Hepatitis C Virus Subgenomic Replicon Research Articles

Divergent activities of interferon-alpha subtypes against intracellular hepatitis C virus replication

Interferon (IFN)-alpha is represented by several structurally related subtypes that show different antiviral and anti-tumor effects. Here, we analyzed differential effects of IFN-alpha subtypes on intracellular hepatitis C virus (HCV) replication using HCV subgenomic replicon system as a model. Huh7 and HeLa cells supporting expression of HCV replicon were treated with various concentrations of five recombinant human IFN-alpha subtypes 1, 2, 5, 8, and 10, and with IFN-alpha con1. The effects of IFNs on various cell-signaling pathways were assayed by using ISRE-, GAS-, AP1-, NF-kappa B-, CRE-, and SRE-luciferase reporter plasmids. Each IFN-alpha subtype suppressed HCV replication in a dose-dependent manner. Among them, IFN-alpha8 was the most effective, while IFN-alpha1 was the least effective with 50% inhibitory concentrations of 0.123IU/ml versus 0.375IU/ml, respectively. These differential effects against HCV replication did not correlate with levels of the IFN-responsive ISRE or GAS reporter activities, nor they did activate the other reporters, AP1, NF-kappa B, CRE and SRE. There were divergent effects of IFN-alpha subtypes against HCV replication that may be through JAK-STAT-independent pathways. Exploring further mechanisms of action may elucidate IFN-mediated cellular antiviral mechanisms.

Activation of Vγ9Vδ2 T cells by non-peptidic antigens induces the inhibition of subgenomic HCV replication

Hepatitis C virus (HCV) has evolved complex strategies to evade host immune responses and establish chronic infection. Since human Vγ9Vδ2 T lymphocytes play a critical role in the immune response against viruses, we analyzed their antiviral functions on Huh7 hepatoma cells carrying the subgenomic HCV replicon (Rep60 cells). In a transwell culture system, Rep60 cells were co-cultured with either PBMCs or highly purified γδ T cells stimulated by non-peptidic antigens. Vγ9Vδ2 T cell activation was associated with a dramatic reduction of HCV RNA levels. Neutralizing antibodies targeting IFN-γ revealed a critical role for this cytokine in the inhibition of HCV replication. Interestingly, drugs already in clinical use, such as Phosphostim and Zoledronate, known to activate γδ T cells, were shown to induce the inhibition of HCV replication mediated by Vγ9Vδ2 T cells of HCV patients. Our data suggest that the therapeutic activation of Vγ9Vδ2 T lymphocytes may represent an additional strategy to inhibit HCV replication and to restore a Th1-oriented immune response in HCV-infected patients.

Hepatitis C Virus Nonstructural Proteins Inhibit Apolipoprotein B100 Secretion

Host genes involved in lipid metabolism are differentially regulated during the early stages of hepatitis C virus (HCV) infection. The majority of lipids synthesized in the liver are exported to other tissues in the form of lipoproteins. The formation of these lipoproteins is dependent upon the association of triglycerides with apolipoprotein B100. Using the HCV subgenomic replicon expression system, we show that secretion of apoB100 is significantly reduced. Inhibition of apoB100 degradation by ALLN did not improve secretion. Triglyceride levels as well as microsomal triglyceride transfer protein mRNA and activity levels were reduced in replicon-expressing cells, indicating potential reasons for the observed decrease. Further evidence is presented for the interaction between the HCV nonstructural protein 5A and apoB100. These results provide further insight into the alteration of lipid metabolism by HCV.

Functional Analysis of RNA Binding by the Hepatitis C Virus RNA-dependent RNA Polymerase

Protein-RNA interaction plays a critical role in regulating RNA synthesis by the hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp). RNAs of 7 nucleotides (nt) or longer had affinities 5-fold better than an RNA of 5 nt, suggesting a minimal length required for binding. To identify RNA contact sites on the HCV RdRp, a biotinylated 7-nt RNA capable of directing de novo initiation was used in a process that coupled reversible formaldehyde cross-linking, RNA affinity chromatography, and mass spectrometry. By this process, we identified 18 peptides cross-linked to the 7-nt RNA. When these identified peptides were overlaid on the three-dimensional structures of NS5B, most mapped to the fingers subdomain, connecting loops between fingers and thumb subdomains and in the putative RNA binding channel. Two of the identified peptides resided in the active site cavity of the RdRp. Recombinant HCV RdRp with single residue changes in likely RNA contact sites were generated and characterized for effects on HCV RdRp activity. Mutant proteins had significant effects on cross-linking to 7-nt RNA and reduced RNA synthesis in vitro by 2- to 20-fold compared with wild type protein. When the mutations were tested for the replication of HCV RNA in the context of the cells transfected with the HCV subgenomic replicon, all except one prevented colony formation, indicating a defect in HCV RNA replication. These biochemical and functional analyses identified a number of residues in the HCV RdRp that are important for HCV RNA synthesis.

