Mechanism of action of a pestivirus antiviral compound

Abstract

We report here the discovery of a small molecule inhibitor of pestivirus replication. The compound, designated VP32947, inhibits the replication of bovine viral diarrhea virus (BVDV) in cell culture at a 50% inhibitory concentration of approximately 20 nM. VP32947 inhibits both cytopathic and noncytopathic pestiviruses, including isolates of BVDV-1, BVDV-2, border disease virus, and classical swine fever virus. However, the compound shows no activity against viruses from unrelated virus groups. Time of drug addition studies indicated that VP32947 acts after virus adsorption and penetration and before virus assembly and release. Analysis of viral macromolecular synthesis showed VP32947 had no effect on viral protein synthesis or polyprotein processing but did inhibit viral RNA synthesis. To identify the molecular target of VP32947, we isolated drug-resistant (DR) variants of BVDV-1 in cell culture. Sequence analysis of the complete genomic RNA of two DR variants revealed a single common amino acid change located within the coding region of the NS5B protein, the viral RNA-dependent RNA polymerase. When this single amino acid change was introduced into an infectious clone of drug-sensitive wild-type (WT) BVDV-1, replication of the resulting virus was resistant to VP32947. The RNA-dependent RNA polymerase activity of the NS5B proteins derived from WT and DR viruses expressed and purified from recombinant baculovirus-infected insect cells confirmed the drug sensitivity of the WT enzyme and the drug resistance of the DR enzyme. This work formally validates NS5B as a target for antiviral drug discovery and development. The utility of VP32947 and similar compounds for the control of pestivirus diseases, and for hepatitis C virus drug discovery efforts, is discussed.

Figure 1

Effect of time of VP32947 addition on inhibition of BVDV growth. VP32947 was added to BVDV-infected MDBK cell cultures to a final concentration of 167 nM at the indicated times postinfection. For the “minus 1 h” time point, VP32947 was included during the 1-h virus adsorption period. All cultures were harvested at 14 h postinfection, and virus yield was quantified by plaque assay. The data are presented as the percent of virus produced in the absence of drug.

Figure 2

Effect of VP32947 on viral RNA synthesis in WT virus-infected cells. Incorporation of ³H-uridine into viral RNA in the absence (○) or presence (●) of 167 nM VP32947 was determined as described in Materials and Methods.

Figure 3

Effect of VP32947 on DR virus growth. The growth of WT (●), DR clone 2 (▾), DR clone 3 (■), and F224S DR () viruses in the presence of increasing concentrations of VP32947 was assessed in a virus cytopathic effect assay. Data are plotted as the percent of control virus cytopathic effect.

Figure 4

Effect of VP32947 on viral RNA synthesis in F224S DR virus-infected cells. (A) Viral RNA synthesis was determined in cell cultures in the absence (○) or presence (●) of 167 nM VP32947 as described in Materials and Methods section. (B) MDBK cells were mock-infected or were infected with either WT or F224S DR virus. After 6 h, actinomycin D was added to 5 μg/ml without (−) or with (+) 167 nM VP32947. After an additional 2 h, ³H-uridine (Amersham) was added to the cultures. Cells were harvested 3 h later, and total RNA was extracted and subjected to urea-agarose gel electrophoresis followed by fluorography (21).

Figure 5

Purified NS5B proteins from WT and F224S DR viruses. NS5B proteins were purified from Sf9 cells infected with recombinant baculoviruses expressing either the WT or F224S DR NS5B proteins as described in Materials and Methods. Aliquots of the final dialyzed preparations were subjected to SDS/10% PAGE, and the resolved proteins were visualized by silver staining. M, molecular mass standards (numbers at left in kilodaltons).

Figure 6

Inhibition of NS5B polymerase activity by VP32947. The RdRp activity of purified baculovirus-expressed WT (●) and F224S DR (▴) NS5B proteins was measured in the presence of increasing concentrations of VP32947 as described in Materials and Methods.