PCR-based in vitro synthesis of hepatitis C virus NS3 protease for rapid phenotypic resistance testing of protease inhibitors.
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.
Center for Emerging Infectious Diseases and State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.
Jinjuan
J
Qiao
Junping
J
Yu
Hang
H
Yang
Hongping
H
Wei
D000998:Antiviral Agents / Q000378:metabolism*; D024882:Drug Resistance, Viral; D004926:Escherichia coli / Q000235:genetics; D005470:Fluorometry; D016174:Hepacivirus / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods*; D011480:Protease Inhibitors / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000235:genetics* / Q000378:metabolism*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D000998:Antiviral Agents; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases