Substitutions at NS3 Residue 155, 156, or 168 of Hepatitis C Virus Genotypes 2 to 6 Induce Complex Patterns of Protease Inhibitor Resistance

Jensen, Sanne B.; Serre, Stéphanie B. N.; Humes, Daryl; Ramírez, Santseharay; Li, Yiping; Bukh, Jens; Gottwein, Judith M.

doi:10.1128/aac.01953-15

Cited by 42 publications

(49 citation statements)

References 59 publications

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“…Huh7.5 cells (3.5 ϫ 10 5 /well) were plated in a 6-well plate, incubated overnight, and infected with stocks of HCV genotype 1 to 6 recombinants for 24 h. PIs purchased from Acme Bioscience were dissolved in dimethyl sulfoxide (Sigma) (21,28 50 of faldaprevir (BI 201335), paritaprevir (ABT-450), or deldeprevir (ACH-2684) was initiated when ϳ1% to 20% cells were infected. Cultures were retreated with the PI upon cell splitting every 2 to 3 days; cells plated on a chamber slide after treatment and incubated overnight were immunostained as described above in order to evaluate viral spread.…”

Section: Methodsmentioning

confidence: 99%

“…Substitutions that occurred in nontreated cultures were not studied. Most recombinants encoding the R155K and A156S substitutions have been published (28). For final plasmid preparations (Qiagen Plasmid Maxi kit or Plasmid Midi kit), the complete HCV genome was sequenced (Macrogen).…”

Section: Methodsmentioning

confidence: 99%

“…For each virus and compound, a concentration-response curve was generated from which the EC 50 was determined as described previously (21,27). Fold resistance was calculated by relating the EC 50 obtained for the variant to the EC 50 obtained for the original virus included in the same experiment, as described by us recently (28).…”

Section: Methodsmentioning

confidence: 99%

“…Genotype 2a (isolate JFH1) recombinants with substitutions of A, G, E, or V for D168, selected in 2a (JFH1) treated with newer PIs (Fig. 2), showed different degrees of fitness impairment but were all genetically stable (28). Overall, viral fitness depended on the NS3P position, the specific substitution, and the recombinant (Fig.…”

Section: Ns3p Substitutions In Escape Variantsmentioning

confidence: 99%

“…1) on the fitness of the recombinants in which they were identified. We examined 60 recombinants with single NS3P substitutions, including previously tested R155K and A156S recombinants (28), and 17 recombinants encoding combinations of NS3P substitutions (Fig. 3).…”

Section: Ns3p Substitutions In Escape Variantsmentioning

confidence: 99%

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

Serre

Jensen

Ghanem

et al. 2016

Antimicrob Agents Chemother

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bHepatitis 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. With more than 100 million chronic infections causing approximately 500,000 deaths annually, hepatitis C virus (HCV) is a major global health and economic burden (1, 2). The six epidemiologically important genotypes differ in ϳ30% of their sequence and in their sensitivity to antiviral regimens (3-6). In Europe, the Americas, Asia, and Australasia, genotypes 1, 2, and 3 are most prevalent. While genotypes 4, 5, and 6 are more restricted to specific geographical regions in Africa and Asia, they account for 20% of global HCV infections and have spread beyond these primary geographical locations (1, 2, 7).The development of directly acting antivirals (DAAs) has revolutionized HCV therapy. The main components of interferonfree regimens introduced in the clinic are inhibitors of the HCV nonstructural (NS) proteins NS3 protease (NS3P), NS5A,8,9). Even though DAA-based therapy regimens could cure most patients in clinical trials, failure rates of 5 to 10% are to be expected in real life, due primarily to the development of DAA resistance (4,8). Given the large ...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Ns3p Substitutions In Escape Variantsmentioning

confidence: 99%

Section: Ns3p Substitutions In Escape Variantsmentioning

confidence: 99%

See 3 more Smart Citations

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

Serre

Jensen

Ghanem

et al. 2016

Antimicrob Agents Chemother

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Mechanisms of Resistance to Antiviral Agents

Shafer,

Boivin,

Howe

et al. 2023

ClinMicroNow

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Understanding the mechanisms of viral drug resistance is critical for clinical management of individuals receiving antiviral therapy, for developing new antiviral drugs, and for drug resistance surveillance. This chapter reviews the mechanisms of resistance to antiviral drugs used to treat infections by eight common viruses: herpes simplex, cytomegalovirus, varicella‐zoster virus, human immunodeficiency virus type 1, influenza A and B, hepatitis B, hepatitis C, and severe acute respiratory syndrome coronavirus 2. Drug‐resistant virus subpopulations may exist at low levels in clinical isolates or may arise only during drug exposure. The error‐prone polymerases in RNA viruses render the development of resistance more frequent than in DNA viruses. Drug‐resistant viruses are identified by in vitro passage experiments in which wild‐type viruses are cultured in increasing concentrations of an inhibitory drug and by ex vivo analysis of virus isolates obtained from individuals receiving antiviral therapy.

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Evolutionary Pathways to Persistence of Highly Fit and Resistant Hepatitis C Virus Protease Inhibitor Escape Variants

Jensen¹,

Fahnøe²,

Pham³

et al. 2019

Hepatology

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Protease inhibitors (PIs) are important components of treatment regimens for patients with chronic hepatitis C virus (HCV) infection. However, emergence and persistence of antiviral resistance could reduce their efficacy. Thus, defining resistance determinants is highly relevant for efforts to control HCV. Here, we investigated patterns of PI resistance–associated substitutions (RASs) for the major HCV genotypes and viral determinants for persistence of key RASs. We identified protease position 156 as a RAS hotspot for genotype 1‐4, but not 5 and 6, escape variants by resistance profiling using PIs grazoprevir and paritaprevir in infectious cell culture systems. However, except for genotype 3, engineered 156‐RASs were not maintained. For genotypes 1 and 2, persistence of 156‐RASs depended on genome‐wide substitution networks, co‐selected under continued PI treatment and identified by next‐generation sequencing with substitution linkage and haplotype reconstruction. Persistence of A156T for genotype 1 relied on compensatory substitutions increasing replication and assembly. For genotype 2, initial selection of A156V facilitated transition to 156L, persisting without compensatory substitutions. The developed genotype 1, 2, and 3 variants with persistent 156‐RASs had exceptionally high fitness and resistance to grazoprevir, paritaprevir, glecaprevir, and voxilaprevir. A156T dominated in genotype 1 glecaprevir and voxilaprevir escape variants, and pre‐existing A156T facilitated genotype 1 escape from clinically relevant combination treatments with grazoprevir/elbasvir and glecaprevir/pibrentasvir. In genotype 1 infected patients with treatment failure and 156‐RASs, we observed genome‐wide selection of substitutions under treatment. Conclusion: Comprehensive PI resistance profiling for HCV genotypes 1‐6 revealed 156‐RASs as key determinants of high‐level resistance across clinically relevant PIs. We obtained in vitro proof of concept for persistence of highly fit genotype 1‐3 156‐variants, which might pose a threat to clinically relevant combination treatments.

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Substitutions at NS3 Residue 155, 156, or 168 of Hepatitis C Virus Genotypes 2 to 6 Induce Complex Patterns of Protease Inhibitor Resistance

Cited by 42 publications

References 59 publications

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

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

Mechanisms of Resistance to Antiviral Agents

Evolutionary Pathways to Persistence of Highly Fit and Resistant Hepatitis C Virus Protease Inhibitor Escape Variants

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