Replicon Cell Lines Research Articles

Inhibition of WNT signalling interferes with HCV replication

Background and Aims: The tumor relevant wnt pathway activity is associated with higher HCV replication. HCV core and NS5 protein have recently been shown to induce wnt pathway activity, implicating a cause for progression to HCC. We now investigated if wnt pathway activity might affect HCV replication. Methods: For in vitro experiments the HCV replicon system expressing HCV genes NS3 to NS5B alone (Huh5–15) or in combination with firefly luciferase (LucUbiNeo-ET) was used. To promote wnt pathway activity the glycogen synthase kinase 3 beta (GSK3b) inhibitors Kenpaullone or SB216763 were used. Inhibition of the wnt pathway was achieved by use of either Quercetin or 4,5,6,7-tetrabromo-2-azabenzimidazole (TBB), an inhibitor of beta-catenin stabilizing kinase CK2, or by transient knockdown of beta-catenin by siRNA. Gene expression was measured by real time RT-PCR and Western Blot analysis. Wnt pathway activity was measured by a reporter assay for beta catenin related transcription (CRT assay). Results: Our results confirm that activity of the wnt pathway is increased in replicon cell lines compared to the replicon background cell line Huh7 and that the HCV protein NS5A is involved. Additionally we found that NS3B is able to increase Wnt pathway activity. Inhibition of the wnt signaling pathway by quercetin, TBB or siRNA directed against beta-catenin reduced HCV replication significantly without toxic effects on cultured cells. Moreover, activation of wnt pathway activity by inhibitors of GSK3b increased HCV replication. Conclusions: HCV replication induces wnt signaling which in turn seems to promote HCV replication. Therefore, inhibition of the wnt signaling pathway might not only represent a way to interfere with tumor growth but might also become a novel approach for HCV therapy.

Identification of HCV Inhibitors from a Cell-Based Sub-Genomic Replicon Screen

A high throughput screen of the Pfizer compound collection was carried out using a hepatitis C virus (HCV) genotype 1b subgenomic replicon cell line. Those confirmed hits that demonstrated broad spectrum activity without overt cytotoxicity were further evaluated, leading to the identification of a series of pyrrolopyridines with excellent antiviral activity in a fully infectious HCV cell-based assay and pharmacokinetic properties.

Combinations of Lambda Interferon with Direct-Acting Antiviral Agents Are Highly Efficient in Suppressing Hepatitis C Virus Replication

The clinical efficacy of a pegylated form of human lambda 1 interferon (IFN-λ1; also referred to herein as lambda) has been demonstrated in patients chronically infected with hepatitis C virus (HCV) representing genotypes 1 through 4. In these proof-of-concept studies, lambda showed an improved safety profile compared to the pegylated form of alpha interferon (referred to herein as alfa). In the study described in this report, an assessment of the in vitro antiviral activity of type III IFNs toward different HCV replicons revealed that the unpegylated recombinant form of IFN-λ1 (rIFN-λ1) exerted the most robust effect, while rIFN-λ3 exhibited greater activity than rIFN-λ2. More importantly, cross-resistance to rIFN-λ1 was not observed in replicon cell lines known to have reduced susceptibility to investigational direct-acting antiviral (DAA) agents targeting the essential HCV nonstructural protein NS3, NS5A, or NS5B. When combined with either rIFN-α, the NS3 protease inhibitor (NS3 PI) asunaprevir (ASV), the NS5A replication complex inhibitor (NS5A RCI) daclatasvir (DCV), or the NS5B polymerase site I inhibitor (NS5B I) BMS-791325, rIFN-λ1 displayed a mixture of additive and synergistic effects. In three-drug combination studies, inclusion of lambda with ASV and DCV also yielded additive to synergistic effects. In line with these observations, it was demonstrated that a regimen that used a combination of rIFN-λ1 with one or two DAAs was superior to an IFN-free regimen in clearing HCV RNA in genotype 1a cell lines representing wild-type and NS3 protease inhibitor-resistant sequences. Overall, these data support further clinical development of lambda as part of alternative combination treatments with DAAs for patients chronically infected with HCV.