In Vitro Studies of Cross-resistance Mutations against Two Hepatitis C Virus Serine Protease Inhibitors, VX-950 and BILN 2061

VX-950 is a potent, small molecule, peptidomimetic inhibitor of the hepatitis C virus (HCV) NS3.4A serine protease and has recently been shown to possess antiviral activity in a phase I trial in patients chronically infected with genotype 1 HCV. In a previous study, we described in vitro resistance mutations against either VX-950 or another HCV NS3.4A protease inhibitor, BILN 2061. Single amino acid substitutions that conferred drug resistance (distinct for either inhibitor) were identified in the HCV NS3 serine protease domain. The dominant VX-950-resistant mutant (A156S) remains sensitive to BILN 2061. The major BILN 2061-resistant mutants (D168V and D168A) are fully susceptible to VX-950. Modeling analysis suggested that there are different mechanisms of resistance for these mutations induced by VX-950 or BILN 2061. In this study, we identified mutants that are cross-resistant to both HCV protease inhibitors. The cross-resistance conferred by substitution of Ala(156) with either Val or Thr was confirmed by characterization of the purified enzymes and reconstituted replicon cells containing the single amino acid substitution A156V or A156T. Both cross-resistance mutations (A156V and A156T) displayed significantly diminished fitness (or replication capacity) in a transient replicon cell system.

Host sphingolipid biosynthesis as a target for hepatitis C virus therapy

An estimated 170 million individuals worldwide are infected with hepatitis C virus (HCV), a serious cause of chronic liver disease. Current interferon-based therapy for treating HCV infection has an unsatisfactory cure rate, and the development of more efficient drugs is needed. During the early stages of HCV infections, various host genes are differentially regulated, and it is possible that inhibition of host proteins affords a therapeutic strategy for treatment of HCV infection. Using an HCV subgenomic replicon cell culture system, here we have identified, from a secondary fungal metabolite, a lipophilic long-chain base compound, NA255 (1), a previously unknown small-molecule HCV replication inhibitor. NA255 prevents the de novo synthesis of sphingolipids, major lipid raft components, thereby inhibiting serine palmitoyltransferase, and it disrupts the association among HCV nonstructural (NS) viral proteins on the lipid rafts. Furthermore, we found that NS5B protein has a sphingolipid-binding motif in its molecular structure and that the domain was able to directly interact with sphingomyelin. Thus, NA255 is a new anti-HCV replication inhibitor that targets host lipid rafts, suggesting that inhibition of sphingolipid metabolism may provide a new therapeutic strategy for treatment of HCV infection.

Quantitative Analysis of the Hepatitis C Virus Replication Complex

The hepatitis C virus (HCV) encodes a large polyprotein; therefore, all viral proteins are produced in equimolar amounts regardless of their function. The aim of our study was to determine the ratio of nonstructural proteins to RNA that is required for HCV RNA replication. We analyzed Huh-7 cells harboring full-length HCV genomes or subgenomic replicons and found in all cases a >1,000-fold excess of HCV proteins over positive- and negative-strand RNA. To examine whether all nonstructural protein copies are involved in RNA synthesis, we isolated active HCV replication complexes from replicon cells and examined them for their content of viral RNA and proteins before and after treatment with protease and/or nuclease. In vitro replicase activity, as well as almost the entire negative- and positive-strand RNA, was resistant to nuclease treatment, whereas <5% of the nonstructural proteins were protected from protease digest but accounted for the full in vitro replicase activity. In consequence, only a minor fraction of the HCV nonstructural proteins was actively involved in RNA synthesis at a given time point but, due to the high amounts present in replicon cells, still representing a huge excess compared to the viral RNA. Based on the comparison of nuclease-resistant viral RNA to protease-resistant viral proteins, we estimate that an active HCV replicase complex consists of one negative-strand RNA, two to ten positive-strand RNAs, and several hundred nonstructural protein copies, which might be required as structural components of the vesicular compartments that are the site of HCV replication.

