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Protease inhibitor-resistant hepatitis C virus mutants with reduced fitness from impaired production of infectious virus.

ABSTRACT: Several small molecule inhibitors of the hepatitis C virus (HCV) nonstructural protein (NS) 3/4A protease have advanced successfully to clinical trials. However, the selection of drug-resistant mutants is a significant issue with protease inhibitors (PIs). A variety of amino acid substitutions in the protease domain of NS3 can lead to PI resistance. Many of these significantly impair the replication fitness of HCV RNA replicons. However, it is not known whether these mutations also adversely affect infectious virus assembly and release, processes in which NS3 also participates.We studied the impact of 25 previously identified PI-resistance mutations on the capacity of genotype 1a H77S RNA to replicate in cell culture and produce infectious virus.Most PI-resistance mutations resulted in moderate loss of replication competence, although several (V36A/L/M, R109K, and D168E) showed fitness comparable to wild type, whereas others (S138T and A156V) were severely impaired both in RNA replication and infectious virus production. Although reductions in RNA replication capacity correlated with decreased yields of infectious virus for most mutations, a subset of mutants (Q41R, F43S, R155T, A156S, and I170A/T) showed greater impairment in their ability to produce virus than predicted from reductions in RNA replication capacity. Detailed examination of the I170A mutant showed no defect in release of virus from cells and no significant difference in specific infectivity of extracellular virus particles.Replicon-based assays might underestimate the loss of fitness caused by PI-resistance mutations, because some mutations in the NS3 protease domain specifically impair late steps in the viral life cycle that involve intracellular assembly of infectious virus.

SUBMITTER: Shimakami T

PROVIDER: S-EPMC3155954 | biostudies-literature | 2011 Feb

REPOSITORIES: biostudies-literature

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Protease inhibitor-resistant hepatitis C virus mutants with reduced fitness from impaired production of infectious virus.

Shimakami Tetsuro T Welsch Christoph C Yamane Daisuke D McGivern David R DR Yi Minkyung M Zeuzem Stefan S Lemon Stanley M SM

Gastroenterology 20101104 2

<h4>Background & aims</h4>Several small molecule inhibitors of the hepatitis C virus (HCV) nonstructural protein (NS) 3/4A protease have advanced successfully to clinical trials. However, the selection of drug-resistant mutants is a significant issue with protease inhibitors (PIs). A variety of amino acid substitutions in the protease domain of NS3 can lead to PI resistance. Many of these significantly impair the replication fitness of HCV RNA replicons. However, it is not known whether these mu ...[more]

PMID: 21056040

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Project description: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.

Project description: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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Protease inhibitor-resistant hepatitis C virus mutants with reduced fitness from impaired production of infectious virus.

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Protease inhibitor-resistant hepatitis C virus mutants with reduced fitness from impaired production of infectious virus.

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