scholarly journals Relative Replication Capacity and Selective Advantage Profiles of Protease Inhibitor-Resistant Hepatitis C Virus (HCV) NS3 Protease Mutants in the HCV Genotype 1b Replicon System

2007 ◽  
Vol 52 (3) ◽  
pp. 1101-1110 ◽  
Author(s):  
Yupeng He ◽  
Martin S. King ◽  
Dale J. Kempf ◽  
Liangjun Lu ◽  
Hock Ben Lim ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Drug Concentration ◽  
Selective Advantage ◽  
Drug Susceptibility ◽  
Replication Capacity ◽  
Wild Type ◽  
Drug Pressure ◽  
Genotype 1B ◽  
Ns3 Protease

ABSTRACT We characterized the selective advantage profiles of a panel of hepatitis C virus (HCV) NS3 protease mutants with three HCV protease inhibitors (PIs), BILN-2061, ITMN-191, and VX-950, using a genotype 1b HCV replicon system. Selective advantage curves were generated by a novel mathematical method that factors in the degree of drug susceptibility provided by the mutation, the base-level replication capacity of the mutant in the absence of drugs, and the overall viral replication levels as a function of drug concentration. Most of the mutants showed significantly increased selective advantages over the wild-type species upon drug treatment. Each drug is associated with unique selective advantage profiles that reflect its antiviral activity and mutant susceptibility. Five mutants (R155K/Q, A156T, and D168A/V) showed significant levels of selective advantage after treatment with >10 nM (∼7 times the wild-type 50% effective concentration [EC50]) of BILN-2061. R155K displayed dominant levels of selective advantage over the other mutants upon treatment with ITMN-191 over a broad range of concentrations. Upon VX-950 treatment, various mutants (A156T, A156S, R155K, T54A, V170A, V36M/R155K, and R155Q) exhibited high levels of selective advantage in different drug concentration ranges, with A156T and A156S being the dominant mutants at >3 μM (∼10 times the wild-type EC50) of VX-950. This method provides more accurate estimates of the behavior of various mutants under drug pressure than replication capacity analysis. We noted that the R155K mutant shows reduced susceptibility to all three PIs and significant selective advantage, raising concern over the potential emergence of R155K as a multidrug-resistant, highly fit mutant in HCV patients treated with PIs.

scholarly journals Resistance Analysis of the Hepatitis C Virus NS3 Protease Inhibitor Asunaprevir

2012 ◽  
Vol 56 (7) ◽  
pp. 3670-3681 ◽  
Author(s):  
Fiona McPhee ◽  
Jacques Friborg ◽  
Steven Levine ◽  
Chaoqun Chen ◽  
Paul Falk ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Protease Inhibitor ◽  
Clinical Studies ◽  
Replication Capacity ◽  
Replication Complex ◽  
Hcv Genotype ◽  
Genotype 1A ◽  
Genotype 1B ◽  
Ns3 Protease

ABSTRACTAsunaprevir (BMS-650032) is a potent hepatitis C virus (HCV) NS3 protease inhibitor demonstrating efficacy in alfa interferon-sparing, direct-acting antiviral dual-combination regimens (together with the NS5A replication complex inhibitor daclatasvir) in patients chronically infected with HCV genotype 1b. Here, we describe a comprehensivein vitrogenotypic and phenotypic analysis of asunaprevir-associated resistance against genotypes 1a and 1b using HCV replicons and patient samples obtained from clinical studies of short-term asunaprevir monotherapy. During genotype 1a resistance selection using HCV replicons, the primary NS3 protease substitutions identified were R155K, D168G, and I170T, which conferred low- to moderate-level asunaprevir resistance (5- to 21-fold) in transient-transfection susceptibility assays. For genotype 1b, a higher level of asunaprevir-associated resistance was observed at the same selection pressures, ranging from 170- to 400-fold relative to the wild-type control. The primary NS3 protease substitutions identified occurred predominantly at amino acid residue D168 (D168A/G/H/V/Y) and were associated with high-level asunaprevir resistance (16- to 280-fold) and impaired replication capacity. In asunaprevir single-ascending-dose and 3-day multiple-ascending-dose studies in HCV genotype 1a- or 1b-infected patients, the predominant pre-existing NS3 baseline polymorphism was NS3-Q80K. This substitution impacted initial virologic response rates in a single-ascending-dose study, but its effects after multiple doses were more ambiguous. Interestingly, for patient NS3 protease sequences containing Q80 and those containing K80, susceptibilities to asunaprevir were comparable when tested in an enzyme assay. No resistance-associated variants emerged in these clinical studies that significantly impacted susceptibility to asunaprevir. Importantly, asunaprevir-resistant replicons remained susceptible to an NS5A replication complex inhibitor, consistent with a role for asunaprevir in combination therapies.

