scholarly journals Development of Intergenotypic Chimeric Replicons To Determine the Broad-Spectrum Antiviral Activities of Hepatitis C Virus Polymerase Inhibitors

2008 ◽  
Vol 52 (10) ◽  
pp. 3523-3531 ◽  
Author(s):  
Koleen J. Herlihy ◽  
Joanne P. Graham ◽  
Robert Kumpf ◽  
Amy K. Patick ◽  
Rohit Duggal ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Antiviral Activity ◽  
Broad Spectrum ◽  
Inhibitor Binding ◽  
Subgenomic Replicon ◽  
Activity Determination ◽  
Polymerase Inhibitors ◽  
Overlap Extension

ABSTRACT To address the need for broad-spectrum antiviral activity characterization of hepatitis C virus (HCV) polymerase inhibitors, we created a panel of intergenotypic chimeric replicons containing nonstructural (NS) protein NS5B sequences from genotype 2b (GT2b), GT3a, GT4a, GT5a, and GT6a HCV isolates. Viral RNA extracted from non-GT1 HCV patient plasma was subjected to reverse transcription. The NS5B region was amplified by nested PCR and introduced into the corresponding region of the GT1b (Con-1) subgenomic reporter replicon by Splicing by Overlap Extension (SOEing) PCR. Stable cell lines were generated with replication-competent chimeras for in vitro antiviral activity determination of HCV nonnucleoside polymerase inhibitors (NNIs) that target different regions of the protein. Compounds that bind to the NNI2 (thiophene carboxylic acid) or NNI3 (benzothiadiazine) allosteric sites showed 8- to >1,280-fold reductions in antiviral activity against non-GT1 NS5B chimeric replicons compared to that against the GT1b subgenomic replicon. Smaller reductions in susceptibility, ranging from 0.2- to 33-fold, were observed for the inhibitor binding to the NNI1 (benzimidazole) site. The inhibitor binding to the NNI4 (benzofuran) site showed broad-spectrum antiviral activity against all chimeric replicons evaluated in this study. In conclusion, evaluation of HCV NNIs against intergenotypic chimeric replicons showed differences in activity spectrum for inhibitors that target different regions of the enzyme, some of which could be associated with specific residues that differ between GT1 and non-GT1 polymerases. Our study demonstrates the utility of chimeric replicons for broad-spectrum activity determination of HCV inhibitors.

scholarly journals In Vitro Resistance Study of AG-021541, a Novel Nonnucleoside Inhibitor of the Hepatitis C Virus RNA-Dependent RNA Polymerase

2007 ◽  
Vol 52 (2) ◽  
pp. 675-683 ◽  
Author(s):  
Stephanie T. Shi ◽  
Koleen J. Herlihy ◽  
Joanne P. Graham ◽  
Shella A. Fuhrman ◽  
Chau Doan ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
High Throughput Screening ◽  
Subgenomic Replicon ◽  
Replicon Cell ◽  
Resistance Selection ◽  
Polymerase Inhibitors ◽  
Virus Rna ◽  
Allosteric Sites

ABSTRACT A novel class of nonnucleoside hepatitis C virus (HCV) polymerase inhibitors characterized by a dihydropyrone core was identified by high-throughput screening. Crystallographic studies of these compounds in complex with the polymerase identified an allosteric binding site close to the junction of the thumb and finger domains, approximately 30 Å away from the catalytic center. AG-021541, a representative compound from this series, displayed measurable in vitro antiviral activity against the HCV genotype 1b subgenomic replicon with a mean 50% effective concentration of 2.9 μM. To identify mutations conferring in vitro resistance to AG-021541, resistance selection was carried out using HCV replicon cells either by serial passages in increasing concentrations of AG-021541 or by direct colony formation at fixed concentrations of the compound. We identified several amino acid substitutions in the AG-021541-binding region of the polymerase, including M423(T/V/I), M426T, I482(S/T), and V494A, with M423T as the predominant change observed. These mutants conferred various levels of resistance to AG-021541 and structurally related compounds but remained sensitive to interferon and HCV polymerase inhibitors known to interact with the active site or other allosteric sites of the protein. In addition, dihydropyrone polymerase inhibitors retained activity against replicons that contain signature resistance changes to other polymerase inhibitors, including S282T, C316N, M414T, and P495(S/L), indicating their potential to be used in combination therapies with these polymerase inhibitors. AG-021541-resistant replicon cell lines provide a valuable tool for mechanism-of-action studies of dihydropyrone polymerase inhibitors. The clinical relevance of in vitro resistance to HCV polymerase inhibitors remains to be investigated.

scholarly journals Interleukin-1 Inhibits Hepatitis C Virus Subgenomic RNA Replication by Activation of Extracellular Regulated Kinase Pathway

2003 ◽  
Vol 77 (9) ◽  
pp. 5493-5498 ◽  
Author(s):  
Haizhen Zhu ◽  
Chen Liu
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Antiviral Activity ◽  
Interleukin 1 ◽  
Rna Replication ◽  
Inhibitory Effect ◽  
Chronic Infections ◽  
Subgenomic Replicon ◽  
Subgenomic Rna ◽  
Interferon Stimulated Genes

