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  • Development and application of a fast, reproducible assay to measure HCV NS3 protease activity using Escherichia coli lysate.

Development and application of a fast, reproducible assay to measure HCV NS3 protease activity using Escherichia coli lysate.

Journal of virological methods (2015-09-24)
Bin Han, Hadas Dvory-Sobol, Andrew Greenstein, Joseph F McCarville, Magdeleine Hung, Xiaohong Liu, Michael D Miller, Hongmei Mo
ABSTRACT

The hepatitis C virus (HCV) NS3/4A protease is a key target of efforts to develop direct-acting antiviral inhibitors for treatment of chronic HCV infection. In vitro analyses of the effects of NS3/4A mutations and polymorphisms on protease inhibitor (PI) susceptibility are essential to nonclinical and clinical compound characterization, but can be hampered by time and technical limitations of current in vitro methods using replicon or purified protein systems. We have developed a fast and simple method utilizing full-length NS3/4A protease inducibly expressed in Escherichia coli cells. Minimally processed E. coli whole cell lysate was used for analyzing NS3/4A protease activity and inhibition by antiviral compounds. Assay conditions were optimized to develop a reproducible assay that can be used for efficient analysis of NS3 protease mutants with poor replication capacity in the replicon system. IC50 fold-changes for NS3 mutants relative to their wild-types generated by this NS3 assay are comparable to those observed in the replicon system, with an R(2) of 0.82 for the values obtained by the two methods. In addition, we demonstrate that this assay can be successfully used for population and clonal phenotyping of patient samples and characterization of PIs against the NS3/4A protease from HCV genotypes 1-6.

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