scholarly journals Hepatitis C Virus Genotype 1 to 6 Protease Inhibitor Escape Variants:In VitroSelection, Fitness, and Resistance Patterns in the Context of the Infectious Viral Life Cycle

2016 ◽  
Vol 60 (6) ◽  
pp. 3563-3578 ◽  
Author(s):  
Stéphanie B. N. Serre ◽  
Sanne B. Jensen ◽  
Lubna Ghanem ◽  
Daryl G. Humes ◽  
Santseharay Ramirez ◽  
...  
Keyword(s):  
Life Cycle ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Hot Spots ◽  
Viral Life Cycle ◽  
Viral Fitness ◽  
Cross Resistance ◽  
Genotype 1 ◽  
Genotype 2 ◽  
Ns3 Protease

Hepatitis C virus (HCV) NS3 protease inhibitors (PIs) are important components of novel HCV therapy regimens. Studies of PI resistance initially focused on genotype 1. Therefore, knowledge about the determinants of PI resistance for the highly prevalent genotypes 2 to 6 remains limited. Using Huh7.5 cell culture-infectious HCV recombinants with genotype 1 to 6 NS3 protease, we identified protease positions 54, 155, and 156 as hot spots for the selection of resistance substitutions under treatment with the first licensed PIs, telaprevir and boceprevir. Treatment of a genotype 2 isolate with the newer PIs vaniprevir, faldaprevir, simeprevir, grazoprevir, paritaprevir, and deldeprevir identified positions 156 and 168 as hot spots for resistance; the Y56H substitution emerged for three newer PIs. Substitution selection also depended on the specific recombinant. The substitutions identified conferred cross-resistance to several PIs; however, most substitutions selected under telaprevir or boceprevir treatment conferred less resistance to certain newer PIs. In a single-cycle production assay, across genotypes, PI treatment primarily decreased viral replication, which was rescued by PI resistance substitutions. The substitutions identified resulted in differential effects on viral fitness, depending on the original recombinant and the substitution. Across genotypes, fitness impairment induced by resistance substitutions was due primarily to decreased replication. Most combinations of substitutions that were identified increased resistance or fitness. Combinations of resistance substitutions with fitness-compensating substitutions either rescued replication or compensated for decreased replication by increasing assembly. This comprehensive study provides insight into the selection patterns and effects of PI resistance substitutions for HCV genotypes 1 to 6 in the context of the infectious viral life cycle, which is of interest for clinical and virological HCV research.

scholarly journals Preclinical Characterization of BMS-791325, an Allosteric Inhibitor of Hepatitis C Virus NS5B Polymerase

2014 ◽  
Vol 58 (6) ◽  
pp. 3485-3495 ◽  
Author(s):  
Julie A. Lemm ◽  
Mengping Liu ◽  
Robert G. Gentles ◽  
Min Ding ◽  
Stacey Voss ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Nucleoside Analogs ◽  
Synergistic Activity ◽  
Genotype 1 ◽  
Allosteric Inhibitor ◽  
Genotype 2 ◽  
Hcv Genotype 1 ◽  
Ns3 Protease