ABSTRACT Interleukin-1 (IL-1) plays an important role in the inflammatory process. Some studies have demonstrated that IL-1 production was impaired in patients with chronic infections of hepatitis C virus (HCV), implying that IL-1 may play a role in viral clearance. Using an HCV subgenomic replicon cell line, we demonstrate that IL-1 can effectively inhibit HCV subgenomic RNA replication and viral protein expression, suggesting that IL-1 has direct antiviral activity. The inhibitory effect is associated with the extracellular regulatory kinase (ERK) activation. In addition, we also show that IL-1 can induce one of the interferon-stimulated genes (ISGs), 1-8U, which exhibits antiviral activity. However, it has no effect on the other ISG, 6-16, suggesting that IL-1 induces novel antiviral pathways within a cell.

scholarly journals Combinations of Cyclophilin Inhibitor NIM811 with Hepatitis C Virus NS3-4A Protease or NS5B Polymerase Inhibitors Enhance Antiviral Activity and Suppress the Emergence of Resistance

2008 ◽  
Vol 52 (9) ◽  
pp. 3267-3275 ◽  
Author(s):  
Joanna E. Mathy ◽  
Sue Ma ◽  
Teresa Compton ◽  
Kai Lin
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Antiviral Activity ◽  
Host Factors ◽  
Polymerase Inhibitors ◽  
Emergence Of Resistance ◽  
Ns5b Polymerase ◽  
Specific Inhibitors ◽  
Cyclophilin Inhibitor

ABSTRACT Chronic hepatitis C virus (HCV) infection remains a major global health burden while current interferon-based therapy is suboptimal. Efforts to develop more effective antiviral agents mainly focus on two viral targets: NS3-4A protease and NS5B polymerase. However, resistant mutants against these viral specific inhibitors emerge quickly both in vitro and in patients, particularly in the case of monotherapy. An alternative and complementary strategy is to target host factors such as cyclophilins that are also essential for viral replication. Future HCV therapies will most likely be combinations of multiple drugs of different mechanisms to maximize antiviral activity and to suppress the emergence of resistance. Here, the effects of combining a host cyclophilin inhibitor NIM811 with other viral specific inhibitors were investigated in vitro using HCV replicon. All of the combinations led to more pronounced antiviral effects than any single agent, with no significant increase of cytotoxicity. Moreover, the combination of NIM811 with a nucleoside (NM107) or a non-nucleoside (thiophene-2-carboxylic acid) polymerase inhibitor was synergistic, while the combination with a protease inhibitor (BILN2061) was additive. Resistant clones were selected in vitro with these inhibitors. Interestingly, it was much more difficult to develop resistance against NIM811 than viral specific inhibitors. No cross-resistance was observed among these inhibitors. Most notably, NIM811 was highly effective in blocking the emergence of resistance when used in combination with viral protease or polymerase inhibitors. Taken together, these results illustrate the significant advantages of combining inhibitors targeting both viral and host factors as key components of future HCV therapies.

Binding Mode Determination of Benzimidazole Inhibitors of the Hepatitis C Virus RNA Polymerase by a Structure and Dynamics Strategy

2004 ◽  
Vol 116 (33) ◽  
pp. 4406-4411 ◽  
Author(s):  
Steven R. LaPlante ◽  
Araz Jakalian ◽  
Norman Aubry ◽  
Yves Bousquet ◽  
Jean-Marie Ferland ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
Binding Mode ◽  
Hepatitis C Virus Rna ◽  
Structure And Dynamics ◽  
Virus Rna

scholarly journals Development and characterization of a transient-replication assay for the genotype 2a hepatitis C virus subgenomic replicon

2005 ◽  
Vol 86 (11) ◽  
pp. 3075-3080 ◽  
Author(s):  
Paul Targett-Adams ◽  
John McLauchlan
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Osteosarcoma Cell ◽  
Dependent Manner ◽  
Entry Site ◽  
Subgenomic Replicon ◽  
Replication Assay ◽  
Cell Extracts ◽  
Genotype 1B

Dicistronic, subgenomic hepatitis C virus (HCV) replicons were constructed containing sequences from JFH1, a genotype 2a strain, that also incorporated the firefly luciferase gene under the control of the HCV internal ribosome entry site element. Luciferase activity in Huh-7 cell extracts containing in vitro-transcribed subgenomic JFH1 RNA was monitored over a 72 h period to examine early stages of HCV replication in the absence of any selective pressure. Enzyme activities produced by the replicon were almost 200-fold greater than those generated from corresponding genotype 1b replicons and correlated with an accumulation of NS5A protein and replicon RNA. Transient replication was sensitive to IFN treatment in a dose-dependent manner and, in addition to Huh-7 cells, the U2OS human osteosarcoma cell line supported efficient replication of the JFH1 replicon. Thus, this system based on JFH1 sequences offers improvements over prior genotype 1b replicons for quantitative measurement of viral RNA replication.

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