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Antimicrobial Agents and Chemotherapy, February 2008, p. 393-401, Vol. 52, No. 2
0066-4804/08/$08.00+0     doi:10.1128/AAC.00961-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

NS3 Peptide, a Novel Potent Hepatitis C Virus NS3 Helicase Inhibitor: Its Mechanism of Action and Antiviral Activity in the Replicon System

Institute of Biochemistry and Biophysics PAS, Warsaw, Poland,¹ National Institute of Chemistry, SI-1001, Lubljana, Slovenia,² Institute of Environmental Protection, John Paul II Catholic University of Lublin, Lublin, Poland³

Received 25 July 2007/ Returned for modification 17 September 2007/ Accepted 12 November 2007

Hepatitis C virus (HCV) chronic infections represent one of the major and still unresolved health problems because of low efficiency and high cost of current therapy. Therefore, our studies centered on a viral protein, the NS3 helicase, whose activity is indispensable for replication of the viral RNA, and on its peptide inhibitor that corresponds to a highly conserved arginine-rich sequence of domain 2 of the helicase. The NS3 peptide (p14) was expressed in bacteria. Its 50% inhibitory activity in a fluorometric helicase assay corresponded to 725 nM, while the ATPase activity of NS3 was not affected. Nuclear magnetic resonance (NMR) studies of peptide-protein interactions using the relaxation filtering technique revealed that p14 binds directly to the full-length helicase and its separately expressed domain 1 but not to domain 2. Changes in the NMR chemical shift of backbone amide nuclei (¹H and ¹⁵N) of domain 1 or p14, measured during complex formation, were used to identify the principal amino acids of both domain 1 and the peptide engaged in their interaction. In the proposed interplay model, p14 contacts the clefts between domains 1 and 2, as well as between domains 1 and 3, preventing substrate binding. This interaction is strongly supported by cross-linking experiments, as well as by kinetic studies performed using a fluorometric assay. The antiviral activity of p14 was tested in a subgenomic HCV replicon assay that showed that the peptide at micromolar concentrations can reduce HCV RNA replication.

Published ahead of print on 26 November 2007.