Modulation of alpha interferon anti-hepatitis C virus activity by ISG15

ISG15 has recently been reported to possess antiviral properties against viruses, both in vivo and in vitro. Knock-down of ISG15 gene expression by small interfering RNA followed by alpha interferon (IFN-α) treatment in Huh-7 cells resulted in an increased phenotypic sensitivity to IFN-α, as determined by measuring hepatitis C virus (HCV) RNA replication inhibition in stably transfected HCV replicon cells and in cells infected with genotype 1a HCVcc (infectious HCV). This IFN-α-specific effect, which was not observed with IFN-γ, correlated with an increase in expression of the IFN-α-inducible genes IFI6, IFITM3, OAS1 and MX1, whereas the expression of the non-IFN-α-inducible genes PTBP-1 and JAK1 remained unchanged. It has previously been reported that, unlike ISG15 knock-down, increased sensitivity to IFN-α after knock-down of USP18 occurs through the prolonged phosphorylation of STAT-1. Combination knock-down of ISG15 and USP18 resulted in a moderate increase in IFN-α-inducible gene expression compared with single ISG15 or USP18 knock-down. Furthermore, the phenotype of increased gene expression after ISG15 knock-down and IFN-α treatment was also observed in non-hepatic cell lines A549 and HeLa. Taken together, these results reveal a novel function for ISG15 in the regulation of the IFN-α pathway and its antiviral effect.

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Combination of a Hepatitis C Virus NS3-NS4A Protease Inhibitor and Alpha Interferon Synergistically Inhibits Viral RNA Replication and Facilitates Viral RNA Clearance in Replicon Cells

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.48.12.4784-4792.2004 ◽

2004 ◽

Vol 48 (12) ◽

pp. 4784-4792 ◽

Cited By ~ 65

Author(s):

Kai Lin ◽

Ann D. Kwong ◽

Chao Lin

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Adverse Effects ◽

Protease Inhibitor ◽

Standard Of Care ◽

Rna Replication ◽

Viral Rna ◽

Alpha Interferon ◽

Hcv Rna

ABSTRACT The present standard of care for hepatitis C virus (HCV) infection is pegylated alpha interferon (IFN-α) in combination with ribavirin. However, specific antivirals such as HCV NS3-NS4A protease inhibitors are now in clinical development, and these agents can potentially be used in combination with the present treatments. Therefore, it is important to investigate the potential benefits or adverse effects of these new combinations by using available in vitro HCV culture systems first. In the present study we demonstrate that the combination of a specific HCV NS3-NS4A protease inhibitor and IFN-α synergistically inhibits HCV RNA replication in replicon cells, with little or no increase in cytotoxicity. Furthermore, the benefit of the combination was sustained over time, such that a greater than 3-log reduction in HCV RNA levels was achieved following 9 days of treatment. The viral RNA appeared to be cleared from the replicon cells after 14 days of treatment, and no viral RNA rebound was observed upon withdrawal of the inhibitors. In each case, the antiviral effects obtained with higher concentrations of either the protease inhibitor alone or IFN-α alone can be achieved by a combination of both agents at lower concentrations, which may potentially reduce the risk of possible adverse effects associated with high doses of either agent.

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VX-950, a Novel Hepatitis C Virus (HCV) NS3-4A Protease Inhibitor, Exhibits Potent Antiviral Activities in HCV Replicon Cells

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.50.5.1813-1822.2006 ◽

2006 ◽

Vol 50 (5) ◽

pp. 1813-1822 ◽

Cited By ~ 148

Author(s):

Kai Lin ◽

Robert B. Perni ◽

Ann D. Kwong ◽

Chao Lin

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Antiviral Agents ◽

Alpha Interferon ◽

Hcv Rna ◽

Resistance Mutations ◽

In Vitro Characterization ◽

Antiviral Activities

ABSTRACT The NS3-4A serine protease of hepatitis C virus (HCV) is essential for viral replication and therefore has been one of the most attractive targets for developing specific antiviral agents against HCV. VX-950, a highly selective, reversible, and potent peptidomimetic inhibitor of the HCV NS3-4A protease, is currently in clinical development for the treatment of hepatitis C. In this report, we describe the in vitro characterization of anti-HCV activities of VX-950 in subgenomic HCV replicon cells. Incubation with VX-950 resulted in a time- and dose-dependent reduction of HCV RNA and proteins in replicon cells. Moreover, following a 2-week incubation with VX-950, a reduction in HCV RNA levels of 4.7 log10 was observed, and this reduction resulted in elimination of HCV RNA from replicon cells, since there was no rebound in replicon RNA after withdrawal of the inhibitor. The combination of VX-950 and alpha interferon was additive to moderately synergistic in reducing HCV RNA in replicon cells with no significant increase in cytotoxicity. The benefit of the combination was sustained over time: a 4-log10 reduction in HCV RNA level was achieved following a 9-day incubation with VX-950 and alpha interferon at lower concentrations than when either VX-950 or alpha interferon was used alone. The combination of VX-950 and alpha interferon also suppressed the emergence of in vitro resistance mutations against VX-950 in replicon cells.

