Mechanism of action of a pestivirus antiviral compound

We report here the discovery of a small molecule inhibitor of pestivirus re plication. The compound, designated VP32947, inhibits the replication of bo vine viral diarrhea virus (BVDV) in cell culture at a 50% inhibitory concen tration of approximately 20 nM. VP32947 inhibits both cytopathic and noncyt opathic pestiviruses, including isolates of BVDV-1, BVDV-2, border disease virus, and classical swine fever virus. However, the compound shows no acti vity against viruses from unrelated virus groups. Time of drug addition stu dies indicated that VP32947 acts after virus adsorption and penetration and before virus assembly and release. Analysis of viral macromolecular synthe sis showed VP32947 had no effect on viral protein synthesis or polyprotein processing but did inhibit viral RNA synthesis. To identify the molecular t arget of VP32947, we isolated drug-resistant (DR) variants of BVDV-1 in cel l culture. Sequence analysis of the complete genomic RNA of two DR variants revealed a single common amino acid change located within the coding regio n of the NS5B protein, the viral RNA-dependent RNA polymerase. When this si ngle amino acid change was introduced into an infectious clone of drug-sens itive wild-type (WT) BVDV-1, replication of the resulting virus was resista nt to VP32947. The RNA-dependent RNA polymerase activity of the NS5B protei ns derived from WT and DR viruses expressed and purified from recombinant b aculovirus-infected insect cells confirmed the drug sensitivity of the WT e nzyme and the drug resistance of the DR enzyme. This work formally validate s NS5B as a target for antiviral drug discovery and development. The utilit y of VP32947 and similar compounds for the control of pestivirus diseases, and for hepatitis C virus drug discovery efforts, is discussed.