Cell culture models
Once potent ligands for a viral protein are identified, further advancement depends on demonstrating activity in cells. Unfortunately, reproducible in vitro viral replication assays for HCV have not been reported. There are scattered reports that a very low level of genome replication, or even virus production, can be observed under certain circumstances . However, recently specific sequences yielding relatively reproducible replication, at consistently detectable levels have been reported . In the coming years these may allow routine assays suitable for compound evaluation to be developed, but to date drug discovery must rely on other cell culture models.
The protease is uniquely suited for the development of surrogate cellular assays. Several variations of these assays have been developed in which the protease cleaves a fusion protein at an engineered NS3-specific cleavage site, directly activating a reporter gene such as secreted alkaline phosphatase (SEAP) or releasing a transcription factor which can subsequently direct reporter gene expression [58-61]. Detection of engineered target proteins by
Western blot has also been reported [62, 63]. These assays have been widely used over the last several years but have recently been largely supplanted by the subgenomic replicon.
An important advance in HCV research was the report in 1999 that subgenomic constructs encoding only the nonstructural proteins together with an antibiotic resistance gene, flanked by authentic 5' and 3' sequences (Figure 2.1B), could replicate stably in the Huh-7 cell line isolated from human liver . While monocistronic systems do exist, most replicons are bicistronic; that is, they encode two protein chains, with translation of the resistance gene directed by the HCV IRES and translation of the HCV non-structural region directed by the encephalomyocarditis virus (EMCV) IRES . Replicon cell lines are typically generated by transfection of the subgenomic RNA into Huh-7 human liver cells and selection for colonies, which are able to grow in the presence of the antibiotic neomycin. The first replicons were generated using a consensus genotype 1b sequence  or a genotype 1a strain [65, 66]. Isolation of stable replicon cell lines was at first a long and very inefficient process, but it was soon found that the HCV sequences in stable replicon-containing cell lines had mutations, which significantly improved efficiency of replication [56, 66]. Fresh cells transfected with adapted sequences yield colonies at a frequency of 0.2-10%, whereas the original genotype 1b sequence yielded only 0.0005% . The adaptive mutations map principally to the NS3 helicase domain and the NS5A sequence , but the mechanism by which they improve replication efficiency is still being investigated. With adapted replicons, replication efficiency is high enough so that assays do not need to be performed with stable replicon cell lines. Huh-7 cells can be transfected and the level of replication in the cell population evaluated after 24-96 h [67, 68].
Interestingly, it has been found that although a full-length genome containing the original genotype 1b replicon sequence was infectious in chimpanzees, sequences containing the adaptive mutations were not . Thus, although the subgenomic replicon is believed to carry out many authentic functions of the HCV life cycle, there are significant differences in the constraints on sequences for the cell culture model and authentic infections. Very recently, a genotype 2a replicon was reported . Since genotype 2 differs significantly in sequence from genotype 1, comparing the properties of this new replicon to the established genotype 1 replicons should yield new insights into HCV replication.
Academic research using the replicon has yielded a number of insights into the biology of HCV, but it has also been widely adopted for pharmaceutical research. Compounds can by tested in 96- or 384-well plate format. RNA copy numbers can be accurately quantified using real-time polymerase chain reaction (PCR). This procedure requires that RNA be isolated from cells at the completion of the assay, and is time-consuming and expensive. Alternative detection procedures have been described in which the level of NS3 protease activity is detected after compound incubation and cell lysis , or in which the concentration of the neomycin resistance protein is quantified by (ELISA) . Recently, replicons further engineered to contain a luciferase reporter gene in the first reading frame have been developed . These allow the level of replication to be determined using a simple luminescence readout, significantly increasing throughput of the assay. With these improvements the replicon is not only used as the cell culture assay for inhibitors of defined enzymatic targets, but can also be the subject of high-throughput screening, to yield compounds which appear to target proteins of poorly understood function such as NS5A .
