title: Selection and characterization of drug-resistant HCV replicons in vitro with a flow cytometry-based assay.
abstract: Because HCV RNA-dependent RNA polymerase is error-prone and the viral RNA has a high turnover rate, the genetic diversity of HCV is very high both in vitro and in vivo. The mutation rate in long-term replicon cultures approaches 3.0 x 10(-3) base substitutions/site/year in this in vitro replication model. A direct consequence of the high mutation rate is the rapid emergence of drug-resistant variants, both in cell culture and in patients. Selectable replicons have been used extensively to isolate and characterize drug-resistant HCV genomes in vitro. Typically, replicon cells are plated at a low density and then subjected to a double selection by G418 and escalating dosages of a compound of choice. Here we describe an alternative screening assay that takes advantage of an HCV replicon that is amenable to live-cell sorting with a suitable flow cytometer. We also present a strategy for determining the relative contribution to the resistance by viral genome and host cells. We use selection and characterization of Cyclosporine A (CsA)-resistant replicons as a example to present the protocols, but this method can easily be adapted for the selection of replicon cells resistant to other chemical compounds as long as the compound does not fluoresce at the same wavelength as the fluorescent reporter protein in the replicon.
authors.affiliation: Department of Biological Science, Florida State University, Tallahassee, FL, USA.
authors.forename: Jason M
authors.initials: JM
authors.lastname: Robotham
authors.forename: Heather B
authors.initials: HB
authors.lastname: Nelson
authors.forename: Hengli
authors.initials: H
authors.lastname: Tang
mesh_terms: D000818:Animals; D002469:Cell Separation / Q000379:methods*; D016572:Cyclosporine / Q000494:pharmacology; D004279:DNA, Viral / Q000378:metabolism; D024882:Drug Resistance, Viral / Q000235:genetics*; D018274:Electroporation; D005434:Flow Cytometry / Q000379:methods*; D005838:Genotype; D049452:Green Fluorescent Proteins / Q000378:metabolism; D016174:Hepacivirus / Q000187:drug effects* / Q000235:genetics* / Q000378:metabolism; D016898:Interferon-alpha / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D012367:RNA, Viral / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012093:Replicon / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic; D017361:Viral Nonstructural Proteins / Q000235:genetics; D012324:RNA-Dependent RNA Polymerase
chemical_list: D016572:Cyclosporine; D004279:DNA, Viral; D049452:Green Fluorescent Proteins; D016898:Interferon-alpha; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase