PCR-based in vitro synthesis of hepatitis C virus NS3 protease for rapid phenotypic resistance testing of protease inhibitors

Abstract

Protease inhibitors (PIs) targeting the hepatitis C virus (HCV) NS3 protease, such as telaprevir, have significantly improved the sustained virologic response (SVR) rates of HCV genotype 1 antiviral therapy. Given the expanding antiviral therapy regimen, fast HCV PI resistance assays are urgently needed. In this view, we have developed a novel phenotypic resistance test for HCV PIs based on in vitro synthesis of patient-derived HCV NS3 protease and subsequent enzymatic testing in a fluorescent readout. The enzymatically active HCV NS3 proteases were synthesized from PCR-derived templates by an Escherichia coli S30 extract system. Tests of the protease genes with known mutations for telaprevir resistance showed that the phenotypic resistance test was fast, with a total turnaround time of <10 h, and was fully in agreement with the previous resistance results. The initial tests with 38 treatment-naive serum samples showed that the method was significantly less laborious and faster than currently available phenotypic resistance assays of HCV NS3 PIs.

FIG 1

Scheme of PCR-based in vitro-synthesized NS3 protease phenotyping assay.

FIG 2

Proteolytic activity of the lysates in commercial in vitro expression systems (A) and the initial reaction rates of the protease synthesized in vitro by E. coli S30 lysate for different times at different temperatures (B).

FIG 3

The v_i/v₀ ratios of the in vitro-synthesized NS3 proteases determined under different concentrations of telaprevir.

FIG 4

The v_i/v₀ ratios of the in vitro-synthesized NS3 proteases determined with 100 nM telaprevir. The cutoff value is the mean plus 3 standard deviations of the v_i/v₀ ratio of Con1 (wild-type control). Each control and sample were tested three times.