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NIM811, a Cyclophilin Inhibitor, Exhibits Potent In Vitro Activity against Hepatitis C Virus Alone or in Combination with Alpha Interferon

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00310-06 ◽

2006 ◽

Vol 50 (9) ◽

pp. 2976-2982 ◽

Cited By ~ 111

Author(s):

Sue Ma ◽

Joanna E. Boerner ◽

ChoiLai TiongYip ◽

Beat Weidmann ◽

Neil S. Ryder ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Clinical Investigation ◽

Term Treatment ◽

Alpha Interferon ◽

Hcv Rna ◽

Long Term Treatment ◽

Antiviral Targets

ABSTRACT Host factors involved in viral replication are potentially attractive antiviral targets that are complementary to specific inhibitors of viral enzymes, since resistant mutations against the latter are likely to emerge during long-term treatment. It has been reported recently that cyclosporine, which binds to a family of cellular proteins, cyclophilins, inhibits hepatitis C virus (HCV) replication in vitro. Here, the activities of various cyclosporine derivatives were evaluated in the HCV replicon system. There was a strong correlation between the anti-HCV activity and cyclophilin-binding affinity of these compounds. Of these, NIM811 has been selected as a therapeutic candidate for HCV infection, since it binds to cyclophilins with higher affinity than cyclosporine but is devoid of the significant immunosuppressive activity associated with cyclosporine. NIM811 induced a concentration-dependent reduction of HCV RNA in the replicon cells with a 50% inhibitory concentration of 0.66 μM at 48 h. Furthermore, a greater than three-log10 viral RNA reduction was achieved after treating the cells with as little as 1 μM of NIM811 for 9 days. In addition, the combination of NIM811 with alpha interferon significantly enhanced anti-HCV activities without causing any increase of cytotoxicity. Taken together, these promising in vitro data warrant clinical investigation of NIM811, an inhibitor of novel mechanism, for the treatment of hepatitis C.

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Modeling Subgenomic Hepatitis C Virus RNA Kinetics during Treatment with Alpha Interferon

Journal of Virology ◽

10.1128/jvi.02612-08 ◽

2009 ◽

Vol 83 (13) ◽

pp. 6383-6390 ◽

Cited By ~ 42

Author(s):

Harel Dahari ◽

Bruno Sainz ◽

Alan S. Perelson ◽

Susan L. Uprichard

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Viral Rna ◽

Alpha Interferon ◽

Hcv Rna ◽

Second Phase ◽

Inhibition Kinetics ◽

Rna Levels

ABSTRACT Although replicons have been used to demonstrate hepatitis C virus (HCV) inhibition by alpha interferon (IFN-α), the detailed inhibition kinetics required to mathematically model HCV RNA decline have been lacking. Therefore, we measured genotype 1b subgenomic replicon (sg1b) RNA levels under various IFN-α concentrations to assess the inhibition kinetics of intracellular HCV RNA. During nine days of IFN-α treatment, sg1b RNA decreased in a biphasic, dose-dependent manner. Using frequent measurements to dissect these phases during IFN-α treatments of 100 and 250 U/ml revealed that the first-phase sg1b RNA decline began ∼12 h posttreatment, continued for 2 to 4 days, and then exhibited a distinct flat or slower second phase. Based on these data, we developed a mathematical model of IFN-α-induced intracellular sg1b RNA decline, and we show that the mechanism(s) mediating IFN-α inhibition of HCV acts primarily by reducing sg1b RNA amplification, with an additional effect on HCV RNA stability/degradation detectable at a dose of 250 U/ml IFN-α. While the extremely slow or flat second phase of viral RNA inhibition observed in vitro, in which there is little or no cell death, supports the in vivo modeling prediction that the more profound second-phase decline observed in IFN-α-treated patients reflects immune-mediated death/loss of productively infected cells, the second-phase decline in viral RNA with a dose of 250 U/ml IFN-α suggests that a further inhibition of intracellular HCV RNA levels may contribute as well. As such, dissection of HCV IFN-α inhibition kinetics in vitro has brought us closer to understanding the mechanism(s) by which IFN-α may be inhibiting HCV in vivo.

