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Antimicrobial Agents and Chemotherapy, October 2009, p. 4103-4114, Vol. 53, No. 10
0066-4804/09/$08.00+0     doi:10.1128/AAC.00074-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Identification of Inhibitors of Yellow Fever Virus Replication Using a Replicon-Based High-Throughput Assay{triangledown}

Chinmay G. Patkar,1,{dagger} Martha Larsen,2 Michael Owston,1 Janet L. Smith,2 and Richard J. Kuhn1*

Markey Center for Structural Biology, Department of Biological Sciences, 915 W. State Street, Purdue University, West Lafayette, Indiana 47907-2054,1 Life Sciences Institute, University of Michigan, 210 Washtenaw Avenue, Ann Arbor, Michigan 48109-22162

Received 19 January 2009/ Returned for modification 20 February 2009/ Accepted 15 July 2009

Flaviviruses cause severe disease in humans and are a public health priority worldwide. However, no effective therapies or drugs are commercially available yet. Several flavivirus replicon-based assays amenable to high-throughput screening of inhibitors have been reported recently. We developed and performed a replicon-based high-throughput assay for screening small-molecule inhibitors of yellow fever virus (YFV) replication. This assay utilized packaged pseudoinfectious particles containing a YFV replicon that expresses Renilla luciferase in a replication-dependent manner. Several small-molecule compounds with inhibitory activity at micromolar concentrations were identified in the high-throughput screen. These compounds were subsequently tested for their inhibitory activities against YFV replication and propagation in low-throughput assays. Furthermore, YFV mutants that escaped inhibition by two of the compounds were isolated, and in both cases, the mutations were mapped to the NS4B coding region, suggesting a novel inhibitory target for these compounds. This study opens up new avenues for pursuing the nonenzymatic nonstructural proteins as targets for antivirals against YFV and other flaviviruses.

* Corresponding author. Mailing address: Markey Center for Structural Biology, Department of Biological Sciences, 915 W. State St., Purdue University, West Lafayette, IN 47907-2054. Phone: (765) 494-1164. Fax: (765) 496-1189. E-mail: kuhnr{at}purdue.edu

{triangledown} Published ahead of print on 3 August 2009.

{dagger} Present address: UMass Medical School, 55 Lake Avenue North, Worcester, MA 01655.

Antimicrobial Agents and Chemotherapy, October 2009, p. 4103-4114, Vol. 53, No. 10
0066-4804/09/$08.00+0     doi:10.1128/AAC.00074-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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