ABSTRACTBMS-791325 is an allosteric inhibitor that binds to thumb site 1 of the hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase. BMS-791325 inhibits recombinant NS5B proteins from HCV genotypes 1, 3, 4, and 5 at 50% inhibitory concentrations (IC50) below 28 nM. In cell culture, BMS-791325 inhibited replication of HCV subgenomic replicons representing genotypes 1a and 1b at 50% effective concentrations (EC50s) of 3 nM and 6 nM, respectively, with similar (3 to 18 nM) values for genotypes 3a, 4a, and 5a. Potency against genotype 6a showed more variability (9 to 125 nM), and activity was weaker against genotype 2 (EC50, 87 to 925 nM). Specificity was demonstrated by the absence of activity (EC50s of >4 μM) against a panel of mammalian viruses, and cytotoxic concentrations (50%) were >3,000-fold above the HCV EC50. Resistance substitutions selected by BMS-791325 in genotype 1 replicons mostly mapped to a single site, NS5B amino acid 495 (P495A/S/L/T). Additive or synergistic activity was observed in combination studies using BMS-791325 with alfa interferon plus ribavirin, inhibitors of NS3 protease or NS5A, and other classes of NS5B inhibitor (palm site 2-binding or nucleoside analogs). Plasma and liver exposuresin vivoin several animal species indicated that BMS-791325 has a hepatotropic disposition (liver-to-plasma ratios ranging from 1.6- to 60-fold across species). Twenty-four hours postdose, liver exposures across all species tested were ≥10-fold above the inhibitor EC50s observed with HCV genotype 1 replicons. These findings support the evaluation of BMS-791325 in combination regimens for the treatment of HCV. Phase 3 studies are ongoing.

773 HEPATITIS C VIRUS AND LIPID DROPLETS: ROLE OF SEIPIN IN LIPID DROPLET MATURATION AND VIRAL LIFE CYCLE

2011 ◽  
Vol 54 ◽  
pp. S311 ◽  
Author(s):  
S. Clement ◽  
C. Fauvelle ◽  
S. Pascarella ◽  
S. Conzelmann ◽  
V. Kaddai ◽  
...  
Keyword(s):  
Life Cycle ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Lipid Droplet ◽  
Lipid Droplets ◽  
Viral Life Cycle

scholarly journals Preclinical Characterization of the Novel Hepatitis C Virus NS3 Protease Inhibitor GS-9451

2013 ◽  
Vol 58 (2) ◽  
pp. 647-653 ◽  
Author(s):  
Huiling Yang ◽  
Margaret Robinson ◽  
Amoreena C. Corsa ◽  
Betty Peng ◽  
Guofeng Cheng ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Antiviral Activity ◽  
Protease Inhibitor ◽  
Hcv Infection ◽  
Cross Resistance ◽  
Protease Gene ◽  
Ns5a Inhibitor ◽  
Ns3 Protease

ABSTRACTGS-9451 is a selective hepatitis C virus (HCV) NS3 protease inhibitor in development for the treatment of genotype 1 (GT1) HCV infection. Key preclinical properties of GS-9451, includingin vitroantiviral activity, selectivity, cross-resistance, and combination activity, as well as pharmacokinetic properties, were determined. In multiple GT1a and GT1b replicon cell lines, GS-9451 had mean 50% effective concentrations (EC50s) of 13 and 5.4 nM, respectively, with minimal cytotoxicity; similar potency was observed in chimeric replicons encoding the NS3 protease gene of GT1 clinical isolates. GS-9451 was less active in GT2a replicon cells (EC50= 316 nM). Additive to synergisticin vitroantiviral activity was observed when GS-9451 was combined with other agents, including alpha interferon, ribavirin, and the polymerase inhibitors GS-6620 and tegobuvir (GS-9190), as well as the NS5A inhibitor ledipasvir (GS-5885). GS-9451 retained wild-type activity against multiple classes of NS5B and NS5A inhibitor resistance mutations. GS-9451 was stable in hepatic microsomes and hepatocytes from human and three other tested species. Systemic clearance was low in dogs and monkeys but high in rats. GS-9451 showed good oral bioavailability in all three species tested. In rats, GS-9451 levels were ∼40-fold higher in liver than plasma after intravenous dosing, and elimination of GS-9451 was primarily through biliary excretion. Together, these results are consistent with the antiviral activity observed in a recent phase 1b study. The results ofin vitrocross-resistance and combination antiviral assays support the ongoing development of GS-9451 in combination with other agents for the treatment of chronic HCV infection.

scholarly journals Selection of Replicon Variants Resistant to ACH-806, a Novel Hepatitis C Virus Inhibitor with No Cross-Resistance to NS3 Protease and NS5B Polymerase Inhibitors

