A cellular replicon-based phenotyping assay to determine susceptibility of hepatitis C virus clinical isolates to NS3/4A protease inhibitors
Vijgen, Leen Verbeeck, Jannick Van Kerckhove, Barbara Berke, Jan Martin Koletzki, Diana Fanning, Gregory Lenz, Oliver #
Walker, John M
Methods in Molecular Biology vol:1030
Antiviral Methods and Protocols edition:2nd pages:105-117
A hepatitis C virus (HCV) replicon-based protease phenotyping assay has been developed that allows determining the susceptibility of a patient’s HCV protease sequence to HCV protease inhibitors. In brief, HCV protease sequences amplified from clinical samples are cloned in a transient HCV genotype 1b replicon backbone, containing a luciferase reporter gene. These protease chimeric replicons are replication-competent when electroporated into susceptible Huh7-Lunet cells. Replication can be quantified by measuring the enzymatic activity of the luciferase protein. This assay is reproducible and robust, and has a high overall success rate for determining the phenotypic susceptibility of HCV genotype 1a and 1b patient-derived protease domains to HCV protease inhibitors. In addition, the HCV genotype 1b protease shuttle backbone also supports efficient replication of HCV genotype 4 protease sequences.