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The effects of the conserved extreme 3' end sequence of hepatitis C virus (HCV) RNA on the in vitro stabilization and translation of the HCV RNA genome

Journal of Hepatology ◽

10.1034/j.1600-0641.2000.033004632.x ◽

2000 ◽

Vol 33 (4) ◽

pp. 632-639 ◽

Cited By ~ 4

Author(s):

Jane W. S. Fang ◽

Richard W. Moyer

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Hcv Rna ◽

Rna Genome

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Genotypic and Phenotypic Analyses of Hepatitis C Virus from Patients Treated with JTK-853 in a Three-Day Monotherapy

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01432-12 ◽

2012 ◽

Vol 57 (1) ◽

pp. 436-444 ◽

Cited By ~ 1

Author(s):

Naoki Ogura ◽

Yukiyo Toyonaga ◽

Izuru Ando ◽

Kunihiro Hirahara ◽

Tsutomu Shibata ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Amino Acid ◽

Viral Resistance ◽

Hcv Rna ◽

Amino Acid Substitutions ◽

Sequencing Analysis ◽

Resistance Mutations ◽

In The Wild

ABSTRACTJTK-853, a palm site-binding NS5B nonnucleoside polymerase inhibitor, shows antiviral activityin vitroand in hepatitis C virus (HCV)-infected patients. Here, we report the results of genotypic and phenotypic analyses of resistant variants in 24 HCV genotype 1-infected patients who received JTK-853 (800, 1,200, or 1,600 mg twice daily or 1,200 mg three times daily) in a 3-day monotherapy. Viral resistance in NS5B was investigated using HCV RNA isolated from serum specimens from the patients. At the end of treatment (EOT) with JTK-853, the amino acid substitutions M414T (methionine [M] in position 414 at baseline was replaced with threonine [T] at EOT), C445R (cysteine [C] in position 445 at baseline was replaced with arginine [R] at EOT), Y448C/H (tyrosine [Y] in position 448 at baseline was replaced with cysteine [C] or histidine [H] at EOT), and L466F (leucine [L] in position 466 at baseline was replaced with phenylalanine [F] at EOT), which are known to be typical resistant variants of nonnucleoside polymerase inhibitors, were observed in a clonal sequencing analysis. These substitutions were also selected by a treatment with JTK-853in vitro, and the 50% effective concentration of JTK-853 in the M414T-, C445F-, Y448H-, and L466V-harboring replicons attenuated the susceptibility by 44-, 5-, 6-, and 21-fold, respectively, compared with that in the wild-type replicon (Con1). These findings suggest that amino acid substitutions of M414T, C445R, Y448C/H, and L466F are thought to be viral resistance mutations in HCV-infected patients receiving JTK-853 in a 3-day monotherapy.

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Alpha Interferon Inhibits Hepatitis C Virus Replication in Primary Human Hepatocytes Infected In Vitro

Journal of Virology ◽

10.1128/jvi.76.16.8189-8199.2002 ◽

2002 ◽

Vol 76 (16) ◽

pp. 8189-8199 ◽

Cited By ~ 87

Author(s):

Valérie Castet ◽

Chantal Fournier ◽

Alexandre Soulier ◽

Rozenn Brillet ◽

Joliette Coste ◽

...

Keyword(s):

Chronic Hepatitis ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Chronic Hepatitis C ◽

Primary Cultures ◽

Indirect Evidence ◽

Human Hepatocytes ◽

Alpha Interferon ◽

Healthy Human

ABSTRACT Chronic hepatitis C is a common cause of liver disease, the complications of which include cirrhosis and hepatocellular carcinoma. Treatment of chronic hepatitis C is based on the use of alpha interferon (IFN-α). Recently, indirect evidence based on mathematical modeling of hepatitis C virus (HCV) dynamics during human IFN-α therapy suggested that the major initial effect of IFN-α is to block HCV virion production or release. Here, we used primary cultures of healthy, uninfected human hepatocytes to show that: (i) healthy human hepatocytes can be infected in vitro and support HCV genome replication, (ii) hepatocyte treatment with IFN-α results in expression of IFN-α-induced genes, and (iii) IFN-α inhibits HCV replication in infected human hepatocytes. These results show that IFN-α acts primarily through its nonspecific antiviral effects and suggest that primary cultures of human hepatocytes may provide a good model to study intrinsic HCV resistance to IFN-α.

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