Generation of stable cell lines expressing Lamivudine-resistant hepatitis B virus for antiviral-compound screening

Abstract

Lamivudine [beta-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.

FIG. 1.

Map of pCMV-HBV-neo construct used to generate stable cell lines rtL180M/M204V and rtM204I. (A) Schematic diagram of the replication-competent HBV construct shows the cytomegalovirus immediate early (CMV-IE) promoter, HBV sequences, NsiI recognition site, ampicillin resistance (amp R) marker and neomycin resistance (neomycin R) marker. (B) Diagram depicting organization of open reading frames in HBV sequences. CMV, cytomegalovirus; Pol, polymerase; X, X gene; S, surface. (C) Nucleotide sequence of wild-type and mutant viruses. Altered nucleotides are depicted in bold.

FIG. 2.

Integration of HBV sequences into genomic DNA of HepG2, rtM204I, and rtL180M/M204V cell lines. Genomic DNA from confluent monolayers was isolated, digested with NsiI, and analyzed by Southern blotting for the presence of HBV sequences.