Mutations Conferring Resistance to a Hepatitis C Virus (HCV) RNA-Dependent RNA Polymerase Inhibitor Alone or in Combination with an HCV Serine Protease Inhibitor In Vitro

Compounds A-782759 (an N-1-aza-4-hydroxyquinolone benzothiadiazine) and BILN-2061 are specific anti-hepatitis C virus (HCV) agents that inhibit the RNA-dependent RNA polymerase and the NS3 serine protease, respectively. Both compounds display potent activity against HCV replicons in tissue culture. In order to characterize the development of resistance to these anti-HCV agents, HCV subgenomic 1b-N replicon cells were cultured with A-782759 alone or in combination with BILN-2061 at concentrations 10 times above their corresponding 50% inhibitory concentrations in the presence of neomycin. Single substitutions in the NS5B polymerase gene (H95Q, N411S, M414L, M414T, or Y448H) resulted in substantial decreases in susceptibility to A-782759. Similarly, replicons containing mutations in the NS5B polymerase gene (M414L or M414T), together with single mutations in the NS3 protease gene (A156V or D168V), conferred high levels of resistance to both A-782759 and BILN-2061. However, the A-782759-resistant mutants remained susceptible to nucleoside and two other classes of nonnucleoside NS5B polymerase inhibitors, as well as interferon. In addition, we found that the frequency of replicons resistant to both compounds was significantly lower than the frequency of resistance to the single compound. Furthermore, the dually resistant mutants displayed significantly reduced replication capacities compared to the wild-type replicon. These findings provide strategic guidance for the future treatment of HCV infection.

Synthesis and anti-viral activity of a series of sesquiterpene lactones and analogues in the subgenomic HCV replicon system

Hepatitis C virus (HCV) infection is a severe liver disease that often leads to liver cirrhosis and hepatocellular carcinoma (HCC). Current therapy is inadequate to conquer this viral disease. In this study, we identified parthenolide (1), an active component in feverfew, a popular remedy for fever and migraine, as a lead compound with an EC50 value of 2.21μM against HCV replication in a subgenomic RNA replicon assay system. Parthenolide is able to potentiate the interferon α-exerted anti-HCV effect. Several commercially available sesquiterpene lactones (2–5) structurally analogous to parthenolide and a series of synthesized Michael-type adducts of parthenolide (12–18) also exhibit micromolar concentrations for anti-HCV activities. Structure–activity relationship was elucidated to reveal that the spatial arrangement of the terpenoid skeleton fused with an α-methylene-γ-lactone moiety produces maximal anti-HCV activity. In addition, a strong anti-HCV potency indicates a possibility of secondary amino adducts (12–18) converting back to parthenolide or being replaced by the nucleophilic residues of proteins inside cells. This work shows that screening of natural products is a viable and fast way for identifying novel molecular diversity as potential drug leads.

Review HCV Antiviral Resistance: The Impact of in vitro Studies on the Development of Antiviral Agents Targeting the Viral NS5B Polymerase

The high prevalence of the disease caused by hepatitis C virus (HCV) and the limited efficacy of interferon-based therapies have stimulated the search for safer and more effective drugs. The development of inhibitors of the HCV NS5B RNA polymerase represents a promising strategy for identifying novel anti-HCV therapeutics. However, the high genetic diversity, mutation rate and turnover of HCV are expected to favour the emergence of drug resistance, limiting the clinical usefulness of polymerase inhibitors. Thus, the characterization of the drug-resistance profile of these antiviral agents is considered crucial for identifying the inhibitors with a higher probability of clinical success. In the absence of an efficient in vitro infection system, HCV sub-genomic replicons have been used to study viral resistance to both nucleoside and non-nucleoside NS5B inhibitors. While these studies suggest that drug-resistant viruses are likely to evolve in vivo, they provide a wealth of information that should help in the identification of inhibitors with improved and distinct resistance profiles that might be used for combination therapy.

