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Antimicrobial Agents and Chemotherapy, January 2003, p. 244-254, Vol. 47, No. 1
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.1.244-254.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Ribonucleoside Analogue That Blocks Replication of Bovine Viral Diarrhea and Hepatitis C Viruses in Culture

Lieven J. Stuyver,1* Tony Whitaker,1 Tamara R. McBrayer,1 Brenda I. Hernandez-Santiago,2 Stefania Lostia,1 Phillip M. Tharnish,1 Mangala Ramesh,1 Chung K. Chu,3 Robert Jordan,4 Junxing Shi,1 Suguna Rachakonda,1 Kyoichi A. Watanabe,1 Michael J. Otto,1 and Raymond F. Schinazi2

Pharmasset Inc., Tucker, Georgia 30084,1 Department of Pediatrics, Emory School of Medicine-Veterans Affairs Medical Center, Decatur, Georgia 30033,2 College of Pharmacy, University of Georgia, Athens, Georgia 30602,3 Jefferson Center for Biomedical Research, Doylestown, Pennsylvania 189014

Received 17 June 2002/ Returned for modification 12 September 2002/ Accepted 17 October 2002

A base-modified nucleoside analogue, ß-D-N4-hydroxycytidine (NHC), was found to have antipestivirus and antihepacivirus activities. This compound inhibited the production of cytopathic bovine viral diarrhea virus (BVDV) RNA in a dose-dependant manner with a 90% effective concentration (EC90) of 5.4 µM, an observation that was confirmed by virus yield assays (EC90 = 2 µM). When tested for hepatitis C virus (HCV) replicon RNA reduction in Huh7 cells, NHC had an EC90 of 5 µM on day 4. The HCV RNA reduction was incubation time and nucleoside concentration dependent. The in vitro antiviral effect of NHC was additive with recombinant alpha interferon-2a and could be prevented by the addition of exogenous cytidine and uridine but not of other natural ribo- or 2'-deoxynucleosides. When HCV RNA replicon cells were cultured in the presence of increasing concentrations of NHC (up to 40 µM) for up to 45 cell passages, no resistant replicon was selected. Similarly, resistant BVDV could not be selected after 20 passages. NHC was phosphorylated to the triphosphate form in Huh7 cells, but in cell-free HCV NS5B assays, synthetic NHC-triphosphate (NHC-TP) did not inhibit the polymerization reaction. Instead, NHC-TP appeared to serve as a weak alternative substrate for the viral polymerase, thereby changing the mobility of the product in polyacrylamide electrophoresis gels. We speculate that incorporated nucleoside analogues with the capacity of changing the thermodynamics of regulatory secondary structures (with or without introducing mutations) may represent an important class of new antiviral agents for the treatment of RNA virus infections, especially HCV.

* Corresponding author. Mailing address: Pharmasset Inc., 1860 Montreal Rd., Tucker, GA 30084. Phone: (678) 395-0050. Fax: (678) 395-0030. E-mail: lstuyver{at}pharmasset.com.

Antimicrobial Agents and Chemotherapy, January 2003, p. 244-254, Vol. 47, No. 1
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.1.244-254.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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