title: Development of a replicon-based phenotypic assay for assessing the drug susceptibilities of HCV NS3 protease genes from clinical isolates.
abstract: Hepatitis C virus (HCV) protease inhibitors targeting HCV NS3 can efficiently suppress HCV replication. However, the selection of resistance has been observed both in vitro and in vivo. Here, we describe a new method for efficient analysis of the drug susceptibility of the NS3 protease genes from patient isolates. Luciferase-reporter 1b replicon shuttle vectors that allow cloning of either the HCV full-length NS3/4A gene or the NS3 protease domain gene only were created. Initially, chimeric replicons carrying patient-derived full-length NS3/4A failed to replicate in cell culture. However, the poor replication efficiency of the NS3/4A shuttle vector was enhanced by approximately 100-fold when the NS3 helicase domains of clinical isolates were substituted for that of the 1b Con1 lab strain. Chimeric replicons carrying only the patient-derived NS3 protease domains replicated at levels sufficient for phenotypic analysis in 20/20 clinical isolates. EC(50) values for the NS3 inhibitor BILN-2061 ranged from 0.2 to 1.1nM for 20 genotype 1 patient isolates. Significantly reduced susceptibility to BILN-2061 was observed with mutant/wild type mixtures of 5%/95% for the D168V or 50%/50% for A156T resistance mutations in the NS3. These shuttle vectors can be used to evaluate candidate drugs and assist in the development of new drugs targeting the NS3 protease.
authors.affiliation: Department of Clinical Virology, Gilead Sciences, Inc., 333 Lakeside Dr., Foster City, CA 94404, USA.
authors.forename: Xiaoping
authors.initials: X
authors.lastname: Qi
authors.forename: Andrew
authors.initials: A
authors.lastname: Bae
authors.forename: Shan
authors.initials: S
authors.lastname: Liu
authors.forename: Huiling
authors.initials: H
authors.lastname: Yang
authors.forename: Siu-Chi
authors.initials: SC
authors.lastname: Sun
authors.forename: Jeanette
authors.initials: J
authors.lastname: Harris
authors.forename: William
authors.initials: W
authors.lastname: Delaney
authors.forename: Michael
authors.initials: M
authors.lastname: Miller
authors.forename: Hongmei
authors.initials: H
authors.lastname: Mo
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology*; D024882:Drug Resistance, Viral; D017930:Genes, Reporter; D005822:Genetic Vectors; D016174:Hepacivirus / Q000187:drug effects* / Q000235:genetics; D006801:Humans; D020128:Inhibitory Concentration 50; D008156:Luciferases / Q000378:metabolism; D008826:Microbial Sensitivity Tests / Q000379:methods*; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D000998:Antiviral Agents; D008156:Luciferases; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases