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Journal of Virology, June 2003, p. 6753-6760, Vol. 77, No. 12
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.12.6753-6760.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Specific Inhibition of Bovine Viral Diarrhea Virus Replicase

Jin-Hua Sun, Julie A. Lemm, Donald R. O'Boyle II, Jason Racela, Richard Colonno, and Min Gao*

Department of Virology, Bristol-Myers Squibb Pharmaceutical Research Institute, Wallingford, Connecticut 06492

Received 17 December 2002/ Accepted 27 March 2003

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 ~2.2 µM, 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 µM 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 µM), 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.

* Corresponding author. Mailing address: Department of Virology, Bristol-Myers Squibb Pharmaceutical Research Institute, 5 Research Parkway, Wallingford, CT 06492. Phone: (203) 677-6692. Fax: (203) 667-6088. E-mail: min.gao{at}bms.com.

Journal of Virology, June 2003, p. 6753-6760, Vol. 77, No. 12
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.12.6753-6760.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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