scholarly journals Viral Resistance in Hepatitis C Virus Genotype 1-Infected Patients Receiving the NS3 Protease Inhibitor Faldaprevir (BI 201335) in a Phase 1b Multiple-Rising-Dose Study

2013 ◽  
Vol 57 (10) ◽  
pp. 4928-4936 ◽  
Author(s):  
Kristi L. Berger ◽  
Lisette Lagacé ◽  
Ibtissem Triki ◽  
Mireille Cartier ◽  
Martin Marquis ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Protease Inhibitor ◽  
Genotype 1 ◽  
Resistance Mutations ◽  
Virus Genotype ◽  
Drug Pressure ◽  
Phase 1B ◽  
Ns3 Protease ◽  
Emergence Of Resistance

ABSTRACTFaldaprevir (BI 201335) is a selective NS3/4A protease inhibitor under development for the treatment of chronic hepatitis C virus (HCV) infection. NS3/4A genotyping and NS3 protease phenotyping analyses were performed to monitor the emergence of resistance in patients with HCV genotype 1 infection receiving faldaprevir alone or combined with pegylated interferon alfa 2a and ribavirin (PegIFN-RBV) during a phase 1b study. Among all baseline variants, a maximum 7-fold reduction inin vitrosensitivity to faldaprevir was observed for a rare NS3 (V/I)170T polymorphism. During faldaprevir monotherapy in treatment-naive patients, virologic breakthrough was common (77%, 20/26) and was associated with the emergence of resistance mutations predominantly carrying NS3 substitutions R155K in GT1a and D168V in GT1b. D168V conferred a greater reduction in faldaprevir sensitivity (1,800-fold) than R155K (330-fold); however, D168V was generally less fit than R155K in the absence of selective drug pressure. Treatment-experienced patients treated with faldaprevir-PegIFN-RBV triple therapy showed higher viral load reductions, lower rates of breakthrough (8%, 5/62), and less frequent emergence of resistance-associated variants compared with faldaprevir monotherapy. (This study has been registered at ClinicalTrials.gov under registration no. NCT00793793.)

scholarly journals Characterization of V36C, a Novel Amino Acid Substitution Conferring Hepatitis C Virus (HCV) Resistance to Telaprevir, a Potent Peptidomimetic Inhibitor of HCV Protease

2010 ◽  
Vol 54 (6) ◽  
pp. 2681-2683 ◽  
Author(s):  
Laetitia Barbotte ◽  
Abdelhakim Ahmed-Belkacem ◽  
Stéphane Chevaliez ◽  
Alexandre Soulier ◽  
Christophe Hézode ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Enzyme Assay ◽  
Inhibitory Concentration ◽  
Fold Increase ◽  
Replication Capacity ◽  
Wild Type ◽  
Hcv Protease ◽  
Moderate Resistance

ABSTRACT We characterized a novel substitution conferring moderate resistance to telaprevir, a peptidomimetic inhibitor of hepatitis C virus protease. V36C conferred a 4.0-fold increase in the telaprevir 50% inhibitory concentration in an enzyme assay and a 9.5-fold increase in the replicon model. The replication capacity of a replicon harboring V36C was close to that of the wild-type protease. This case emphasizes the complexity of hepatitis C virus resistance to protease inhibitors.

scholarly journals Phenotypic Characterization of Resistant Val36 Variants of Hepatitis C Virus NS3-4A Serine Protease

2007 ◽  
Vol 52 (1) ◽  
pp. 110-120 ◽  
Author(s):  
Yi Zhou ◽  
Doug J. Bartels ◽  
Brian L. Hanzelka ◽  
Ute Müh ◽  
Yunyi Wei ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Serine Protease ◽  
Phenotypic Characterization ◽  
Replication Capacity ◽  
Wild Type ◽  
Protease Domain

ABSTRACT In patients chronically infected with hepatitis C virus (HCV) strains of genotype 1, rapid and dramatic antiviral activity has been observed with telaprevir (VX-950), a highly selective and potent inhibitor of the HCV NS3-4A serine protease. HCV variants with substitutions in the NS3 protease domain were observed in some patients during telaprevir dosing. In this study, purified protease domain proteins and reconstituted HCV subgenomic replicons were used for phenotypic characterization of many of these substitutions. V36A/M or T54A substitutions conferred less than eightfold resistance to telaprevir. Variants with double substitutions at Val36 plus Thr54 had ∼20-fold resistance to telaprevir, and variants with double substitutions at Val36 plus Arg155 or Ala156 had >40-fold resistance to telaprevir. An X-ray structure of the HCV strain H protease domain containing the V36M substitution in a cocomplex with an NS4A cofactor peptide was solved at a 2.4-Å resolution. Except for the side chain of Met36, the V36M variant structure is identical to that of the wild-type apoenzyme. The in vitro replication capacity of most variants was significantly lower than that of the wild-type replicon in cells, which is consistent with the impaired in vivo fitness estimated from telaprevir-dosed patients. Finally, the sensitivity of these replicon variants to alpha interferon or ribavirin remained unchanged compared to that of the wild-type.

scholarly journals Structural characterization of the Hepatitis C Virus NS3 protease from genotype 3a: The basis of the genotype 1b vs. 3a inhibitor potency shift

Virology ◽  
2010 ◽  
Vol 405 (2) ◽  
pp. 424-438 ◽  
Author(s):  
Mariana Gallo ◽  
Matthew James Bottomley ◽  
Matteo Pennestri ◽  
Tommaso Eliseo ◽  
Maurizio Paci ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Structural Characterization ◽  
Genotype 1B ◽  
Ns3 Protease ◽  
Genotype 3A

scholarly journals Genotypic and Phenotypic Analyses of Hepatitis C Virus Variants Observed in Clinical Studies of VX-222, a Nonnucleoside NS5B Polymerase Inhibitor

2014 ◽  
Vol 58 (9) ◽  
pp. 5456-5465 ◽  
Author(s):  
Min Jiang ◽  
Eileen Z. Zhang ◽  
Andrzej Ardzinski ◽  
Ann Tigges ◽  
Andrew Davis ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Clinical Studies ◽  
Phase 1 ◽  
Replication Capacity ◽  
Hcv Rna ◽  
Genotype 1 ◽  
Wild Type ◽  
Ns5b Polymerase Inhibitor ◽  
Hcv Ns5b

ABSTRACTVX-222, a thiophene-2-carboxylic acid derivative, is a selective nonnucleoside inhibitor of the hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase. In phase 1 and 2 clinical studies, VX-222 demonstrated effective antiviral efficacy, with substantial reductions in plasma HCV RNA in patients chronically infected with genotype 1 HCV. To characterize the potential for selection of VX-222-resistant variants in HCV-infected patients, the HCV NS5B gene was sequenced at baseline and during and after 3 days of VX-222 dosing (monotherapy) in a phase 1 study. Variants with the substitutions L419C/I/M/P/S/V, R422K, M423I/T/V, I482L/N/T, A486S/T/V, and V494A were selected during VX-222 dosing, and their levels declined over time after the end of dosing. Phenotypic analysis of these variants was conducted using HCV replicons carrying site-directed mutations. Of the 17 variants, 14 showed reduced susceptibility to VX-222 compared with the wild type, with the L419C/S and R422K variants having higher levels of resistance (>200-fold) than the rest of the variants (6.8- to 76-fold). The M423I and A486S variants remained susceptible to VX-222. The 50% effective concentration (EC50) for the L419P variant could not be obtained due to the poor replication of this replicon. The majority of the variants (15/17) were less fit than the wild type. A subset of the variants, predominately the L419S and R422K variants, were observed when the efficacy and safety of VX-222- and telaprevir-based regimens given for 12 weeks were investigated in genotype 1 HCV-infected patients in a phase 2 study. The NS3 and NS5B variants selected during the dual combination therapy showed reduced susceptibility to both telaprevir and VX-222 and had a lower replication capacity than the wild type. The phase 1b study has the ClinicalTrials.gov identifier NCT00911963, and the phase 2a study has ClinicalTrials.gov identifier NCT01080222.

scholarly journals Efficient Hepatitis C Virus Genotype 1b Core-NS5A Recombinants Permit Efficacy Testing of Protease and NS5A Inhibitors

2017 ◽  
Vol 61 (6) ◽  
Author(s):  
Long V. Pham ◽  
Santseharay Ramirez ◽  
Thomas H. R. Carlsen ◽  
Yi-Ping Li ◽  
Judith M. Gottwein ◽  
...  
Keyword(s):  
Cell Culture ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Resistance Testing ◽  
Replication Capacity ◽  
Genotype 1 ◽  
Virus Genotype ◽  
Culture Systems ◽  
Genotype 1B ◽  
Subtype 1B

