Development of a replicon-based phenotypic assay for assessing the drug susceptibilities of HCV NS3 protease genes from clinical isolates.
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.
Qi
Xiaoping
X
Department of Clinical Virology, Gilead Sciences, Inc., 333 Lakeside Dr., Foster City, CA 94404, USA.
Bae
Andrew
A
Liu
Shan
S
Yang
Huiling
H
Sun
Siu-Chi
SC
Harris
Jeanette
J
Delaney
William
W
Miller
Michael
M
Mo
Hongmei
H
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
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