title: In vitro selection and characterization of hepatitis C virus serine protease variants resistant to an active-site peptide inhibitor.
abstract: The hepatitis C virus (HCV) serine protease is necessary for viral replication and represents a valid target for developing new therapies for HCV infection. Potent and selective inhibitors of this enzyme have been identified and shown to inhibit HCV replication in tissue culture. The optimization of these inhibitors for clinical development would greatly benefit from in vitro systems for the identification and the study of resistant variants. We report the use HCV subgenomic replicons to isolate and characterize mutants resistant to a protease inhibitor. Taking advantage of the replicons' ability to transduce resistance to neomycin, we selected replicons with decreased sensitivity to the inhibitor by culturing the host cells in the presence of the inhibitor and neomycin. The selected replicons replicated to the same extent as those in parental cells. Sequence analysis followed by transfection of replicons containing isolated mutations revealed that resistance was mediated by amino acid substitutions in the protease. These results were confirmed by in vitro experiments with mutant enzymes and by modeling the inhibitor in the three-dimensional structure of the protease.
authors.affiliation: IRBM P. Angeletti, 00040 Pomezia, Rome, Italy.
authors.forename: Caterina
authors.initials: C
authors.lastname: Trozzi
authors.forename: Linda
authors.initials: L
authors.lastname: Bartholomew
authors.forename: Alessandra
authors.initials: A
authors.lastname: Ceccacci
authors.forename: Gabriella
authors.initials: G
authors.lastname: Biasiol
authors.forename: Laura
authors.initials: L
authors.lastname: Pacini
authors.forename: Sergio
authors.initials: S
authors.lastname: Altamura
authors.forename: Frank
authors.initials: F
authors.lastname: Narjes
authors.forename: Ester
authors.initials: E
authors.lastname: Muraglia
authors.forename: Giacomo
authors.initials: G
authors.lastname: Paonessa
authors.forename: Uwe
authors.initials: U
authors.lastname: Koch
authors.forename: Raffaele
authors.initials: R
authors.lastname: De Francesco
authors.forename: Christian
authors.initials: C
authors.lastname: Steinkuhler
authors.forename: Giovanni
authors.initials: G
authors.lastname: Migliaccio
mesh_terms: D001665:Binding Sites / Q000187:drug effects; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000235:genetics*; D024882:Drug Resistance, Viral / Q000235:genetics*; D014644:Genetic Variation; D016174:Hepacivirus / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D009154:Mutation; D012093:Replicon; D012641:Selection, Genetic; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000235:genetics*; D014764:Viral Proteins / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000235:genetics*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D015842:Serine Proteinase Inhibitors; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C455041:NS4A cofactor peptide, Hepatitis C virus