HCV Replicon System Research Articles

Emerging Therapeutic Strategies for Hepatitis C Virus Infection

The universal prevalence of hepatitis C virus (HCV) infection, which causes chronic hepatitis, cirrhosis, liver failure, and hepatocellular carcinoma, has become a significant health problem worldwide. Interferon-based therapies, the current standard, IFN-based therapies have limited efficacy and undesirable adverse effects. In addition, neither vaccination against HCV nor specific antiviral reagents for HCV are yet available. Thus, a major medical need still exists for novel and more efficacious anti-HCV reagents showing broad-spectrum clinical efficacy with enhanced tolerability. With the progress in our current understanding of the function and regulation of HCV gene products, the three-dimensional structures of virally encoded enzymes and the recent establishment of the HCV-replicon system, several pharmacological targets are being studied for HCV therapy, including cellular receptors mediating HCV entry, factors facilitating HCV replication and assembly, and intracellular pathways. Recently developed mouse models will be very helpful in evaluating the in vivo efficacy of novel antiviral reagents. Currently many novel antiviral drugs are under evaluation in clinical trials. This review will comprehensively discuss the current treatment options and various novel antiviral reagents available. Ongoing clinical studies of promising lead drugs are also reviewed. Keywords: Hepatitis C virus, therapy, target, inhibitor, effectiveness, safety, resistance, clinical studies

Replication of hepatitis C virus genome

The studies on the mechanism of HCV replication proliferated after the development of cell culture based-subgenomic HCV replicon system and genome-length HCV RNA replication system. Furthermore, these RNA replication systems have been improved to be suitable systems for the screening of anti-HCV reagents by the introduction of reporter genes such as luciferase. Genetic analysis of HCV RNAs obtained in long-term cell culture of HCV replicon or genome-length HCV RNA-harboring cells revealed that genetic mutations in HCV RNAs accumulated in a time-dependent manner. The genetic diversity of HCVs was also enlarged in a time-dependent manner. The appearance of adaptive mutation in HCV replicon or genome-length HCV RNA is one of characteristic features of HCV RNA replication system. Although human hepatoma-derived HuH-7 cell line was mainly used for HCV RNA replication systems, a specific combination of adaptive mutations led to develop the HCV RNA replication systems using a new human hepatoma cell line other than HuH-7.

Functional genetic screens to identify cancer relevant genes

The establishment of HCV replicon systems strongly improved the research on the replication processes but poorly advanced our knowledge on the subcellular localization of the structural glycoproteins, mainly due to their low expression. We sought to verify whether reinforcing E1E2 expression in the context of both HCV genomic and subgenomic replicon from either homologous or heterologous strains leads to formation of supramolecular structures including structural and nonstructural proteins.Robust expression of HCV glycoproteins was achieved by stable expression of E1E2p7 from genotype 1a and 1b.In these cells, E1 and E2 triggered the formation of dot-like structures in which they co-localized with core and the nonstructural proteins NS3 and NS5A. Confocal microscopy analyses suggested that accumulation of HCV proteins occurs in an ER-derived subcompartment. Moreover, by labeling de novo-synthesized HCV RNA, we showed that these structures constitute a site of viral RNA synthesis.Expression in trans of HCV glycoproteins in the context of replicative viral genome or subgenome generates accumulation of structural and nonstructural viral proteins in peculiar cytoplasmic structures. The simultaneous presence of viral RNA, structural and nonstructural protein suggests that these complexes represent not only sites of HCV replication but also potential places of viral pre-budding.

Design and evaluation of a potential mutagen for Hepatitis C virus

Pyrrolopyrimidine nucleoside 1 was designed and synthesized as a potential mutagen for HCV. An in vitro HCV NS5B enzymatic assay indicated that pyrrolopyrimidine triphosphate acts as a CTP analog rather than a UTP analog. The SATE-prodrug of pyrrolopyrimidine monophosphate showed a weak inhibitory activity in an HCV replicon system (EC 50 = 60 μM) and did not exhibit cytotoxicity (CC 50 > 100 μM). Investigation of phosphorylation events using nucleoside kinases and LC–MS analysis revealed that the second phosphorylation step, from monophosphate ester to diphosphate ester, is unfavorable.

