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Antimicrobial Agents and Chemotherapy, October 2008, p. 3523-3531, Vol. 52, No. 10
0066-4804/08/$08.00+0     doi:10.1128/AAC.00533-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Development of Intergenotypic Chimeric Replicons To Determine the Broad-Spectrum Antiviral Activities of Hepatitis C Virus Polymerase Inhibitors{triangledown} ,{dagger}

Koleen J. Herlihy, Joanne P. Graham, Robert Kumpf, Amy K. Patick, Rohit Duggal, and Stephanie T. Shi*

Pfizer Global Research and Development, La Jolla Laboratories, 10724 Science Center Drive, San Diego, California 92121

Received 24 April 2008/ Returned for modification 11 June 2008/ Accepted 29 July 2008

To address the need for broad-spectrum antiviral activity characterization of hepatitis C virus (HCV) polymerase inhibitors, we created a panel of intergenotypic chimeric replicons containing nonstructural (NS) protein NS5B sequences from genotype 2b (GT2b), GT3a, GT4a, GT5a, and GT6a HCV isolates. Viral RNA extracted from non-GT1 HCV patient plasma was subjected to reverse transcription. The NS5B region was amplified by nested PCR and introduced into the corresponding region of the GT1b (Con-1) subgenomic reporter replicon by Splicing by Overlap Extension (SOEing) PCR. Stable cell lines were generated with replication-competent chimeras for in vitro antiviral activity determination of HCV nonnucleoside polymerase inhibitors (NNIs) that target different regions of the protein. Compounds that bind to the NNI2 (thiophene carboxylic acid) or NNI3 (benzothiadiazine) allosteric sites showed 8- to >1,280-fold reductions in antiviral activity against non-GT1 NS5B chimeric replicons compared to that against the GT1b subgenomic replicon. Smaller reductions in susceptibility, ranging from 0.2- to 33-fold, were observed for the inhibitor binding to the NNI1 (benzimidazole) site. The inhibitor binding to the NNI4 (benzofuran) site showed broad-spectrum antiviral activity against all chimeric replicons evaluated in this study. In conclusion, evaluation of HCV NNIs against intergenotypic chimeric replicons showed differences in activity spectrum for inhibitors that target different regions of the enzyme, some of which could be associated with specific residues that differ between GT1 and non-GT1 polymerases. Our study demonstrates the utility of chimeric replicons for broad-spectrum activity determination of HCV inhibitors.

* Corresponding author. Mailing address: Department of Cancer Biology, Pfizer Global Research and Development, La Jolla Laboratories, 10724 Science Center Drive, San Diego, CA 92121. Phone: (858) 526-4906. Fax: (858) 526-4349. E-mail: stephanie.shi{at}pfizer.com

{triangledown} Published ahead of print on 11 August 2008.

{dagger} Supplemental material for this article may be found at http://aac.asm.org/.

Antimicrobial Agents and Chemotherapy, October 2008, p. 3523-3531, Vol. 52, No. 10
0066-4804/08/$08.00+0     doi:10.1128/AAC.00533-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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