PCR-based in vitro synthesis of HCV NS3 protease for rapid phenotypic resistance testing of protease inhibitors.

Protease inhibitors (PIs) targeting hepatitis C virus (HCV) NS3, such as telaprevir, have significantly improved sustained virologic response (SVR) rates of HCV genotype 1 antiviral therapy. Given the expanding antiviral therapy, fast HCV PIs 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 E. coli S30 Extract System. Tests of the protease genes with known resistant mutations to telaprevir showed that the phenotypic resistance test was fast with a total turnaround time of less than 10 h and fully in agreement with the previous resistance results. Initial tests of 38 treatment-na？ve sera showed the method was significantly less laborious and faster than currently available phenotypic resistance assays of HCV NS3 PIs.

http://www.ncbi.nlm.nih.gov/pubmed/24452171