Specific inhibition of bovine viral diarrhea virus replicase

Abstract

Compound-1453 was identified and characterized as a specific inhibitor of bovine viral diarrhea virus (BVDV). The concentration of compound-1453 which results in 50% protection from virus-induced cytopathic effect is approximately 2.2 microM, with a therapeutic index of 60, and it is not active against a panel of RNA and DNA viruses. A time-of-addition experiment suggested that compound-1453 targets a stage of the viral life cycle after viral entry. To determine the target of compound-1453, resistant virus was generated. Resistant variants grew efficiently in the presence or absence of 33 micro M compound-1453 and exhibited replication efficiency in the presence of compound-1453 approximately 1,000-fold higher than that of the wild-type (wt) virus. Functional mapping and sequence analysis of resistant cDNAs revealed a single amino acid substitution (Glu to Gly) at residue 291 in the NS5B polymerase in all eight independently generated cDNA clones. Recombinant virus containing this single mutation retained the resistance phenotype and a replication efficiency similar to that of the original isolated resistant virus. Since compound-1453 did not inhibit BVDV polymerase activity in vitro (50% inhibitory concentration > 300 microM), we developed a membrane-based assay that consisted of a BVDV RNA replicase complex isolated from virus-infected cells. Compound-1453 inhibited the activity of the wt, but not the drug-resistant, replicase in the membrane assay at concentrations similar to those observed in the viral infection assay. This work presents a novel inhibitor of a viral RNA-dependent RNA replicase.

FIG. 1.

Inhibition of BVDV replication by compound-1453. (A) Structure of compound-1453. (B) Plates (96-well) of MDBK cells were infected with BVDV (500 PFU/well) or mock infected in the presence of various concentrations of compound-1453; 4 days later, percentages of protection (closed circles) and cytotoxicity (open circles) were calculated through the MTT-based colorimetric assay.

FIG. 2.

Effect of compound-1453 on BVDV growth. (A) One-step growth curve of BVDV. MDBK cells were infected with BVDV at an MOI of 3; at the indicated times (1, 3, 5, 7, 9, 11, 13, or 23 h p.i.), infected cells were harvested and titers of progeny virus were determined by plaque assay. (B) Effect of time of compound-1453 addition on inhibition of BVDV growth. Cells were infected with BVDV at an MOI of 3, and compound-1453 was added at 1, 3, 5, 7, 9, 11, or 13 h p.i. at a final concentration of 33 μM. At 23 h p.i., infected cells were harvested and titers of progeny virus were determined in the absence of compound.

FIG. 3.

Effect of compound-1453 on BVDV RNA synthesis. (A) Flowchart of the experiment. (B) Effect of Act. D (0.5 μg/ml) and compound-1453 (33 μM) on cellular RNA synthesis. (C) Effect of compound-1453 on [³H]uridine incorporation into viral RNA. Data represent the mean of three repeats for each sample.

FIG. 4.

Effect of time of compound-1453 addition on BVDV NS3 protein expression. MDBK cells were mock infected (A) or infected with wt (B, E, F, G, H, and I) or 1453^r (C and D) virus at an MOI of 3. Compound-1453 was added at 0 (D and E), 2 (F), 5 (G), 7 (H), and 9 (I) h p.i., and using anti-NS3 MAb 20.10.6, cells were processed for indirect immunofluorescence at 23 h p.i.

FIG. 5.

Effect of compound-1453 on viral growth. A one-step growth curve was performed in MDBK cells. Cells were infected with virus at an MOI of 3 and incubated in the absence (A) or presence (B) of 33 μM compound-1453. Virus samples were collected at the indicated times, and titers were determined by plaque assay.

FIG. 6.

Analysis of RNA products in the membrane-based BVDV polymerase assay. (A) [³³P]CTP-labeled RNA products from mock-infected (lanes 1, 2, and 5) or virus-infected (lanes 3, 4, and 6) cell membranes were phenol-chloroform extracted and ethanol precipitated and then either directly loaded (lanes 1 to 4) or treated with S1 nuclease (lanes 5 and 6) before loading on a 1% agarose gel. Positions of the RNA size marker are indicated on the left. A position (12K) indicated by an arrow on the right was extrapolated on the basis of the positions of the RNA size markers. (B) RNA fragments used in a reverse RNase protection assay. + and − refer to positive- and negative-sense polarities of RNA relative to the BVDV genome. Numbers refer to nucleotide positions in the BVDV genome. (C) RNA products after RNase digestion. Lane 1, the RNA product from the membrane assay (no RNase digestion); lane 2, the RNA product from the membrane digested with RNase; lanes 3, 4, 5, and 6, RNA products after hybridization with the protective RNAs indicated in panel B and digestion with RNase. The positions of protective RNA (according to the results of toluidine blue staining) are indicated by arrows.

FIG. 7.

Effect of compound-1453 on the membrane-based BVDV wt (A) and 1453^r (B) replicase activities. The same amount of protein (5 μg) from each membrane preparation was used for the assay. Each concentration of compound-1453 was run in duplicate as indicated above the autoradiogram. RNA products from these reactions were analyzed by electrophoresis on a 1% agarose gel. Sizes of RNA markers are indicated on the right.