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Antimicrobial Agents and Chemotherapy, June 2003, p. 1936-1942, Vol. 47, No. 6
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.6.1936-1942.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Generation of Stable Cell Lines Expressing Lamivudine-Resistant Hepatitis B Virus for Antiviral-Compound Screening

Kathie-Anne Walters,1 Graham A. Tipples,2 Marchelle I. Allen,3 Lynn D. Condreay,3 William R. Addison,1 and Lorne Tyrrell1*

Department of Medical Microbiology and Immunology, Glaxo Wellcome-Heritage Research Institute, University of Alberta, Edmonton, Alberta, Canada T6G 2H7,1 National Microbiology Laboratory, Winnipeg, Manitoba, Canada R3E 3R2,2 Department of Virology, GlaxoSmithKline, Research Triangle Park, North Carolina 277093

Received 30 October 2002/ Returned for modification 6 January 2003/ Accepted 17 March 2003

Lamivudine [ß-L-(-)-2',3'-dideoxy-3'-thiacytidine] is a potent inhibitor of hepadnavirus replication and is used both to treat chronic hepatitis B virus (HBV) infections and to prevent reinfection of transplanted livers. Unfortunately, lamivudine-resistant HBV variants do arise during prolonged therapy, indicating a need for additional antiviral drugs. Replication-competent HBV constructs containing the reverse transcriptase domain L180M/M204V and M204I (rtL180M/M204V and rtM204I) mutations associated with lamivudine resistance were used to produce stable cell lines that express the resistant virus. These cell lines contain stable integrations of HBV sequences and produce both intracellular and extracellular virus. HBV produced by these cell lines was shown to have a marked decrease in sensitivity to lamivudine, with 450- and 3,000-fold shifts in the 50% inhibitory concentrations for the rtM204I and rtL180M/M204V viruses, respectively, compared to that for the wild-type virus. Drug assays indicated that the lamivudine-resistant virus exhibited reduced sensitivity to penciclovir [9-(4-hydroxy-3-hydroxymethyl-but-1-yl) guanine] but was still inhibited by the nucleoside analogues CDG (carbocyclic 2'-deoxyguanosine) and abacavir {[1S,4R]-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol}. Screening for antiviral compounds active against the lamivudine-resistant HBV can now be done with relative ease.

* Corresponding author. Mailing address: Department of Medical Microbiology and Immunology, Glaxo Wellcome-Heritage Research Institute, University of Alberta, Edmonton, Alberta, Canada T6G 2H7. Phone: (780) 492-9728. Fax: (780) 492-9828. E-mail: lorne.tyrrell{at}ualberta.ca.

Antimicrobial Agents and Chemotherapy, June 2003, p. 1936-1942, Vol. 47, No. 6
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.6.1936-1942.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.





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