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

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.

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Comparison of Daclatasvir Resistance Barriers on NS5A from Hepatitis C Virus Genotypes 1 to 6: Implications for Cross-Genotype Activity

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02788-14 ◽

2014 ◽

Vol 58 (9) ◽

pp. 5155-5163 ◽

Cited By ~ 50

Author(s):

Chunfu Wang ◽

Lingling Jia ◽

Donald R. O'Boyle ◽

Jin-Hua Sun ◽

Karen Rigat ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Antiviral Activity ◽

Rank Order ◽

Replicon Cell ◽

Hcv Genotypes ◽

Resistance Selection ◽

Virus Genotypes ◽

Hepatitis C Virus Genotypes

ABSTRACTA comparison of the daclatasvir (DCV [BMS-790052]) resistance barrier on authentic or hybrid replicons containing NS5A from hepatitis C virus (HCV) genotypes 1 to 6 (GT-1 to -6) was completed using a replicon elimination assay. The data indicated that genotype 1b (GT-1b) has the highest relative resistance barrier and genotype 2a (GT-2a M31) has the lowest. The rank order of resistance barriers to DCV was 1b > 4a ≥ 5a > 6a ≅ 1a > 2a JFH > 3a > 2a M31. Importantly, DCV in combination with a protease inhibitor (PI) eliminated GT-2a M31 replicon RNA at a clinically relevant concentration. Previously, we reported the antiviral activity and resistance profiles of DCV on HCV genotypes 1 to 4 evaluated in the replicon system. Here, we report the antiviral activity and resistance profiles of DCV against hybrid replicons with NS5A sequences derived from HCV GT-5a and GT-6a clinical isolates. DCV was effective against both GT-5a and -6a hybrid replicon cell lines (50% effective concentrations [EC50s] ranging from 3 to 7 pM for GT-5a, and 74 pM for GT-6a). Resistance selection identified amino acid substitutions in the N-terminal domain of NS5A. For GT-5a, L31F and L31V, alone or in combination with K56R, were the major resistance variants (EC50s ranging from 2 to 40 nM). In GT-6a, Q24H, L31M, P32L/S, and T58A/S were identified as resistance variants (EC50s ranging from 2 to 250 nM). Thein vitrodata suggest that DCV has the potential to be an effective agent for HCV genotypes 1 to 6 when used in combination therapy.

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Mutations in NS5B Polymerase of Hepatitis C Virus: Impacts on in Vitro Enzymatic Activity and Viral RNA Replication in the Subgenomic Replicon Cell Culture

Virology ◽

10.1006/viro.2002.1461 ◽

2002 ◽

Vol 297 (2) ◽

pp. 298-306 ◽

Cited By ~ 35

Author(s):

I.Wayne Cheney ◽

Suhaila Naim ◽

Vicky C.H. Lai ◽

Shannon Dempsey ◽

Daniel Bellows ◽

...

Keyword(s):

Cell Culture ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Enzymatic Activity ◽

Rna Replication ◽

Viral Rna ◽

Subgenomic Replicon ◽

Replicon Cell ◽

Ns5b Polymerase

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

Journal of General Virology ◽

10.1099/vir.0.81334-0 ◽

2005 ◽

Vol 86 (11) ◽

pp. 3075-3080 ◽

Cited By ~ 63

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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Quantitation of hepatitis C virus RNA production in two human bone marrow-derived B-cell lines infected in vitro

Research in Virology ◽

10.1016/s0923-2516(97)89901-3 ◽

1997 ◽

Vol 148 (2) ◽

pp. 147-151 ◽

Cited By ~ 16

Author(s):

S. Iacovacci ◽

L. Bertolini ◽

A. Manzin ◽

M.B. Valli ◽

M. Battaglia ◽

...