Intracellular accumulation of hepatitis C virus proteins in a human hepatoma cell line

The establishment of HCV replicon systems strongly improved the research on the replication processes but poorly advanced our knowledge on the subcellular localization of the structural glycoproteins, mainly due to their low expression. We sought to verify whether reinforcing E1E2 expression in the context of both HCV genomic and subgenomic replicon from either homologous or heterologous strains leads to formation of supramolecular structures including structural and nonstructural proteins. Robust expression of HCV glycoproteins was achieved by stable expression of E1E2p7 from genotype 1a and 1b. In these cells, E1 and E2 triggered the formation of dot-like structures in which they co-localized with core and the nonstructural proteins NS3 and NS5A. Confocal microscopy analyses suggested that accumulation of HCV proteins occurs in an ER-derived subcompartment. Moreover, by labeling de novo-synthesized HCV RNA, we showed that these structures constitute a site of viral RNA synthesis. Expression in trans of HCV glycoproteins in the context of replicative viral genome or subgenome generates accumulation of structural and nonstructural viral proteins in peculiar cytoplasmic structures. The simultaneous presence of viral RNA, structural and nonstructural protein suggests that these complexes represent not only sites of HCV replication but also potential places of viral pre-budding.

Novel Alpha Interferon (IFN-α) Variant with Improved Inhibitory Activity against Hepatitis C Virus Genotype 1 Replication Compared to IFN-α2b Therapy in a Subgenomic Replicon System

Hepatitis C virus (HCV) treatment is based on the association of pegylated alpha interferon (IFN-alpha) and ribavirin. To improve the level of sustained virological response to treatment, especially in patients infected with HCV genotype 1, new IFNs with improved efficacy and toxicity profiles may be developed. In this report, we show that, in the BM4-5 cell line harboring an HCV subgenomic replicon, a novel and naturally occurring human IFN-alpha17 variant, GEA007.1, which was discovered by using an original population genetics-based drug discovery approach, inhibits HCV genotype 1 RNA replication more efficiently than does IFN-alpha2b. Moreover, we show that complete viral clearance is obtained in BM4-5 cells after long-term treatment with GEA007.1, while HCV subgenomic RNA is still detected in cells treated with other IFN-alpha variants or with standard IFN-alpha2b. Eventually, we demonstrate that the better inhibitory activity of GEA007.1 compared to that of standard IFN-alpha is likely to be due to stronger and faster activation of the JAK-STAT signaling pathway and to broader expression of IFN-alpha-responsive genes in cells. Our results demonstrate a superior inhibitory activity of GEA007.1 over that of IFN-alpha2b in the HCV replicon system. Clinical trials are required to determine whether GEA007.1 could be a potent "next generation" IFN for the treatment of HCV infection, especially in nonresponders or relapsing patients infected with HCV genotype 1 who currently represent a clinical unmet need.

Antiviral treatment of hepatitis C

Therapy of hepatitis C virus (HCV) infection may prevent progression to cirrhosis, hepatocellular carcinoma and end-stage liver disease. The cornerstone of treatment has long been standard IFN-α, the use of which was associated with a sustained biochemical and viral response in only a small proportion of patients. More recently, the success of interferon-based regimens has substantially improved due to the combination with the guanosine analogue ribavirin and to the advent of pegylated interferon formulations. However, even the most up-to-date regimens fail to cure the infection in many cases and are limited by side effects and high costs. A better understanding of the HCV genomic organisation, the elucidation of the three-dimensional structures of virally encoded enzymes and the recent development of a HCV-replicon system in human hepatoma (Huh-7) cells have led to significant advances in the development of new antiviral compounds, many of which are under evaluation in clinical trials. The aim of this review is to trace a brief overview of the progress made by interferon-based treatments for hepatitis C since their introduction in the early 1990s, and to highlight the results of recent clinical studies concerning new and emerging drugs.

Selective inhibitors of hepatitis C virus replication

Worldwide over 170 million people are chronically infected with the hepatitis C virus and hence at high risk to develop fatal liver disease. There is no vaccine available and the standard therapy [(pegylated) interferon alfa plus ribavirin] is only effective in 50–60% of patients and is associated with important side-effects. The discovery of novel antiviral strategies to selectively inhibit HCV replication has long been hindered by the lack of convenient cell culture models for the propagation of HCV. This hurdle has been overcome first with the establishment of the HCV replicon system in 1999 and, in 2005, with the development of robust HCV cell culture models. In recent years also mouse models have been elaborated that will be instrumental in assessing the in vivo efficacy of novel drugs. The viral serine protease and the viral RNA dependent RNA polymerase have shown to be excellent targets for selective anti-HCV therapy. Clinical studies with a limited number of HCV protease and polymerase inhibitors resulted in encouraging results. However, and not unexpected, preclinical evidence suggest that the virus may become rapidly resistant to such inhibitors. Combination therapy of drugs with different mode of action and resistance profiles may thus be required. Alternative strategies, such as the use of non-immunosuppressive cyclosporin A analogues with potent anti-HCV activity, may prove important, in particular since such compounds may have a resistance profile that is very different from that of protease or polymerase inhibitors.

