Journal
GASTROENTEROLOGY
Volume 140, Issue 3, Pages 1032-U488Publisher
W B SAUNDERS CO-ELSEVIER INC
DOI: 10.1053/j.gastro.2010.11.036
Keywords
Hepatitis C Virus; Virology; Drug Resistance Mutations; Antiviral Agents
Categories
Funding
- Faculty of Health Sciences, University of Copenhagen
- Lundbeck Foundation
- Danish Cancer Society
- Novo Nordisk Foundation
- Danish Council for Independent Research, Medical Science
- A. P. Moller and Chastine Mc-Kinney Mollers Medical Research Foundation
- Hvidovre Hospital Research Foundation
- Aage Thuesen Bruun and Emmy Katy Bruun's memorial foundation
- Leo Nielsen and Karen Margethe Nielsens Foundation for Basic Medical Research
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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-alpha. METHODS: Huh7.5 hepatoma cells were transfected with RNA of genotype 1-7 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 1-7 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 (EC50) 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-alpha, all NS5A recombinants had EC50 values of similar to 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 1-7 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.
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