Keyword(s):

Bone Marrow ◽

Hepatitis C Virus ◽

Hepatitis C ◽

B Cell ◽

Cell Lines ◽

Human Bone ◽

Human Bone Marrow ◽

Hepatitis C Virus Rna ◽

Virus Rna

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The C-Terminal Hydrophobic Domain of Hepatitis C Virus RNA Polymerase NS5B Can Be Replaced with a Heterologous Domain of Poliovirus Protein 3A

Journal of Virology ◽

10.1128/jvi.02072-05 ◽

2006 ◽

Vol 80 (22) ◽

pp. 11343-11354 ◽

Cited By ~ 4

Author(s):

Haekyung Lee ◽

Ying Liu ◽

Edison Mejia ◽

Aniko V. Paul ◽

Eckard Wimmer

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Amino Acid ◽

Rna Polymerase ◽

Decisive Role ◽

Amino Acid Residues ◽

Subgenomic Replicon ◽

Hydrophobic Domain ◽

Virus Rna ◽

Membrane Bound

ABSTRACT Replication of the plus-stranded RNA genome of hepatitis C virus (HCV) occurs in a membrane-bound replication complex consisting of viral and cellular proteins and viral RNA. NS5B, the RNA polymerase of HCV, is anchored to the membranes via a C-terminal 20-amino-acid-long hydrophobic domain, which is flanked on each side by a highly conserved positively charged arginine. Using a genotype 1b subgenomic replicon (V. Lohmann, F. Korner, J. O. Koch, U. Herian, L. Theilmann, and R. Bartensclager, Science 285:110-113, 1999), we determined the effect of mutations of some highly conserved residues in this domain. The replacement of arginine 570 with alanine completely abolished the colony-forming ability by the replicon, while a R591A change was found to be highly detrimental to replication, viability, and membrane binding by the mutant NS5B protein. Mutations of two other highly conserved amino acids (L588A and P589A) reduced but did not eliminate colony formation. It was of interest, if specific amino acid residues play a role in membrane anchoring of NS5B and replication, to determine whether a complete exchange of the NS5B hydrophobic domain with a domain totally unrelated to NS5B would ablate replication. We selected the 22-amino-acid-long hydrophobic domain of poliovirus polypeptide 3A that is known to adopt a transmembrane configuration, thereby anchoring 3A to membranes. Surprisingly, either partial or full replacement of the NS5B hydrophobic domain with the anchor sequences of poliovirus polypeptide 3A resulted in the replication of replicons whose colony-forming abilities were reduced compared to that of the wild-type replicon. Upon continued passage of the replicon in Huh-7 cells in the presence of neomycin, the replication efficiency of the replicon increased. However, the sequence of the poliovirus polypeptide 3A hydrophobic domain, in the context of the subgenomic HCV replicon, was stably maintained throughout 40 passages. Our results suggest that anchoring NS5B to membranes is necessary but that the amino acid sequence of the anchor per se does not require HCV origin. This suggests that specific interactions between the NS5B hydrophobic domain and other membrane-bound factors may not play a decisive role in HCV replication.

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Detection of hepatitis C virus RNA in alpha interferon derived from in vitro culture of leukocytes of human origin

Biologicals ◽

10.1006/biol.2001.0275 ◽

2001 ◽

Vol 29 (1) ◽

pp. 45-53 ◽

Cited By ~ 1

Author(s):

D. Forčić ◽

R. Zgorelec ◽

M. Šantak ◽

T. Košutić ◽

R. Mažuran

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

In Vitro Culture ◽

Alpha Interferon ◽

Human Origin ◽

Hepatitis C Virus Rna ◽

Virus Rna

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00533-08 ◽

2008 ◽

Vol 52 (10) ◽

pp. 3523-3531 ◽

Cited By ~ 40

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.