2008 ◽  
Vol 52 (6) ◽  
pp. 2043-2052 ◽  
Author(s):  
Wengang Yang ◽  
Yongsen Zhao ◽  
Joanne Fabrycki ◽  
Xiaohong Hou ◽  
Xingtie Nie ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Amino Acid ◽  
Protease Inhibitors ◽  
Cross Resistance ◽  
Polymerase Inhibitors ◽  
Ns3 Protease Inhibitors ◽  
Ns3 Protease ◽  
Ns5b Polymerase ◽  
Selection Of

ABSTRACT We have discovered a novel class of compounds active against hepatitis C virus (HCV), using a surrogate cellular system, HCV replicon cells. The leading compound in the series, ACH-806 (GS-9132), is a potent and specific inhibitor of HCV. The selection of resistance replicon variants against ACH-806 was performed to map the mutations conferring resistance to ACH-806 and to determine cross-resistance profiles with other classes of HCV inhibitors. Several clones emerged after the addition of ACH-806 to HCV replicon cells at frequencies and durations similar to that observed with NS3 protease inhibitors and NS5B polymerase inhibitors. Phenotypic analyses of these clones revealed that they are resistant to ACH-806 but remain sensitive to other classes of HCV inhibitors. Moreover, no significant change in the susceptibility to ACH-806 was found when the replicon cellular clones resistant to NS3 protease inhibitors and NS5B polymerase inhibitors were examined. Sequencing of the entire coding region of ACH-806-resistant replicon variants yielded several consensus mutations. Reverse genetics identified two single mutations in NS3, a cysteine-to-serine mutation at amino acid 16 and an alanine-to-valine mutation at amino acid 39, that are responsible for the resistance of the replicon variants to ACH-806. Both mutations are located at the N terminus of NS3 where extensive interactions with the central hydrophobic region of NS4A exist. These data provide evidence that ACH-806 inhibits HCV replication by a novel mechanism.

scholarly journals Epidemic history of hepatitis C virus infection in two remote communities in Nigeria, West Africa

2012 ◽  
Vol 93 (7) ◽  
pp. 1410-1421 ◽  
Author(s):  
Joseph C. Forbi ◽  
Michael A. Purdy ◽  
David S. Campo ◽  
Gilberto Vaughan ◽  
Zoya E. Dimitrova ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
20Th Century ◽  
Hypervariable Region ◽  
Hcv Infection ◽  
Genotype 1 ◽  
Remote Communities ◽  
African Countries ◽  
Genotype 2 ◽  
History Of

We investigated the molecular epidemiology and population dynamics of HCV infection among indigenes of two semi-isolated communities in North-Central Nigeria. Despite remoteness and isolation, ~15 % of the population had serological or molecular markers of hepatitis C virus (HCV) infection. Phylogenetic analysis of the NS5b sequences obtained from 60 HCV-infected residents showed that HCV variants belonged to genotype 1 (n = 51; 85 %) and genotype 2 (n = 9; 15 %). All sequences were unique and intermixed in the phylogenetic tree with HCV sequences from people infected from other West African countries. The high-throughput 454 pyrosequencing of the HCV hypervariable region 1 and an empirical threshold error correction algorithm were used to evaluate intra-host heterogeneity of HCV strains of genotype 1 (n = 43) and genotype 2 (n = 6) from residents of the communities. Analysis revealed a rare detectable intermixing of HCV intra-host variants among residents. Identification of genetically close HCV variants among all known groups of relatives suggests a common intra-familial HCV transmission in the communities. Applying Bayesian coalescent analysis to the NS5b sequences, the most recent common ancestors for genotype 1 and 2 variants were estimated to have existed 675 and 286 years ago, respectively. Bayesian skyline plots suggest that HCV lineages of both genotypes identified in the Nigerian communities experienced epidemic growth for 200–300 years until the mid-20th century. The data suggest a massive introduction of numerous HCV variants to the communities during the 20th century in the background of a dynamic evolutionary history of the hepatitis C epidemic in Nigeria over the past three centuries.