Hepatitis C virus NS3 protein interacts with ELKS-δ and ELKS-α, members of a novel protein family involved in intracellular transport and secretory pathways

The NS3 protein of hepatitis C virus (HCV) has a serine protease activity in its N-terminal region, which plays a crucial role in virus replication. This region has also been reported to interact not only with its viral cofactor NS4A, but also with a number of host-cell proteins, which suggests a multifunctional feature of NS3. By means of yeast two-hybrid screening using an N-terminal region of NS3 as bait, a human cDNA encoding a region of ELKS-delta, a member of a novel family of proteins involved in intracellular transport and secretory pathways, was molecularly cloned. Using co-immunoprecipitation, GST pull-down and confocal and immunoelectron microscopic analyses, it was shown that full-length NS3 interacted physically with full-length ELKS-delta and its splice variant, ELKS-alpha, both in the absence and presence of NS4A, in cultured human cells, including Huh-7 cells harbouring an HCV subgenomic RNA replicon. The degree of binding to ELKS-delta varied with different sequences of the N-terminal 180 residues of NS3. Interestingly, NS3, either full-length or N-terminal fragments, enhanced secretion of secreted alkaline phosphatase (SEAP) from the cells, and the increase in SEAP secretion correlated well with the degree of binding between NS3 and ELKS-delta. Taken together, these results suggest the possibility that NS3 plays a role in modulating host-cell functions such as intracellular transport and secretion through its binding to ELKS-delta and ELKS-alpha, which may facilitate the virus life cycle and/or mediate the pathogenesis of HCV.

Screening for hepatitis C virus antiviral activity with a cell-based secreted alkaline phosphatase reporter replicon system

We describe a phased screening system for discovery of compounds with antiviral activity against hepatitis C virus (HCV). The primary assay utilizes dicistronic subgenomic HCV replicons in which the upstream cistron was modified to express the human immunodeficiency virus (HIV) tat protein. When these replicons are stably transfected into Huh-7-derived cells that express secreted alkaline phosphatase (SEAP) under transcriptional control of the HIV long terminal repeat promoter, there is a strong correlation between intracellular HCV RNA abundance and the activity of SEAP secreted into the culture medium. Thus, active compounds are easily identified by direct enzymatic quantification of SEAP in the medium without post-assay processing. Compounds that reduce SEAP activity without causing cellular toxicity are next evaluated in a second Huh-7-derived cell line constitutively expressing SEAP under control of the tat-HIV promoter axis, independent of HCV RNA replication. This specificity control identifies compounds that cause reductions in SEAP that are unrelated to suppression of HCV RNA replication. Compounds showing HCV-specific activity in primary assays are next evaluated by real-time RT-PCR to directly quantify reductions in HCV RNA. We have found excellent agreement between the SEAP and RT-PCR assays. This phased system provides an efficient and cost-effective screen for compounds with antiviral activity against HCV.

Inhibition of hepatitis C virus translation and subgenomic replication by siRNAs directed against highly conserved HCV sequence and cellular HCV cofactors

Small interfering RNAs (siRNAs) are an efficient tool to specifically inhibit gene expression by RNA interference. Since hepatitis C virus (HCV) replicates in the cytoplasm of liver cells without integration into the host genome, RNA-directed antiviral strategies are likely to successfully block the HCV replication cycle. Additional benefit might arise from inhibition of cellular cofactors of HCV replication, such as proteasome alpha-subunit 7 (PSMA7) or Hu antigen R (HuR). In this study, we investigated direct and cofactor-mediated inhibition of HCV by a panel of DNA-based retroviral vectors expressing siRNAs against highly conserved HCV sequences or the putative HCV cofactors PSMA7 and HuR. Effects were determined in HCV IRES-mediated translation assays and subgenomic HCV replicon cells. PSMA7- and HuR-directed siRNAs successfully inhibited expression of the endogenous genes, and PSMA7 and HuR silencing significantly diminished HCV replicon RNA and NS5B protein levels. HCV-directed siRNAs substantially inhibited HCV IRES-mediated translation and subgenomic HCV replication. Combinations of PSMA7- and HuR-directed siRNAs with HCV-directed siRNAs revealed additive HCV RNA inhibitory effects in monocistronic replicon cells. A dual approach of direct- and cofactor-mediated inhibition of HCV replication might avoid selection of mutants and thereby become a powerful strategy against HCV.

