Recombinant HCV variants with NS5A from genotypes 1-7 have different sensitivities to an NS5A inhibitor but not interferon-a

Troels K H Scheel; Judith M Gottwein; Lotte S Mikkelsen; Tanja B Jensen; Jens Bukh

doi:10.1053/j.gastro.2010.11.036

Recombinant HCV variants with NS5A from genotypes 1-7 have different sensitivities to an NS5A inhibitor but not interferon-a

Troels K H Scheel, Judith M Gottwein, Lotte S Mikkelsen, Tanja B Jensen, Jens Bukh

119 Citationer (Scopus)

Abstract

Background & Aims: Heterogeneity in the hepatitis C virus (HCV) protein NS5A influences its sensitivity to interferon-based therapy. Furthermore, NS5A is an important target for development of HCV-specific inhibitors. We aimed to develop recombinant infectious cell culture systems that express NS5A from isolates of the 7 major HCV genotypes, and determining their sensitivity to a specific NS5A inhibitor and to interferon-α. Methods: Huh7.5 hepatoma cells were transfected with RNA of genotype 17 NS5A recombinants. Viability was determined by measuring HCV replication and infectivity titers. Putative adaptive mutations were analyzed by reverse genetics. The activity of antiviral agents was determined in high-throughput infection assays. Results: Cells infected with viable HCV that expressed NS5A of genotypes 17 produced relatively high viral titers; most NS5A recombinants required introduction of specific adaptive mutations. The efficacy of the NS5A inhibitor BMS-790052 varied greatly, based on NS5A isolate, with median effective concentration (EC ₅₀) values ranging from 0.009 nmol/L to 14 nmol/L; the high sensitivity of genotype 1b NS5A to BMS-790052 reflected observations from clinical studies. Specific residues in NS5A domain I were associated with >100-fold variations in sensitivity between isolates of the same HCV subtype. The Y/T2065H mutation conferred resistance to BMS-790052 that varied among NS5A isolates. When infected cultures were incubated with interferon-α, all NS5A recombinants had EC₅₀ values of ∼0.2 IU/mL, including an NS5A genotype 1b mutant with a putative sensitive-type, interferon sensitivity determining region. Conclusions: We developed efficient in vitro systems in which recombinant viruses express HCV genotypes 17 NS5A; these permit genotype- and isolate-specific analyses of NS5A and the effects of antiviral compounds and resistance mutations. These culture systems will facilitate development of specific inhibitors against NS5A of different HCV variants.

Originalsprog	Engelsk
Tidsskrift	Gastroenterology Today
Vol/bind	140
Udgave nummer	3
Sider (fra-til)	1032-42
Antal sider	11
ISSN	0969-0131
DOI	https://doi.org/10.1053/j.gastro.2010.11.036
Status	Udgivet - mar. 2011

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10.1053/j.gastro.2010.11.036

http://www.gastrojournal.org/article/S0016508510017269/pdf

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title = "Recombinant HCV variants with NS5A from genotypes 1-7 have different sensitivities to an NS5A inhibitor but not interferon-a",

abstract = "Background & Aims: Heterogeneity in the hepatitis C virus (HCV) protein NS5A influences its sensitivity to interferon-based therapy. Furthermore, NS5A is an important target for development of HCV-specific inhibitors. We aimed to develop recombinant infectious cell culture systems that express NS5A from isolates of the 7 major HCV genotypes, and determining their sensitivity to a specific NS5A inhibitor and to interferon-α. Methods: Huh7.5 hepatoma cells were transfected with RNA of genotype 17 NS5A recombinants. Viability was determined by measuring HCV replication and infectivity titers. Putative adaptive mutations were analyzed by reverse genetics. The activity of antiviral agents was determined in high-throughput infection assays. Results: Cells infected with viable HCV that expressed NS5A of genotypes 17 produced relatively high viral titers; most NS5A recombinants required introduction of specific adaptive mutations. The efficacy of the NS5A inhibitor BMS-790052 varied greatly, based on NS5A isolate, with median effective concentration (EC 50) values ranging from 0.009 nmol/L to 14 nmol/L; the high sensitivity of genotype 1b NS5A to BMS-790052 reflected observations from clinical studies. Specific residues in NS5A domain I were associated with >100-fold variations in sensitivity between isolates of the same HCV subtype. The Y/T2065H mutation conferred resistance to BMS-790052 that varied among NS5A isolates. When infected cultures were incubated with interferon-α, all NS5A recombinants had EC50 values of ∼0.2 IU/mL, including an NS5A genotype 1b mutant with a putative sensitive-type, interferon sensitivity determining region. Conclusions: We developed efficient in vitro systems in which recombinant viruses express HCV genotypes 17 NS5A; these permit genotype- and isolate-specific analyses of NS5A and the effects of antiviral compounds and resistance mutations. These culture systems will facilitate development of specific inhibitors against NS5A of different HCV variants.",

keywords = "Antiviral Agents, Cell Line, Tumor, Cell Survival, Dose-Response Relationship, Drug, Enzyme Inhibitors, Genotype, Hepacivirus, High-Throughput Screening Assays, Humans, Imidazoles, Interferon-alpha, Mutation, Phenotype, Recombinant Proteins, Time Factors, Viral Nonstructural Proteins, Virus Replication",

author = "Scheel, {Troels K H} and Gottwein, {Judith M} and Mikkelsen, {Lotte S} and Jensen, {Tanja B} and Jens Bukh",

year = "2011",

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doi = "10.1053/j.gastro.2010.11.036",