Inhibition of Both Protease and Helicase Activities of Hepatitis C Virus NS3 by an Ethyl Acetate Extract of Marine Sponge Amphimedon sp

Combination therapy with ribavirin, interferon, and viral protease inhibitors could be expected to elicit a high level of sustained virologic response in patients infected with hepatitis C virus (HCV). However, several severe side effects of this combination therapy have been encountered in clinical trials. In order to develop more effective and safer anti-HCV compounds, we employed the replicon systems derived from several strains of HCV to screen 84 extracts from 54 organisms that were gathered from the sea surrounding Okinawa Prefecture, Japan. The ethyl acetate-soluble extract that was prepared from marine sponge Amphimedon sp. showed the highest inhibitory effect on viral replication, with EC50 values of 1.5 and 24.9 µg/ml in sub-genomic replicon cell lines derived from genotypes 1b and 2a, respectively. But the extract had no effect on interferon-inducing signaling or cytotoxicity. Treatment with the extract inhibited virus production by 30% relative to the control in the JFH1-Huh7 cell culture system. The in vitro enzymological assays revealed that treatment with the extract suppressed both helicase and protease activities of NS3 with IC50 values of 18.9 and 10.9 µg/ml, respectively. Treatment with the extract of Amphimedon sp. inhibited RNA-binding ability but not ATPase activity. These results suggest that the novel compound(s) included in Amphimedon sp. can target the protease and helicase activities of HCV NS3.

Establishment of a robust hepatitis C virus replicon cell line over-expressing P-glycoprotein that facilitates analysis of P-gp drug transporter effects on inhibitor antiviral activity

P-glycoprotein (P-gp) is an active efflux pump affecting the pharmacokinetic (PK) profiles of drugs that are P-gp substrates. The Caco-2 bi-directional assay is widely used to identify drug-P-gp interactions in vitro. For molecules exhibiting non-classical drug properties however, ambiguous results limit its use in lead optimization. The goal of this study was to develop a robust cell-based assay system to directly measure the role of P-gp-driven efflux in reducing the potency of hepatitis C virus (HCV) replication inhibitors. Vinblastine (Vin) was employed to select for a Vin-resistant HCV replicon (313-11) from the parental cell line (377-2). The 313-11 cell line was >50-fold resistant to Vin and over-expressed P-gp, as determined by Western immunoblots. Increased expression of P-gp was mediated by up-regulation of the MDR1 transcript. The reduced potency of different classes of HCV replication inhibitors in the 313-11 P-gp cell line was restored in the presence of known P-gp inhibitors. Addition of the P-gp inhibitor, tariquidar, increased the uptake of a radiolabeled HCV replication inhibitor by 14-fold in the 313-11 replicon cell line. Finally, a positive correlation was demonstrated between potency in the 313-11 replicon and the bi-directional Caco-2 efflux ratio for a panel of HCV protease inhibitors. In conclusion, a robust P-gp HCV replicon cell-based assay has been developed to measure the effect of the P-gp efflux pump on the potency of different classes of HCV replication inhibitors. This system establishes a direct correlation between antiviral activity and the effect of P-gp efflux in a single cell line.

Free fatty acids induce ER stress and block antiviral activity of interferon alpha against hepatitis C virus in cell culture

BackgroundHepatic steatosis is recognized as a major risk factor for liver disease progression and impaired response to interferon based therapy in chronic hepatitis C (CHC) patients. The mechanism of response to interferon-alpha (IFN-α) therapy under the condition of hepatic steatosis is unexplored. We investigated the effect of hepatocellular steatosis on hepatitis C virus (HCV) replication and IFN-α antiviral response in a cell culture model.MethodsSub-genomic replicon (S3-GFP) and HCV infected Huh-7.5 cells were cultured with a mixture of saturated (palmitate) and unsaturated (oleate) long-chain free fatty acids (FFA). Intracytoplasmic fat accumulation in these cells was visualized by Nile red staining and electron microscopy then quantified by microfluorometry. The effect of FFA treatment on HCV replication and IFN-α antiviral response was measured by flow cytometric analysis, Renilla luciferase activity, and real-time RT-PCR.ResultsFFA treatment induced dose dependent hepatocellular steatosis and lipid droplet accumulation in the HCV replicon cells was confirmed by Nile red staining, microfluorometry, and by electron microscopy. Intracellular fat accumulation supports replication more in the persistently HCV infected culture than in the sub-genomic replicon (S3-GFP) cell line. FFA treatment also partially blocked IFN-α response and viral clearance by reducing the phosphorylation of Stat1 and Stat2 dependent IFN-β promoter activation. We show that FFA treatment induces endoplasmic reticulum (ER) stress response and down regulates the IFNAR1 chain of the type I IFN receptor leading to defective Jak-Stat signaling and impaired antiviral response.ConclusionThese results suggest that intracellular fat accumulation in HCV cell culture induces ER stress, defective Jak-Stat signaling, and attenuates the antiviral response, thus providing an explanation to the clinical observation regarding how hepatocellular steatosis influences IFN-α response in CHC.

