make it a good tool for the analysis of virus/host interaction, together with the development of new anti-HCV strategies for the different bbHCV strains. We have also established hepatocyte lines from IPS-1-ko mice that support HCV replication and infection. These cell lines will be very useful in identifying other species’ restriction factors and viral determinants required for the further establishment of a robust and efficient HCV life cycle in mouse hepatocytes. Further development of hCD81-transgenic IPS-1-ko mice may serve as a good model for the study of immunological responses against HCV infection. This mouse model can be used as a backbone for any further future models supporting robust HCV infectivity for the study of HCV pathogenesis, propagation and vaccine development.
The author wish to thank Dr. Jin Takahashi from the Japanese NIH, NIID for his help designing figure 5 in this paper.
The authors declare no financial or commercial conflict of interest.