Synthesis of 5‐Haloethynyl‐ and 5‐(1,2‐Dihalo)vinyluracil Nucleosides: Antiviral Activity and Cellular Toxicity.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Hepatology highlights

Hepatology highlights

Dissecting the Interferon-Induced Inhibition of Hepatitis C Virus Replication by Using a Novel Host Cell Line

The Hepatitis C virus (HCV), a member of the family Flaviviridae, is a major cause of chronic liver disease. Patients are currently treated with alpha interferon (IFN-alpha) that is given alone or in combination with ribavirin. Unfortunately, this treatment is ineffective in eliminating the virus in a large proportion of individuals. IFN-induced antiviral activities have been intensively studied in the HCV replicon system. It was found that both IFN-alpha and IFN-gamma inhibit HCV replicons, but the underlying mechanisms have not yet been identified. Of note is that nearly all of these studies were performed with the human hepatoma cell line Huh-7. Here, we report that genotypes 1b and 2a replicons also replicate in the human hepatoblastoma cell line HuH6. Similar to what has been described for Huh-7 cells, we observed that efficient HCV replication in HuH6 cells depends on the presence of cell culture-adaptive mutations and the permissiveness of the host cell. However, three major differences exist: in HuH6 cells, viral replication is (i) independent from ongoing cell proliferation, (ii) less sensitive to certain antiviral compounds, and (iii) highly resistant to IFN-gamma. The latter is not due to a general defect in IFN signaling, as IFN-gamma induces the nuclear translocation of signal transducer and activator of transcription 1 (STAT1), the enhanced transcription of several IFN-regulated genes, and the inhibition of unrelated viruses such as influenza A virus and Semliki Forest virus. Taken together, the results establish HuH6 replicon cells as a valuable tool for IFN studies and for the evaluation of antiviral compounds.

Synthesis of 5-haloethynyl- and 5-(1,2-dihalo)vinyluracil nucleosides: Antiviral activity and cellular toxicity

In this article, we report the synthesis of hitherto unknown 5-haloethynyl and 5-(1,2-dihalo)vinyluracil nucleosides in the 2′-deoxy, 3′-deoxy- and ribosyl series, and we discuss their in vitro anti-HIV and anti-HCV activities and cellular toxicitites. As a result, on the basis of their selectivity index (SI) obtained with the HCV replicon system, but also on their cytotoxicity on peripheral blood mononuclear, CEM and VERO cell lines, the best compounds were the 5-bromoethynyluridine (SI = 3.2) and the 5-(1-chloro-2-iodo)vinyluridine (SI > 2.8).

997. Loop Composition as Well as Orientation of the Guide Strand in shRNA May Dramatically Modulate Activity of the shRNA: Effect on the Development of RNAi-Based Inhibitors

RNA interference (RNAi) is a new promising technology for therapeutic drug development. It can be initiated either by chemically synthesized double-stranded RNA delivered to a cell, or by transcription of a short hairpin RNA in the cell nucleus from a DNA template. It has been realized that many RNA sequences although |[ldquo]|active|[rdquo]| as siRNAs, are often inactive as shRNAs. The composition of the loop sequence in shRNA has been suggested to play a role in determining its activity by affecting shRNA transport from the cell nucleus to the cytoplasm (Kawasaki and Taira, Nucleic Acids Res. 2003, 31(2):700-7). In the present study we identified several siRNA sequences that efficiently inhibit HCV replication in the replicon system. In subsequent attempts to generate active shRNAs spanning these validated sequences, we tested the effect of two different loop sequences, as well as the guide strand orientation on the shRNA activity. Functional tests were performed in the HCV replicon systems, as well as in a model luciferase-HCV fusion systems. We demonstrate that the context of the loop within the shRNA has a dramatic effect on shRNA activity, and a loop that is |[ldquo]|inactive|[rdquo]| in one shRNA can be fully active in the context of another shRNA. We also demonstrate that the orientation of the guide strand in the shRNAs (sense vs antisense) may dramatically influence the shRNA activity. Changes in the extent and precision of the shRNA processing by Dicer will be presented. The effect of these findings on the development of ddRNAi-based inhibitors will be discussed.