1000. In Vitro and In Vivo Evaluation of Optimized Short-Hairpin RNAs for the Treatment of the Hepatitis C Virus Using Expression Cassettes Producing One, Two or Three Anti HCV shRNAs

Infection with the Hepatitis C virus (HCV) represents a major world-wide health problem. Current therapies consisting of small molecule pharmaceuticals with or without interferon are most often inadequate due to the generation of viral escape mutants. RNA interference (RNAi) based treatments using both short-interfering RNAs (siRNAs) and short-hairpin RNAs (shRNAs) have been proposed as new therapeutic modalities. As HCV is a chronic disease, we are interested in sustained expression of anti-HCV RNAs, and thus have focused on the expression of short-hairpin RNAs (shRNAs) that can elicit an RNAi response against the HCV genome. We aligned multiple HCV sequences and, and identified 30 potential RNAi targets. As a first step, the siRNAs corresponding to these targets were synthesized and tested in vitro against HCV-luciferase fusion reporter constructs, as well as subgenomic and full length HCV replicons. We identified 8 siRNAs that were capable of efficiently silencing the expression of luciferase-HCV fusions and of the HCV replicon. The corresponding shRNAs were cloned in expression cassettes downstream of ubiquitous promoters. We generated expression cassettes capable of expressing either 1, 2 or 3 shRNAs simultaneously. The shRNAs were evaluated in vitro for silencing of an HCV-luciferase fusion reporter, as well as subgenomic and/or full length HCV replicons in the primary hepatocyte cell line Huh7. The shRNAs that inhibited the luciferase reporter by greater than 80% in vitro, were selected for in vivo analysis. Tail vein injection of mice with single shRNA expression cassettes and the HCV-luciferase reporter resulted in up to 70% inhibition of luciferase. Analysis of shRNA expression cassettes that generate two or three shRNAs targeted to different conserved regions of the HCV genome is ongoing and will be reported on.

Nonstructural Proteins 4A and 4B of Hepatitis C Virus Transactivate the Interleukin 8 Promoter

Interleukin 8 (IL-8) is induced in many cell types by various stimuli including virus infection. It was reported that nonstructural protein 5A (NS5A) of hepatitis C virus (HCV) was involved in induction of IL-8 expression at both mRNA and protein levels in cultured human cells. In this study, we aimed to determine whether or not another HCV protein(s) transactivates the IL-8 gene expression, by means of an IL-8 promoter-driven luciferase reporter assay and measurement of endogenous IL-8 mRNA and secreted IL-8 protein levels. We observed that NS4B, and NS4A to a lesser extent, significantly transactivated the IL-8 promoter, which resulted in enhanced production of IL-8 protein. Also, the IL-8 expression was augmented in Huh-7 cells harboring an HCV subgenomic RNA replicon, compared with the control cells. Deletion mutational analysis of the IL-8 promoter revealed the possible involvement of the transcription factor AP-1 in both NS4A- and NS4B-mediated IL-8 gene activation. In addition, the IL-8 gene activation by NS4B, but not that by NS4A, was likely to involve NF-kappaB and/or NFIL-6. The degree of the transactivation by NS4B and NS4A varied with different human cell lines, with HeLa cells showing the strongest activation followed by Huh-7 cells, and with HepG2 cells exhibiting a marginal level of activation. Taken together, our present results suggest the possibility that NS4B and NS4A play an important role in inducing the IL-8 gene expression under certain cellular conditions, which might be one of the strategies to establish persistent HCV infection.

Hepatitis C virus RNA replication is regulated by host geranylgeranylation and fatty acids

Hepatitis C virus (HCV) infection is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Our laboratory has previously demonstrated that high-level HCV replication during acute infection of chimpanzees is associated with the modulation of multiple genes involved in lipid metabolism, and that drugs that regulate cholesterol and fatty acid biosynthesis regulate the replication of the subgenomic HCV replicon in Huh-7 cells. In this article, we demonstrate that Huh-7 cells harboring replicating, full-length HCV RNAs express elevated levels of ATP citrate lyase and acetyl-CoA synthetase genes, both of which are involved in cholesterol and fatty acid biosynthesis. Further, we confirm that the cholesterol-biosynthetic pathway controls HCV RNA replication by regulating the cellular levels of geranylgeranyl pyrophosphate, we demonstrate that the impact of geranylgeranylation depends on the fatty acid content of the cell, and we show that fatty acids can either stimulate or inhibit HCV replication, depending on their degree of saturation. These results illustrate a complex cellular-regulatory network that controls HCV RNA replication, presumably by modulating the trafficking and association of cellular and/or viral proteins with cellular membranes, suggesting that pharmacologic manipulation of these pathways may have a therapeutic effect in chronic HCV infection.