scholarly journals Role of Conserved E2 Residue W420 in Receptor Binding and Hepatitis C Virus Infection

2016 ◽  
Vol 90 (16) ◽  
pp. 7456-7468 ◽  
Author(s):  
Vanessa M. Cowton ◽  
Allan G. N. Angus ◽  
Sarah J. Cole ◽  
Christina K. Markopoulou ◽  
Ania Owsianka ◽  
...  
Keyword(s):  
Cell Culture ◽  
Life Cycle ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Virus Entry ◽  
Viral Life Cycle ◽  
Tryptophan Residue ◽  
Viral Glycoprotein ◽  
Cd81 Binding

ABSTRACTHepatitis C virus (HCV) enters cells via interactions with several host factors, a key one being that between the viral E2 envelope glycoprotein and the CD81 receptor. We previously identified E2 tryptophan residue 420 (W420) as an essential CD81-binding residue. However, the importance of W420 in the context of the native virion is unknown, as those previous studies predate the infectious HCV cell culture (cell culture-derived HCV [HCVcc]) system. Here, we introduced four separate mutations (F, Y, A, or R) at position 420 within the infectious HCVcc JFH-1 genome and characterized their effects on the viral life cycle. While all mutations reduced E2-CD81 binding, only two (W420A and W420R) reduced HCVcc infectivity. Further analyses of mutants with hydrophobic residues (F or Y) found that interactions with the receptors SR-BI and CD81 were modulated, which in turn determined the viral uptake route. Both mutant viruses were significantly less dependent on SR-BI, and its lipid transfer activity, for virus entry. Furthermore, these viruses were resistant to the drug erlotinib, which targets epidermal growth factor receptor (EGFR) (a host cofactor for HCV entry) and also blocks SR-BI-dependent high-density lipoprotein (HDL)-mediated enhancement of virus entry. Together, our data indicate a model where an alteration at position 420 causes a subtle change in the E2 conformation that prevents interaction with SR-BI and increases accessibility to the CD81-binding site, in turn favoring a particular internalization route. These results further show that a hydrophobic residue with a strong preference for tryptophan at position 420 is important, both functionally and structurally, to provide an additional hydrophobic anchor to stabilize the E2-CD81 interaction.IMPORTANCEHepatitis C virus (HCV) is a leading cause of liver disease, causing up to 500,000 deaths annually. The first step in the viral life cycle is the entry process. This study investigates the role of a highly conserved residue, tryptophan residue 420, of the viral glycoprotein E2 in this process. We analyzed the effect of changing this residue in the virus and confirmed that this region is important for binding to the CD81 receptor. Furthermore, alteration of this residue modulated interactions with the SR-BI receptor, and changes to these key interactions were found to affect the virus internalization route involving the host cofactor EGFR. Our results also show that the nature of the amino acid at this position is important functionally and structurally to provide an anchor point to stabilize the E2-CD81 interaction.

scholarly journals In VitroandIn VivoAntiviral Activity and Resistance Profile of the Hepatitis C Virus NS3/4A Protease Inhibitor ABT-450

2014 ◽  
Vol 59 (2) ◽  
pp. 988-997 ◽  
Author(s):  
Tami Pilot-Matias ◽  
Rakesh Tripathi ◽  
Daniel Cohen ◽  
Isabelle Gaultier ◽  
Tatyana Dekhtyar ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Antiviral Agents ◽  
Cross Resistance ◽  
Hcv Rna ◽  
Genotype 1 ◽  
Common Amino Acid ◽  
Direct Acting Antiviral ◽  
Genotype 1A