Synthesis and anti-viral activity of a series of d- and l-2′-deoxy-2′-fluororibonucleosides in the subgenomic HCV replicon system

Based on the discovery of (2′ R)- d-2′-deoxy-2′-fluorocytidine as a potent anti-hepatitis C virus (HCV) agent, a series of d- and l-2′-deoxy-2′-fluororibonucleosides with modifications at 5- and/or 4-positions were synthesized and evaluated for their in vitro activity against HCV and bovine viral diarrhea virus (BVDV). The key step in the synthesis, the introduction of 2′-fluoro group, was achieved by either fluorination of 2,2′-anhydronucleosides with hydrogen fluoride–pyridine or potassium fluoride, or a fluorination of arabinonucleosides with DAST. Among the 27 analogues synthesized, only the 5-fluoro compound, namely (2′ R)- d-2′-deoxy-2′,5-difluorocytidine ( 13), demonstrated potent anti-HCV activity and toxicity to ribosomal RNA. The replacement of the 4-amino group with a thiol group resulted in the loss of activity, while the 4-methylthio substituted analogue ( 25) exhibited inhibition of ribosomal RNA. As N 4-hydroxycytidine (NHC) had previously shown potent anti-HCV activity, we combined the two functionalities of the N 4-hydroxyl and the 2′-fluoro into one molecule, resulting (2′ R)- d-2′-deoxy-2′-fluoro- N 4-hydroxycytidine ( 23). However, this nucleoside showed neither anti-HCV activity nor toxicity. All the l-forms of the analogues were devoid of anti-HCV activity. None of the compounds showed anti-BVDV activity, suggesting that the BVDV system cannot always predict anti-HCV activity.

Effect of interaction between hepatitis C virus NS5A and NS5B on hepatitis C virus RNA replication with the hepatitis C virus replicon.

Hepatitis C virus (HCV) NS5A has been reported to be important for the establishment of replication by adaptive mutations or localization, although its role in viral replication remains unclear. It was previously reported that NS5A interacts with NS5B via two regions of NS5A in the isolate JK-1 and modulates the activity of NS5B RdRp (Y. Shirota et al., J. Biol. Chem., 277:11149-11155, 2002), but the biological significance of this interaction has not been determined. In this study, we addressed the effect of this interaction on HCV RNA replication with an HCV replicon system derived from the isolate M1LE (H. Kishine et al., Biochem. Biophys. Res. Commun., 293:993-999, 2002). We constructed three internal deletion mutants, M1LE/5Adel-1 and M1LE/5Adel-2, each encoding NS5A which cannot bind NS5B, and M1LE/5Adel-3, encoding NS5A that can bind NS5B. After transfection into Huh-7 cells, M1LE/5Adel-3 was replication competent, but both M1LE/5Adel-1 and M1LE/5Adel-2 were not. Next we prepared 20 alanine-substituted clustered mutants within both NS5B-binding regions and examined the effect of these mutants on HCV RNA replication. Only 5 of the 20 mutants were replication competent. Subsequently, we introduced a point mutation, S225P, a deletion of S229, or S232I into NS5A and prepared cured Huh-7 cells that were cured of RNA replication by alpha interferon. Finally, with these point mutations and cured cells, we established a highly improved replicon system. In this system, only the same five mutants were replication competent. These results strongly suggest that the interaction between NS5A and NS5B is critical for HCV RNA replication in the HCV replicon system.

Application of the trak-C™ HCV core assay for monitoring antiviral activity in HCV replication systems

The Ortho ® trak-C™ immunoassay has recently established detection of the HCV core antigen as a viable indirect marker of HCV replication in clinical samples. In this study, trak-C™ is used to monitor HCV replication in three pre-clinical models: the cellular HCV replicon system, transient transfection of HCV genomes, and the murine Alb-uPa/SCID HCV infection model. All of these systems utilize full-length HCV genomes that direct the expression of core, facilitating its detection with monoclonal antibodies. When performed with purified protein, the assay detects HCV core with a lower limit of detection at 1.5 pg, and exhibits linear detection up to 100 pg. When assaying extracts prepared from Huh-7 clone 21-5 cells harboring a full-length HCV replicon, core is detectable from as few as 63 cell equivalents. The assay was used to determine the sensitivity of Huh 21-5 cells to the antiviral effects of interferon (IFN). Inhibition by IFN-α using core detection was comparable to that observed using branched-DNA (bDNA 3.0) detection of HCV RNA. Replication of transfected full-length HCV 1a Con1 genomes in Huh-7 cells was also detectable using the trak-C™ assay. Finally, in the transgenic murine HCV infection model, the course of viral amplification was detected from serum using trak-C™ with kinetics similar to those observed with RNA detection. Given its ease of use and the lack of requirement for RNA purification, the trak-C™ assay has several advantages over RNA-based methods of viral monitoring.

29 Testing the antiviral activity of novel ribavirin derivatives and IFN alpha variants in the HCV replicon system

29 Testing the antiviral activity of novel ribavirin derivatives and IFN alpha variants in the HCV replicon system