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Antimicrobial Agents and Chemotherapy, January 2007, p. 95-102, Vol. 51, No. 1
0066-4804/07/$08.00+0     doi:10.1128/AAC.01008-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Development of a Novel Dicistronic Reporter-Selectable Hepatitis C Virus Replicon Suitable for High-Throughput Inhibitor Screening{triangledown}

Weidong Hao,1 Koleen J. Herlihy,1 Noelle Jie Zhang,1 Shella A. Fuhrman,2,{dagger} Chau Doan,2 Amy K. Patick,1 and Rohit Duggal1*

Department of Virology,1 Department of Biochemistry, Pfizer Global Research and Development, La Jolla Laboratories, San Diego, California 921212

Received 11 August 2006/ Returned for modification 21 September 2006/ Accepted 16 October 2006

Hepatitis C virus (HCV) research and drug discovery have been facilitated by the introduction of cell lines with self-replicating subgenomic HCV replicons. Early attempts to carry out robust, high-throughput screens (HTS) using HCV replicons have met with limited success. Specifically, selectable replicons have required laborious reverse transcription-PCR quantitation, and reporter replicons have generated low signal-to-noise ratios. In this study, we constructed a dicistronic single reporter (DSR)-selectable HCV replicon that contained a humanized Renilla luciferase (hRLuc) gene separated from the selectable Neor marker by a short peptide cleavage site. The mutations E1202G, T1280I, and S2197P were introduced to enhance replicative capability. A dicistronic dual-reporter HCV replicon cell line (DDR) was subsequently created by transfection of Huh-7 cells with the DSR replicon to monitor antiviral activity and by the introduction of the firefly luciferase (FLuc) reporter gene into the host cell genome to monitor cytotoxicity. The DDR cell line demonstrated low signal variation within the HTS format, with a calculated Z' value of 0.8. A pilot HTS consisting of 20 96-well plates with a single concentration (10 µM) of 1,760 different compounds was executed. Hits were defined as compounds that reduced hRLuc and FLuc signals ≥50 and ≤40%, respectively, relative to those in a compound-free control. Good reproducibility was demonstrated, with a calculated confirmation rate of >75%. The development of a robust, high-throughput HCV replicon assay where the effects of inhibitors can be monitored for antiviral activity and cytotoxicity should greatly facilitate HCV drug discovery.

* Corresponding author. Mailing address: Pfizer Global Research and Development, 10777 Science Center Drive, San Diego, CA 92121. Phone: (858) 622-5948. Fax: (858) 526-4463. E-mail: rohit.duggal{at}pfizer.com.

{triangledown} Published ahead of print on 23 October 2006.

{dagger} Present address: 185 Goldenrod Lane, Friendsville, TN 37737-3141.

Antimicrobial Agents and Chemotherapy, January 2007, p. 95-102, Vol. 51, No. 1
0066-4804/07/$08.00+0     doi:10.1128/AAC.01008-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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