In vitro selection and characterization of hepatitis C virus serine protease variants resistant to an active-site peptide inhibitor.
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.
Trozzi
Caterina
C
IRBM P. Angeletti, 00040 Pomezia, Rome, Italy.
Bartholomew
Linda
L
Ceccacci
Alessandra
A
Biasiol
Gabriella
G
Pacini
Laura
L
Altamura
Sergio
S
Narjes
Frank
F
Muraglia
Ester
E
Paonessa
Giacomo
G
Koch
Uwe
U
De Francesco
Raffaele
R
Steinkuhler
Christian
C
Migliaccio
Giovanni
G
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
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