1200 ANTIVIRAL ACTIVITY OF EP-NI266, A POTENT NUCLEOTIDE HCV POLYMERASE INHIBITOR

1198 HCV NS3/4A PROTEASE RESISTANCE-ASSOCIATED VARIANTS (RAVS) IDENTIFIED IN GENOTYPE 1A PATIENTS EXHIBIT DIFFERENCES IN PHENOTYPIC RESISTANCE TO BOCEPREVIR AND TELAPREVIR IN GENOTYPE 1A REPLICON CELLS S. Black, P. McMonagle, R. Chase, S. Curry, R.J.O. Barnard, D.J. Hazuda, A.Y. Howe, J.A. Howe, R.A. Ogert. Merck & Co., Inc., Kenilworth, NJ, USA E-mail: robert.ogert@merck.com Background: The HCV NS3 protease inhibitors boceprevir (BOC) and telaprevir (TPV) in combination with pegylated interferon alpha-2 plus ribavirin (PR) have been approved for treatment of HCV genotype 1 (GT 1) infection. The major RAVs identified in nonsustained virologic response (SVR) GT 1a infected patients were similar for BOC and TPV. These RAVs were analyzed for phenotypic resistance to BOC and TPV using an in-vitro GT 1a replicon assay. Methods: RAVs were detected by population sequencing in samples from SPRINT-2 (treatment naive) and RESPOND-2 (prior nonresponder/relapser) patients. RAVs were engineered into a GT 1a replicon derived from the H77 1a strain. Replicon cell lines were treated with BOC or TPV for 3 days, and HCVRNA was quantified by Taqman real time PCR analysis. Results: For patients treated with BOC, RAVs were detected in 53% of non-SVR patients with NS3 sequences available. In BOC treated, non-SVR HCV GT 1a infected patients; the most common RAVs were V36M, T54S and R155K. T54A and A156S/T were also identified in HCV GT 1a non-SVR treated patients but were observed more frequently in HCV GT 1b. A similar pattern of HCV GT 1a RAVs was identified in TPV treated patients (Bartels DJ, et al. Hepatology. 2011;54(S1):1328). These RAVs were analyzed for resistance to BOC and TPV in a GT 1a replicon assay. T54A and T54S produced similar fold-shifts in EC50 with BOC and TPV. However, V36M, R155K and A156S each produced a larger fold-shift in resistance to TPV than BOC. The fold shift in EC50 for each RAV was 2to 6-fold for BOC and 5to 16-fold for TPV. Also, the V36M + R155K double variant resulted in a >20-fold increase in EC50 for TPV but <10-fold for BOC.

Inhibitors of Alphavirus Entry and Replication Identified with a Stable Chikungunya Replicon Cell Line and Virus-Based Assays

Chikungunya virus (CHIKV), an alphavirus, has recently caused epidemic outbreaks and is therefore considered a re-emerging pathogen for which no effective treatment is available. In this study, a CHIKV replicon containing the virus replicase proteins together with puromycin acetyltransferase, EGFP and Renilla luciferase marker genes was constructed. The replicon was transfected into BHK cells to yield a stable cell line. A non-cytopathic phenotype was achieved by a Pro718 to Gly substitution and a five amino acid insertion within non-structural protein 2 (nsP2), obtained through selection for stable growth. Characterization of the replicon cell line by Northern blotting analysis revealed reduced levels of viral RNA synthesis. The CHIKV replicon cell line was validated for antiviral screening in 96-well format and used for a focused screen of 356 compounds (natural compounds and clinically approved drugs). The 5,7-dihydroxyflavones apigenin, chrysin, naringenin and silybin were found to suppress activities of EGFP and Rluc marker genes expressed by the CHIKV replicon. In a concomitant screen against Semliki Forest virus (SFV), their anti-alphaviral activity was confirmed and several additional inhibitors of SFV with IC50 values between 0.4 and 24 µM were identified. Chlorpromazine and five other compounds with a 10H-phenothiazinyl structure were shown to inhibit SFV entry using a novel entry assay based on a temperature-sensitive SFV mutant. These compounds also reduced SFV and Sindbis virus-induced cytopathic effect and inhibited SFV virion production in virus yield experiments. Finally, antiviral effects of selected compounds were confirmed using infectious CHIKV. In summary, the presented approach for discovering alphaviral inhibitors enabled us to identify potential lead structures for the development of alphavirus entry and replication phase inhibitors as well as demonstrated the usefulness of CHIKV replicon and SFV as biosafe surrogate models for anti-CHIKV screening.

Monoclonal Antibody 2-152a Suppresses Hepatitis C Virus Infection Through Betaine/GABA Transporter-1

We recently established a monoclonal antibody (2-152a MAb) that binds to 3β-hydroxysterol-Δ24-reductase (DHCR24) by immunizing mice with cells (RzM6-LC) persistently expressing hepatitis C virus (HCV). Here, we aimed to analyze the activity of 2-152a MAb against HCV replication and explore the molecular mechanism underlying the antiviral activity. We characterized the effects of 2-152a MAb on HCV replication and performed a microarray analysis of antibody-treated HCV replicon cells. The molecules showing a significant change after the antibody treatment were screened to examine their relationship with HCV replication. The antibody had antiviral activity both in vitro and in vivo (chimeric mice). In the microarray analysis, 2-152a MAb significantly suppressed the expression of betaine/GABA transporter-1 (BGT-1) in 2 HCV replicon cell lines but not in HCV-cured cells. Silencing of BGT-1 expression by small interfering RNA (siRNA) revealed significant suppression of HCV replication and infection without cytotoxicity. Further, BGT-1 expression was significantly increased in the presence of HCV (P < .05). Our results suggest that 2-152a MAb suppresses HCV replication and infection through BGT-1. These findings highlight important roles of BGT-1 in HCV replication and reveal a possible target for anti-HCV therapy.

Creation of a Nonspreading Rift Valley Fever Virus

Rift Valley fever virus (RVFV) is a mosquito-borne zoonotic bunyavirus of the genus Phlebovirus and a serious human and veterinary pathogen. RVFV contains a three-segmented RNA genome, which is comprised of the large (L), medium (M), and small (S) segments. The proteins that are essential for genome replication are encoded by the L and S segments, whereas the structural glycoproteins are encoded by the M segment. We have produced BHK replicon cell lines (BHK-Rep) that maintain replicating L and S genome segments. Transfection of BHK-Rep cells with a plasmid encoding the structural glycoproteins results in the efficient production of RVFV replicon particles (RRPs). To facilitate monitoring of infection, the NSs gene was replaced with an enhanced green fluorescent protein gene. RRPs are infectious for both mammalian and insect cells but are incapable of autonomous spreading, rendering their application outside biosafety containment completely safe. We demonstrate that a single intramuscular vaccination with RRPs protects mice from a lethal dose of RVFV and show that RRPs can be used for rapid virus neutralization tests that do not require biocontainment facilities. The methods reported here will greatly facilitate vaccine and drug development as well as fundamental studies on RVFV biology. Moreover, it may be possible to develop similar systems for other members of the bunyavirus family as well.

Biochemical Evaluation of HCV NS3 Protease Inhibitors

This unit describes assays for characterizing the potency and mechanism of action of NS3 protease inhibitors. Determination of IC(50) values is described using in vitro expressed and purified NS3 protease. This assay can also be used for the rapid exploration of structure-activity relationships. Another protocol describes using the full-length NS3/4A complexes expressed in HCV replicon cell lines for a rapid alternative method for assessing protease activity without requiring conventional protein expression and purification. A method is then provided for determination of inhibitor K(i), which more accurately assesses the potency of inhibitors compared to the IC(50) assay, particularly for potent inhibitors that reach the sensitivity limit for the basic IC(50) assay. The final protocol describes how to determine the reversibility of inhibitor binding to the enzyme, an important parameter that can affect the pharmacodynamic properties of a compound.