Current approaches for developing new anti-HCV agents and analyses of HCV replication using anti-HCV agents

Currently, patients with hepatitis C virus (HCV) are mainly treated with interferon alone or in combination with ribavirin. However, because the virus is not eliminated from approximately one half of the patients by this treatment, alternative approaches to the treatment of HCV infection are needed. Recently, an HCV subgenomic replicon system has been established in which an HCV subgenomic replicon autonomously replicated in cultured cells. It enables us to screen for anti-HCV agents in cell culture system. Taking advantage of this system, we examined the effects of various types of compounds on the replication of HCV. Consequently, we found that a well-known immunosuppressant, cyclosporin A (CsA), had a strong suppressive activity on HCV replication, at least in cell culture system. This anti-HCV activity did not require the immunosuppressive feature of CsA. Through the investigation into the mechanism of anti-HCV effect of CsA, it was suggested that cyclophilin B, one of the cellular target molecules of CsA, played a significant role in HCV replication. Thus, searching for anti-HCV agents may lead to the elucidation of one of the mechanisms of HCV replication.

Hepatitis C virus non-structural protein NS3 interacts with LMP7, a component of the immunoproteasome, and affects its proteasome activity.

NS3, a non-structural protein of the HCV (hepatitis C virus), contains a protease and a helicase domain and plays essential roles in the processing of the viral polyprotein, viral RNA replication and translation. LMP7 (low-molecular-mass protein 7), a component of the immunoproteasome, was identified as an NS3-binding protein from yeast two-hybrid screens, and this interaction was confirmed by in vitro binding and co-immunoprecipitation analysis. The minimal domain of interaction was defined to be between the pro-sequence region of LMP7 (amino acids 1-40) and the protease domain of NS3. To elucidate the biological importance of this interaction, we studied the effect of this interaction on NS3 protease activity and on LMP7 immunoproteasome activity. Recombinant LMP7 did not have any effect on NS3 protease activity in vitro. The peptidase activities of LMP7 immunoproteasomes, however, were markedly reduced when tested in a stable cell line containing a HCV subgenomic replicon. The down-regulation of proteasome peptidase activities could interfere with the processing of viral antigens for presentation by MHC class I molecules, and may thus protect HCV from host immune surveillance mechanisms to allow persistent infection by the virus.

Novel Nonnucleoside Inhibitor of Hepatitis C Virus RNA-Dependent RNA Polymerase

A novel nonnucleoside inhibitor of hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), [(1R)-5-cyano-8-methyl-1-propyl-1,3,4,9-tetrahydropyano[3,4-b]indol-1-yl] acetic acid (HCV-371), was discovered through high-throughput screening followed by chemical optimization. HCV-371 displayed broad inhibitory activities against the NS5B RdRp enzyme, with 50% inhibitory concentrations ranging from 0.3 to 1.8 microM for 90% of the isolates derived from HCV genotypes 1a, 1b, and 3a. HCV-371 showed no inhibitory activity against a panel of human polymerases, including mitochondrial DNA polymerase gamma, and other unrelated viral polymerases, demonstrating its specificity for the HCV polymerase. A single administration of HCV-371 to cells containing the HCV subgenomic replicon for 3 days resulted in a dose-dependent reduction of the steady-state levels of viral RNA and protein. Multiple treatments with HCV-371 for 16 days led to a >3-log10 reduction in the HCV RNA level. In comparison, multiple treatments with a similar inhibitory dose of alpha interferon resulted in a 2-log10 reduction of the viral RNA level. In addition, treatment of cells with a combination of HCV-371 and pegylated alpha interferon resulted in an additive antiviral activity. Within the effective antiviral concentrations of HCV-371, there was no effect on cell viability and metabolism. The intracellular antiviral specificity of HCV-371 was demonstrated by its lack of activity in cells infected with several DNA or RNA viruses. Fluorescence binding studies show that HCV-371 binds the NS5B with an apparent dissociation constant of 150 nM, leading to high selectivity and lack of cytotoxicity in the antiviral assays.

Suppression of hepatitis C virus replicon by TGF-β

Hepatitis C virus (HCV) is one of the major causative agents of liver diseases, such as liver inflammation, fibrosis, cirrhosis, and hepatocellular carcinoma. Using an efficient HCV subgenomic replicon system, we demonstrate that transforming growth factor-beta (TGF-β) suppresses viral RNA replication and protein expression from the HCV replicon. We further show that the anti-viral effect of this cytokine is associated with cellular growth arrest in a manner dependent on Smad signaling, not mitogen-activated protein kinase (MAPK) signaling. These results suggest a novel insight into the mechanisms of liver diseases caused by HCV.