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Journal of Virology, January 2004, p. 938-946, Vol. 78, No. 2
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.2.938-946.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Characterization of the Inhibition of Hepatitis C Virus RNA Replication by Nonnucleosides

Licia Tomei,1 Sergio Altamura,1 Linda Bartholomew,1 Monica Bisbocci,1 Carolyn Bailey,2 Michele Bosserman,2 Antonella Cellucci,1 Eleonora Forte,1 Ilario Incitti,1 Laura Orsatti,1 Uwe Koch,3 Raffaele De Francesco,1 David B. Olsen,2 Steven S. Carroll,2* and Giovanni Migliaccio1*

Departments of Biochemistry,1 Chemistry, Istituto di Ricerche di Biologia Molecolare P. Angeletti (IRBM), Pomezia, Italy,3 Department of Biological Chemistry, Merck Research Laboratories, West Point, Pennsylvania2

Received 8 July 2003/ Accepted 29 September 2003

The RNA-dependent RNA polymerase of hepatitis C virus (HCV) is necessary for the replication of viral RNA and thus represents an attractive target for drug development. Several structural classes of nonnucleoside inhibitors (NNIs) of HCV RNA polymerase have been described, including a promising series of benzothiadiazine compounds that efficiently block replication of HCV subgenomic replicons in tissue culture. In this work we report the selection of replicons resistant to inhibition by the benzothiadiazine class of NNIs. Four different single mutations were identified in separate clones, and all four map to the RNA polymerase gene, validating the polymerase as the antiviral target of inhibition. The mutations (M414T, C451R, G558R, and H95R) render the HCV replicons resistant to inhibition by benzothiadiazines, though the mutant replicons remain sensitive to inhibition by other nucleoside and NNIs of the HCV RNA polymerase. Additionally, cross-resistance studies and synergistic inhibition of the enzyme by combinations of a benzimidazole and a benzothiadiazine indicate the existence of nonoverlapping binding sites for these two structural classes of inhibitors.

* Corresponding author. Mailing address for S. S. Carroll: Department of Biological Chemistry, Merck Research Laboratories, West Point, PA 19486. Phone: (215) 652-4488. Fax: (215) 993-2330. E-mail: steve_carroll{at}merck.com. Mailing address for G. Migliaccio: Department of Biochemistry, Istituto di Ricerche di Biologia Molecolare P. Angeletti (IRBM), Pomezia, Italy. Phone: 3906 91093239. Fax: 3906 91093654. E-mail: giovanni_migliaccio{at}merck.com.

Journal of Virology, January 2004, p. 938-946, Vol. 78, No. 2
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.2.938-946.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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