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T1 - Recombinant HCV variants with NS5A from genotypes 1-7 have different sensitivities to an NS5A inhibitor but not interferon-a

AU - Scheel, Troels K H

AU - Gottwein, Judith M

AU - Mikkelsen, Lotte S

AU - Jensen, Tanja B

AU - Bukh, Jens

PY - 2011/3

Y1 - 2011/3

N2 - Background & Aims: Heterogeneity in the hepatitis C virus (HCV) protein NS5A influences its sensitivity to interferon-based therapy. Furthermore, NS5A is an important target for development of HCV-specific inhibitors. We aimed to develop recombinant infectious cell culture systems that express NS5A from isolates of the 7 major HCV genotypes, and determining their sensitivity to a specific NS5A inhibitor and to interferon-α. Methods: Huh7.5 hepatoma cells were transfected with RNA of genotype 17 NS5A recombinants. Viability was determined by measuring HCV replication and infectivity titers. Putative adaptive mutations were analyzed by reverse genetics. The activity of antiviral agents was determined in high-throughput infection assays. Results: Cells infected with viable HCV that expressed NS5A of genotypes 17 produced relatively high viral titers; most NS5A recombinants required introduction of specific adaptive mutations. The efficacy of the NS5A inhibitor BMS-790052 varied greatly, based on NS5A isolate, with median effective concentration (EC 50) values ranging from 0.009 nmol/L to 14 nmol/L; the high sensitivity of genotype 1b NS5A to BMS-790052 reflected observations from clinical studies. Specific residues in NS5A domain I were associated with >100-fold variations in sensitivity between isolates of the same HCV subtype. The Y/T2065H mutation conferred resistance to BMS-790052 that varied among NS5A isolates. When infected cultures were incubated with interferon-α, all NS5A recombinants had EC50 values of ∼0.2 IU/mL, including an NS5A genotype 1b mutant with a putative sensitive-type, interferon sensitivity determining region. Conclusions: We developed efficient in vitro systems in which recombinant viruses express HCV genotypes 17 NS5A; these permit genotype- and isolate-specific analyses of NS5A and the effects of antiviral compounds and resistance mutations. These culture systems will facilitate development of specific inhibitors against NS5A of different HCV variants.

AB - Background & Aims: Heterogeneity in the hepatitis C virus (HCV) protein NS5A influences its sensitivity to interferon-based therapy. Furthermore, NS5A is an important target for development of HCV-specific inhibitors. We aimed to develop recombinant infectious cell culture systems that express NS5A from isolates of the 7 major HCV genotypes, and determining their sensitivity to a specific NS5A inhibitor and to interferon-α. Methods: Huh7.5 hepatoma cells were transfected with RNA of genotype 17 NS5A recombinants. Viability was determined by measuring HCV replication and infectivity titers. Putative adaptive mutations were analyzed by reverse genetics. The activity of antiviral agents was determined in high-throughput infection assays. Results: Cells infected with viable HCV that expressed NS5A of genotypes 17 produced relatively high viral titers; most NS5A recombinants required introduction of specific adaptive mutations. The efficacy of the NS5A inhibitor BMS-790052 varied greatly, based on NS5A isolate, with median effective concentration (EC 50) values ranging from 0.009 nmol/L to 14 nmol/L; the high sensitivity of genotype 1b NS5A to BMS-790052 reflected observations from clinical studies. Specific residues in NS5A domain I were associated with >100-fold variations in sensitivity between isolates of the same HCV subtype. The Y/T2065H mutation conferred resistance to BMS-790052 that varied among NS5A isolates. When infected cultures were incubated with interferon-α, all NS5A recombinants had EC50 values of ∼0.2 IU/mL, including an NS5A genotype 1b mutant with a putative sensitive-type, interferon sensitivity determining region. Conclusions: We developed efficient in vitro systems in which recombinant viruses express HCV genotypes 17 NS5A; these permit genotype- and isolate-specific analyses of NS5A and the effects of antiviral compounds and resistance mutations. These culture systems will facilitate development of specific inhibitors against NS5A of different HCV variants.

KW - Antiviral Agents

KW - Cell Line, Tumor

KW - Cell Survival

KW - Dose-Response Relationship, Drug

KW - Enzyme Inhibitors

KW - Genotype

KW - Hepacivirus

KW - High-Throughput Screening Assays

KW - Humans

KW - Imidazoles

KW - Interferon-alpha

KW - Mutation

KW - Phenotype

KW - Recombinant Proteins

KW - Time Factors

KW - Viral Nonstructural Proteins

KW - Virus Replication

U2 - 10.1053/j.gastro.2010.11.036

DO - 10.1053/j.gastro.2010.11.036

M3 - Journal article

C2 - 21111742

SN - 0969-0131

VL - 140

SP - 1032

EP - 1042

JO - Gastroenterology Today

JF - Gastroenterology Today

IS - 3

ER -

Recombinant HCV variants with NS5A from genotypes 1-7 have different sensitivities to an NS5A inhibitor but not interferon-a

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