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Journal of Virology, March 2003, p. 3669-3679, Vol. 77, No. 6
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.6.3669-3679.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

In Vitro Selection and Characterization of Hepatitis C Virus Serine Protease Variants Resistant to an Active-Site Peptide Inhibitor

Caterina Trozzi, Linda Bartholomew, Alessandra Ceccacci, Gabriella Biasiol, Laura Pacini, Sergio Altamura, Frank Narjes, Ester Muraglia, Giacomo Paonessa, Uwe Koch, Raffaele De Francesco, Christian Steinkuhler, and Giovanni Migliaccio*

IRBM "P. Angeletti," 00040 Pomezia, Rome, Italy

Received 6 November 2002/ Accepted 13 December 2002

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.

* Corresponding author. Mailing address: IRBM "P. Angeletti," Via Pontina Km 30.600, 00040 Pomezia, Rome, Italy. Phone: 3906 91093239. Fax: 3906 91093654. E-mail: giovanni_migliaccio{at}merck.com.

Journal of Virology, March 2003, p. 3669-3679, Vol. 77, No. 6
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.6.3669-3679.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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