Mechanism of HCV's resistance to IFN-α in cell culture involves expression of functional IFN-α receptor 1

The mechanisms underlying the Hepatitis C virus (HCV) resistance to interferon alpha (IFN-α) are not fully understood. We used IFN-α resistant HCV replicon cell lines and an infectious HCV cell culture system to elucidate the mechanisms of IFN-α resistance in cell culture. The IFN-α resistance mechanism of the replicon cells were addressed by a complementation study that utilized the full-length plasmid clones of IFN-α receptor 1 (IFNAR1), IFN-α receptor 2 (IFNAR2), Jak1, Tyk2, Stat1, Stat2 and the ISRE- luciferase reporter plasmid. We demonstrated that the expression of the full-length IFNAR1 clone alone restored the defective Jak-Stat signaling as well as Stat1, Stat2 and Stat3 phosphorylation, nuclear translocation and antiviral response against HCV in all IFN-α resistant cell lines (R-15, R-17 and R-24) used in this study. Moreover RT-PCR, Southern blotting and DNA sequence analysis revealed that the cells from both R-15 and R-24 series of IFN-α resistant cells have 58 amino acid deletions in the extracellular sub domain 1 (SD1) of IFNAR1. In addition, cells from the R-17 series have 50 amino acids deletion in the sub domain 4 (SD4) of IFNAR1 protein leading to impaired activation of Tyk2 kinase. Using an infectious HCV cell culture model we show here that viral replication in the infected Huh-7 cells is relatively resistant to exogenous IFN-α. HCV infection itself induces defective Jak-Stat signaling and impairs Stat1 and Stat2 phosphorylation by down regulation of the cell surface expression of IFNAR1 through the endoplasmic reticulum (ER) stress mechanisms. The results of this study suggest that expression of cell surface IFNAR1 is critical for the response of HCV to exogenous IFN-α.

Preexisting drug-resistance mutations reveal unique barriers to resistance for distinct antivirals

Clinical trials of direct-acting antiviral agents in patients chronically infected with hepatitis C virus (HCV) have demonstrated that viral resistance is detected rapidly during monotherapy. In patients, HCV does not exist as a single, genetically homogenous virus but rather as a population of variants termed "quasispecies." Preexisting variants resistant to specific antiviral drugs, overlooked in traditional hit-to-lead discovery efforts, may be responsible for these poor clinical outcomes. To enable real-time studies of resistance emergence in live cells, we established fluorescent protein-labeled HCV replicon cell lines. We validated these cell lines by demonstrating that antiviral susceptibility and the selection of signature resistance mutations for various drug classes are similar to traditional replicon cell lines. By quantifying the kinetics and uniformity of replication within colonies of drug-resistant fluorescent replicon cells, we showed that resistance emerged from a single cell and preexisted in a treatment-naive replicon population. Within this population, we determined the relative frequency of preexisting replicons capable of establishing foci during treatment with distinct antivirals. By measuring relative frequency as a function of dose, we quantitatively ranked distinct antiviral molecules on the basis of their distinct barriers to resistance. These insights into RNA virus quasispecies structure provide guidance for selecting clinical drug concentrations and selecting antiviral drug combinations most likely to suppress resistance.

Identification of Alphavirus Inhibitors by Using Virus-Based Assays and a Chikungunya Replicon Cell Line

Identification of Alphavirus Inhibitors by Using Virus-Based Assays and a Chikungunya Replicon Cell Line

Impaired S‐adenosylmethionine metabolism inhibits IFNα‐mediated anti‐HCV gene expression

Hepatitis C virus (HCV) is a major cause of chronic liver disease, which is a huge burden on the United States healthcare system. The FDA-approved standard of treatment is pegylated interferon-alpha (IFNα) in combination with ribavirin; however up to 50% of patients do not respond to this therapy. S-adenosylmethionine (SAM) is the major methyl donor in cellular metabolic reactions and impaired SAM metabolism is a well documented feature of chronic liver injury. The present work was carried out to examine the impact of impaired SAM metabolism on the expression of IFNα-stimulated anti-HCV genes. SAM metabolism was impaired in human hepatoma cells by the pharmacologic inhibition of S-adenosylhomocysteine hydrolase (SAHH) leading to an increase in SAH, which significantly decreased methylation potential by decreasing SAM/SAH ratio. Impaired SAM metabolism was observed to significantly inhibit IFNα-inducible anti-HCV gene expression, including PKR, OAS, and ISG15. Importantly, impaired SAM metabolism hindered the ability of IFNα to suppress viral RNA replication in an HCV replicon cell line, as analyzed by real-time PCR. These data reveal the importance of SAM metabolism in managing HCV infection by the current IFNα treatment regimen and also suggest the use of SAM as adjunct therapy.