ABSTRACTThe development of direct-acting antiviral agents is a promising therapeutic advance in the treatment of hepatitis C virus (HCV) infection. However, rapid emergence of drug resistance can limit efficacy and lead to cross-resistance among members of the same drug class. ABT-450 is an efficacious inhibitor of HCV NS3/4A protease, with 50% effective concentration values of 1.0, 0.21, 5.3, 19, 0.09, and 0.69 nM against stable HCV replicons with NS3 protease from genotypes 1a, 1b, 2a, 3a, 4a, and 6a, respectively.In vitro, the most common amino acid variants selected by ABT-450 in genotype 1 were located in NS3 at positions 155, 156, and 168, with the D168Y variant conferring the highest level of resistance to ABT-450 in both genotype 1a and 1b replicons (219- and 337-fold, respectively). In a 3-day monotherapy study with HCV genotype 1-infected patients, ABT-450 was coadministered with ritonavir, a cytochrome P450 3A4 inhibitor shown previously to markedly increase peak, trough, and overall drug exposures of ABT-450. A mean maximum HCV RNA decline of 4.02 log10was observed at the end of the 3-day dosing period across all doses. The most common variants selected in these patients were R155K and D168V in genotype 1a and D168V in genotype 1b. However, selection of resistant variants was significantly reduced at the highest ABT-450 dose compared to lower doses. These findings were informative for the subsequent evaluation of ABT-450 in combination with additional drug classes in clinical trials in HCV-infected patients. (Study M11-602 is registered at ClinicalTrials.gov under registration no. NCT01074008.)

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 In VitroActivity of Simeprevir against Hepatitis C Virus Genotype 1 Clinical Isolates and Its Correlation with NS3 Sequence and Site-Directed Mutants

2015 ◽  
Vol 59 (12) ◽  
pp. 7548-7557 ◽  
Author(s):  
Thierry Verbinnen ◽  
Bart Fevery ◽  
Leen Vijgen ◽  
Tom Jacobs ◽  
Sandra De Meyer ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Genotype 1 ◽  
Low Level ◽  
Fold Reduction ◽  
Hcv Genotype 1 ◽  
High Level ◽  
Ns3 Protease ◽  
Level Resistance

ABSTRACTSimeprevir (TMC435) is a once-daily, single-pill, oral hepatitis C virus (HCV) NS3 protease inhibitor approved for the treatment of chronic HCV infection. Phenotypic characterization of baseline isolates and isolates from HCV genotype 1-infected patients failing with a simeprevir-based regimen was performed using chimeric replicons carrying patient-derived NS3 protease sequences. Cutoff values differentiating between full susceptibility to simeprevir (≤2.0-fold reduction in simeprevir activity) and low-level versus high-level resistance (≥50-fold reduction in simeprevir activity) were determined. The median simeprevir fold change in the 50% effective concentration (FC) of pretreatment genotype 1a isolates, with and without Q80K, and genotype 1b isolates was 11, 0.9, and 0.4, respectively. Naturally occurring NS3 polymorphisms that reduced simeprevir activity, other than Q80K, were uncommon in the simeprevir studies and generally conferred low-level resistancein vitro. Although the proportion of patients with failure differed by HCV geno/subtype and/or presence of baseline Q80K, the level of simeprevir resistance observed at failure was similarly high irrespective of type of failure, HCV genotype 1 subtype, and presence or absence of baseline Q80K. At the end of the study, simeprevir activity against isolates that lost the emerging amino acid substitution returned to pretreatment values. Activity of simeprevir against clinical isolates and site-directed mutant replicons harboring the corresponding single or double amino acid substitutions correlated well, showing that simeprevir resistance can be attributed to these substitutions. In conclusion, pretreatment NS3 isolates were generally fully susceptible (FC, ≤2.0) or conferred low-level resistance to simeprevirin vitro(FC, >2.0 and <50). Treatment failure with a simeprevir-based regimen was associated with emergence of high-level-resistance variants (FC, ≥50).

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