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Identification of Determinants Involved in Initiation of Hepatitis C Virus RNA Synthesis by Using Intergenotypic Replicase Chimeras

Journal of Virology ◽

10.1128/jvi.00032-07 ◽

2007 ◽

Vol 81 (10) ◽

pp. 5270-5283 ◽

Cited By ~ 80

Author(s):

Marco Binder ◽

Doris Quinkert ◽

Olga Bochkarova ◽

Rahel Klein ◽

Nikolina Kezmic ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Synthesis ◽

Nonstructural Protein ◽

Negative Strand ◽

Virus Rna ◽

Nontranslated Region ◽

Genotype 2 ◽

Positive Strand Rna

ABSTRACT The 5′ nontranslated region (NTR) and the X tail in the 3′ NTR are the least variable parts of the hepatitis C virus (HCV) genome and play an important role in the initiation of RNA synthesis. By using subgenomic replicons of the HCV isolates Con1 (genotype 1) and JFH1 (genotype 2), we characterized the genotype specificities of the replication signals contained in the NTRs. The replacement of the JFH1 5′ NTR and X tail with the corresponding Con1 sequence resulted in a significant decrease in replication efficiency. Exchange of the X tail specifically reduced negative-strand synthesis, whereas substitution of the 5′ NTR impaired the generation of progeny positive strands. In search for the proteins involved in the recognition of genotype-specific initiation signals, we analyzed recombinant nonstructural protein 5B (NS5B) RNA polymerases of both isolates and found some genotype-specific template preference for the 3′ end of positive-strand RNA in vitro. To further address genotype specificity, we constructed a series of intergenotypic replicon chimeras. When combining NS3 to NS5A of Con1 with NS5B of JFH1, we observed more-efficient replication with the genotype 2a X tail, indicating that NS5B recognizes genotype-specific signals in this region. In contrast, a combination of the NS3 helicase with NS5A and NS5B was required to confer genotype specificity to the 5′ NTR. These results present the first genetic evidence for an interaction between helicase, NS5A, and NS5B required for the initiation of RNA synthesis and provide a system for the specific analysis of HCV positive- and negative-strand syntheses.

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Cross-Genotypic Examination of Hepatitis C Virus Polymerase Inhibitors Reveals a Novel Mechanism of Action for Thumb Binders

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.03699-14 ◽

2014 ◽

Vol 58 (12) ◽

pp. 7215-7224 ◽

Cited By ~ 10

Author(s):

Auda A. Eltahla ◽

Enoch Tay ◽

Mark W. Douglas ◽

Peter A. White

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

De Novo ◽

Hcv Rna ◽

Amino Acid Residues ◽

Recombinant Enzymes ◽

Polymerase Inhibitors ◽

Binding Pockets ◽

Direct Acting

ABSTRACTDirect-acting antivirals (DAAs) targeting proteins encoded by the hepatitis C virus (HCV) genome have great potential for the treatment of HCV infections. However, the efficacy of DAAs designed to target genotype 1 (G1) HCV against non-G1 viruses has not been characterized fully. In this study, we investigated the inhibitory activities of nonnucleoside inhibitors (NNIs) against the HCV RNA-dependent RNA polymerase (RdRp). We examined the ability of six NNIs to inhibit G1b, G2a, and G3a subgenomic replicons in cell culture, as well asin vitrotranscription by G1b and G3a recombinant RdRps. Of the six G1 NNIs, only the palm II binder nesbuvir demonstrated activity against G1, G2, and G3 HCV, in both replicon and recombinant enzyme models. The thumb I binder JTK-109 also inhibited G1b and G3a replicons and recombinant enzymes but was 41-fold less active against the G2a replicon. The four other NNIs, which included a palm I binder (setrobuvir), two thumb II binders (lomibuvir and filibuvir), and a palm β-hairpin binder (tegobuvir), all showed at least 40-fold decreases in potency against G2a and G3a replicons and the G3a enzyme. This antiviral resistance was largely conferred by naturally occurring amino acid residues in the G2a and G3a RdRps that are associated with G1 resistance. Lomibuvir and filibuvir (thumb II binders) inhibited primer-dependent but notde novoactivity of the G1b polymerase. Surprisingly, these compounds instead specifically enhanced thede novoactivity at concentrations of ≥100 nM. These findings highlight a potential differential mode of RdRp inhibition for HCV NNIs, depending on their prospective binding pockets, and also demonstrate a surprising enhancement ofde novoactivity for thumb RdRp binders. These results also provide a better understanding of the antiviral coverage for these polymerase inhibitors, which will likely be used in future combinational interferon-free therapies.

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