Hepatitis C virus RNA replication in human stellate cells regulates gene expression of extracellular matrix-related molecules

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease, including chronic hepatitis, fibrosis, and cirrhosis. Fibrosis often develops in HCV-infected livers and ultimately leads to cirrhosis and carcinoma. During fibrosis, hepatic stellate cells (HSC) play important roles in the control of extracellular matrix synthesis and degradation in fibrotic livers. In this study, we established a subgenomic replicon (SGR) cell line with human hepatic stellate cells to investigate the effect of HCV RNA replication on HSC. Isolated SGR clones contained HCV RNA copy numbers ranging from 10 4 to 10 7 per μg total RNA, and long-term culture of low-copy number SGR clones resulted in markedly increased HCV RNA copy numbers. Furthermore, HCV RNA replication affected gene expression of extracellular matrix-related molecules in both hepatic stellate cells and hepatic cells, suggesting that HCV RNA replication and/or HCV proteins directly contribute to liver fibrosis. The HCV RNA-replicating hepatic stellate cell line isolated in this study will be useful for investigating hepatic stellate cell functions and HCV replication machinery .

Differential In Vitro Effects of Intravenous versus Oral Formulations of Silibinin on the HCV Life Cycle and Inflammation

Silymarin prevents liver disease in many experimental rodent models, and is the most popular botanical medicine consumed by patients with hepatitis C. Silibinin is a major component of silymarin, consisting of the flavonolignans silybin A and silybin B, which are insoluble in aqueous solution. A chemically modified and soluble version of silibinin, SIL, has been shown to potently reduce hepatitis C virus (HCV) RNA levels in vivo when administered intravenously. Silymarin and silibinin inhibit HCV infection in cell culture by targeting multiple steps in the virus lifecycle. We tested the hepatoprotective profiles of SIL and silibinin in assays that measure antiviral and anti-inflammatory functions. Both mixtures inhibited fusion of HCV pseudoparticles (HCVpp) with fluorescent liposomes in a dose-dependent fashion. SIL inhibited 5 clinical genotype 1b isolates of NS5B RNA dependent RNA polymerase (RdRp) activity better than silibinin, with IC50 values of 40–85 µM. The enhanced activity of SIL may have been in part due to inhibition of NS5B binding to RNA templates. However, inhibition of the RdRps by both mixtures plateaued at 43–73%, suggesting that the products are poor overall inhibitors of RdRp. Silibinin did not inhibit HCV replication in subgenomic genotype 1b or 2a replicon cell lines, but it did inhibit JFH-1 infection. In contrast, SIL inhibited 1b but not 2a subgenomic replicons and also inhibited JFH-1 infection. Both mixtures inhibited production of progeny virus particles. Silibinin but not SIL inhibited NF-κB- and IFN-B-dependent transcription in Huh7 cells. However, both mixtures inhibited T cell proliferation to similar degrees. These data underscore the differences and similarities between the intravenous and oral formulations of silibinin, which could influence the clinical effects of this mixture on patients with chronic liver diseases.

Hepatocellular carcinoma xenograft supports HCV replication: A mouse model for evaluating antivirals

To develop a hepatocellular carcinoma (HCC) xenograft model for studying hepatitis C virus (HCV) replication in a mice, and antiviral treatment. We developed a stable S3-green fluorescence protein (GFP) cell line that replicated the GFP-tagged HCV sub-genomic RNA derived from a highly efficient JFH1 virus. S3-GFP replicon cell line was injected subcutaneously into γ-irradiated SCID mice. We showed that the S3-GFP replicon cell line formed human HCC xenografts in SCID mice. Cells were isolated from subcutaneous tumors and then serially passaged multiple times in SCID mice by culturing in growth medium supplemented with G-418. The mouse-adapted S3-GFP replicon cells were implanted subcutaneously and also into the liver of SCID mice via intrasplenic infusion to study the replication of HCV in the HCC xenografts. The tumor model was validated for antiviral testing after intraperitoneal injection of interferon-α (IFN-α). A highly tumorigenic S3-GFP replicon cell line was developed that formed subcutaneous tumors within 2 wk and diffuse liver metastasis within 4 wk in SCID mice. Replication of HCV in the subcutaneous and liver tumors was confirmed by cell colony assay, detection of the viral RNA by ribonuclease protection assay and real-time quantitative reverse transcription polymerase chain reaction. High-level replication of HCV sub-genomic RNA in the tumor could be visualized by GFP expression using fluorescence microscopy. IFN-α cleared HCV RNA replication in the subcutaneous tumors within 2 wk and 4 wk in the liver tumor model. A non-infectious mouse model allows us to study replication of HCV in subcutaneous and metastatic liver tumors. Clearance of HCV by IFN-α supports use of this model to test other anti-HCV drugs.

Augmentation of DHCR24 expression by hepatitis C virus infection facilitates viral replication in hepatocytes

We characterized the role of 24-dehydrocholesterol reductase (DHCR24) in hepatitis C virus infection (HCV). DHCR24 is a cholesterol biosynthetic enzyme and cholesterol is a major component of lipid rafts, which is reported to play an important role in HCV replication. Therefore, we examined the potential of DHCR24 as a target for novel HCV therapeutic agents. We examined DHCR24 expression in human hepatocytes in both the livers of HCV-infected patients and those of chimeric mice with human hepatocytes. We targeted DHCR24 with siRNA and U18666A which is an inhibitor of both DHCR24 and cholesterol synthesis. We measured the level of HCV replication in these HCV replicon cell lines and HCV infected cells. U18666A was administrated into chimeric mice with humanized liver, and anti-viral effects were assessed. Expression of DHCR24 was induced by HCV infection in human hepatocytes in vitro, and in human hepatocytes of chimeric mouse liver. Silencing of DHCR24 by siRNA decreased HCV replication in replicon cell lines and HCV JFH-1 strain-infected cells. Treatment with U18666A suppressed HCV replication in the replicon cell lines. Moreover, to evaluate the anti-viral effect of U18666A in vivo, we administrated U18666A with or without pegylated interferon to chimeric mice and observed an inhibitory effect of U18666A on HCV infection and a synergistic effect with interferon. DHCR24 is an essential host factor which augmented its expression by HCV infection, and plays a significant role in HCV replication. DHCR24 may serve as a novel anti-HCV drug target.

Intracellular expression of IRF9 Stat fusion protein overcomes the defective Jak-Stat signaling and inhibits HCV RNA replication

Interferon alpha (IFN-α) binds to a cell surface receptor that activates the Jak-Stat signaling pathway. A critical component of this pathway is the translocation of interferon stimulated gene factor 3 (a complex of three proteins Stat1, Stat2 and IRF9) to the nucleus to activate antiviral genes. A stable sub-genomic replicon cell line resistant to IFN-α was developed in which the nuclear translocation of Stat1 and Stat2 proteins was prevented due to the lack of phosphorylation; whereas the nuclear translocation of IRF9 protein was not affected. In this study, we sought to overcome defective Jak-Stat signaling and to induce an antiviral state in the IFN-α resistant replicon cell line by developing a chimera IRF9 protein fused with the trans activating domain (TAD) of either a Stat1 (IRF9-S1C) or Stat2 (IRF9-S2C) protein. We show here that intracellular expression of fusion proteins using the plasmid constructs of either IRF9-S1C or IRF9-S2C, in the IFN-α resistant cells, resulted in an increase in Interferon Stimulated Response Element (ISRE) luciferase promoter activity and significantly induced HLA-1 surface expression. Moreover, we show that transient transfection of IRF9-S1C or IRF9-S2C plasmid constructs into IFN-α resistant replicon cells containing sub-genomic HCV1b and HCV2a viruses resulted in an inhibition of viral replication and viral protein expression independent of IFN-α treatment. The results of this study indicate that the recombinant fusion proteins of IRF9-S1C, IRF9-S2C alone, or in combination, have potent antiviral properties against the HCV in an IFN-α resistant cell line with a defective Jak-Stat signaling.