ABSTRACT Hepatitis C virus (HCV) strains belong to seven genotypes with numerous subtypes that respond differently to antiviral therapies. Genotype 1, and primarily subtype 1b, is the most prevalent genotype worldwide. The development of recombinant HCV infectious cell culture systems for different variants, permitted by the high replication capacity of strain JFH1 (genotype 2a), has advanced efficacy and resistance testing of antivirals. However, efficient infectious JFH1-based cell cultures of subtype 1b are limited and comprise only the 5′ untranslated region (5′UTR)-NS2, NS4A, or NS5A regions. Importantly, it has not been possible to develop efficient 1b infectious systems expressing the NS3/4A protease, an important target of direct-acting antivirals. We developed efficient infectious JFH1-based cultures with genotype 1b core-NS5A sequences of strains DH1, Con1, and J4 by using previously identified HCV cell culture adaptive substitutions A1226G, R1496L, and Q1773H. These viruses spread efficiently in Huh7.5 cells by acquiring additional adaptive substitutions, and final recombinants yielded peak supernatant infectivity titers of 4 to 5 log10 focus-forming units (FFU)/ml. We subsequently succeeded in adapting a JFH1-based 5′UTR-NS5A DH1 recombinant to efficient growth in cell culture. We evaluated the efficacy of clinically relevant NS3/4A protease and NS5A inhibitors against the novel genotype 1b viruses, as well as against previously developed 1a viruses. The inhibitors were efficient against all tested genotype 1 viruses, with NS5A inhibitors showing half-maximal effective concentrations several orders of magnitude lower than NS3/4A protease inhibitors. In summary, the developed HCV genotype 1b culture systems represent valuable tools for assessing the efficacy of various classes of antivirals and for other virological studies requiring genotype 1b infectious viruses.

scholarly journals Differential Requirements of NS4A for Internal NS3 Cleavage and Polyprotein Processing of Hepatitis C Virus

2007 ◽  
Vol 81 (15) ◽  
pp. 7999-8008 ◽  
Author(s):  
Yi-Hen Kou ◽  
Ming-Fu Chang ◽  
Yi-Ming Wang ◽  
Tzu-Min Hung ◽  
Shin C. Chang
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Nonstructural Proteins ◽  
Polyprotein Processing ◽  
Culture Cells ◽  
Genotype 1B ◽  
Ns3 Protein ◽  
Transforming Activity ◽  
Viral Polyprotein ◽  
Ns3 Protease

ABSTRACT The NS3 protein of hepatitis C virus (HCV) possesses protease activity responsible for the proteolytic cleavage of the viral polyprotein at the junctions of nonstructural proteins downstream of NS3. The NS3 protein was also found to be internally cleaved. In this study, we demonstrated that internal cleavages occurred on the NS3 protein of genotype 1b in the presence of NS4A, both in culture cells and with a mouse model system. No internal cleavage products were detected with the NS3 and NS4A proteins of genotype 2a. Three potential cleavage sites were detected in the NS3 protein (genotype 1b), with IPT402|S being the major one. The internal cleavage requires the polyprotein processing activity of NS3 protease, but when supplemented in trans, the internal cleavage efficiency is reduced. In addition, several mutations in NS4A disrupted the internal cleavage of NS3 but did not affect polyprotein processing, indicating that NS4A contributes differently to these two proteolytic activities. Furthermore, Ile-25, Val-26, and Ile-29 of the NS4A protein, important for the NS4A-dependent internal cleavages, were also shown to be critical for the transforming activity of NS3, but mutations at these critical residues resulted only in a slight increase of HCV replicating efficiency. The internal cleavage-associated enhancement of the transforming activity of NS3 was reduced when a T402A substitution at the major internal cleavage site was introduced. The multiple roles of NS4A in viral multiplication and pathogenesis make NS4A an ideal molecular target for HCV therapy.

scholarly journals Comparative In Vitro Anti-Hepatitis C Virus Activities of a Selected Series of Polymerase, Protease, and Helicase Inhibitors

2008 ◽  
Vol 52 (9) ◽  
pp. 3433-3437 ◽  
Author(s):  
Jan Paeshuyse ◽  
Inge Vliegen ◽  
Lotte Coelmont ◽  
Pieter Leyssen ◽  
Oriana Tabarrini ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Comparative Study ◽  
Hcv Genotype ◽  
Genotype 1B ◽  
Polymerase Iii ◽  
Ns3 Protease

ABSTRACT We report here a comparative study of the anti-hepatitis C virus (HCV) activities of selected (i) nucleoside polymerase, (ii) nonnucleoside polymerase, (iii) α,γ-diketo acid polymerase, (iv) NS3 protease, and (v) helicase inhibitors, as well as (vi) cyclophilin binding molecules and (vii) alpha 2b interferon in four different HCV genotype 1b replicon systems.

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