Besides humans, the only known host for HCV is the chimpanzee. A critical early development in HCV research was the discovery that specific cloned RNA genomes could be used to infect chimpanzees [74, 75]. Genomes containing point mutations in nonstructural proteins, including the NS3 protease, which abrogated activity were noninfectious, validating these proteins as essential viral targets . However, for ethical as well as practical reasons the chimpanzee cannot be used routinely to test candidate drugs. To overcome this limitation, two quite elaborate animal models have been developed which use infected human liver cells grafted into severe combined immunodeficiency (SCID) mice. In the Trimera model, mice first receive lethal doses of radiation and are then given bone marrow transplants and grafts of infected human liver tissue . The KMT Hepatech model uses SCID mice which express a urokinase-type plasminogen activator transgene in the liver, resulting in death of most mouse liver cells . Their survival then depends on engrafted human liver cells. Replicating virus can be detected in both models, and they have further been validated using candidate antivirals: a small molecule directed against the IRES and an antibody directed against E2 in the case of the Trimera model  and interferon-a  and the protease inhibitor BILN 2061  in the KMT model. However, both models use immunodeficient animals, and the duration and severity of infection is not well controlled. Thus many questions regarding the kinetics of viral infection or the interaction of the virus and immune system cannot be addressed. In the future, these models may prove useful in helping to validate agents directed against non-traditional targets, such as the poorly understood NS4B or NS5A, but they are not believed to be critical for the advancement of protease or polymerase inhibitors. In some cases, candidate HCV drugs have been tested in primates infected with related viruses such as GVB-B .
A protease-specific model has also been reported in which a replication-defective adenovirus encoding an NS3 protease-SEAP fusion protein is injected into mouse tail veins, resulting in expression of the fusion protein in the liver [82, 83]. Protease activity can be detected both by measuring activity of liberated SEAP or by protease-induced liver damage. Protease activity was found to be reduced by administration of protease inhibitors. This model can be used to show that candidate inhibitors have adequate pharmacokinetic properties in mice to function in the intended target organ, but it is not a true disease model.
A possible limitation of these mouse models is suggested by the recently recognized fact that, in addition to the liver, HCV does replicate in other cell types, including peripheral blood mononuclear cells (PBMCs) and dendritic cells [84-87]. In fact, the quasispecies distribution in non-liver cells is different, suggesting that the virus evolves distinct populations for each cell type [23, 88]. Elimination of virus from these non-liver "reservoirs" could possibly be the key to achieving a sustained viral response. In the long-term, full understanding of HCV will require a model in which the full virus life cycle can be studied in an immuno-competent animal. Alternatively, more intensive study of related animal viruses such as GBV-B may yield insights into the viral life cycle, which could be applied to HCV.
As has been observed with inhibitors of HIV, resistance to anti-HCV drugs is expected when these agents are tested in the clinic. The replicon can be used to identify and characterize potential resistance mutations. Assays to measure inhibition of replication are normally run for 24-72 h, but if cells are cultured for a longer time in the presence of high concentrations of inhibitor, colonies eventually emerge which can grow efficiently. RNA isolated from these colonies will usually contain mutations in the gene for the targeted protein. For example, several independent studies with the protease inhibitor BILN 2061, or a close analogue, have identified resistance mutations in the protease active site (vide infra). Since each isolated sequence usually contains additional mutations due to natural drift of the sequence on long-term culture, resistance is confirmed by engineering the specific resistance mutation into the initial sequence and confirming the decreased sensitivity to the inhibitor for the resulting replicon, as well as for the isolated enzyme containing the same mutation. However, the replicon is under fewer constraints than the full virus sequence, since it only models a part of the viral life cycle. Viruses encoding replicon resistance mutations may not be viable. Sequences encoding resistance mutations have not yet been used to infect chimpanzees, and it will probably be only after long-term clinical trials that authentic resistance mutations can be identified and compared to those that arise in replicon studies. It is encouraging that treatment with sufficiently high concentrations of protease inhibitors or interferon  appears to completely suppress the emergence of resistance and in fact results in "curing" cells of the replicon, such that no HCV replication can be detected even after removal of the drug.
The replicon can also be used to evaluate combinations of inhibitors. Additivity or slight synergies have been reported for interferon-protease inhibitor [90-92], interferon-polymerase inhibitor , and protease-polymerase inhibitor  combinations. Additional advantages are found on longer-term treatment with drug combinations, since as found for HIV these combinations very effectively prevent the emergence of resistance [91, 92]. Any colonies that do form must have sequences encoding separate mutations for both drugs in the combination .
INHIBITORS OF THE NS3/4A SERINE PROTEASE
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