In vitro susceptibilities of wild-type or drug-resistant hepatitis B virus to (-)-beta-D-2,6-diaminopurine dioxolane and 2'-fluoro-5-methyl-beta-L-arabinofuranosyluracil

Abstract

Prolonged treatment of chronic hepatitis B virus (HBV) infection with lamivudine ([-]-beta-L-2',3'-dideoxy-3' thiacytidine) or famciclovir may select for viral mutants that are drug resistant due to point mutations in the polymerase gene. Determining whether such HBV mutants are sensitive to new antiviral agents is therefore important. We used a transient transfection system to compare the sensitivities of wild-type HBV and four lamivudine- and/or famciclovir-resistant HBV mutants to adefovir [9-(2-phosphonyl-methoxyethyl)-adenine; PMEA] and the nucleoside analogues (-)-beta-D-2, 6-diaminopurine dioxolane (DAPD) and 2'-fluoro-5-methyl-beta-L-arabinofuranosyluracil (L-FMAU). The drug-resistant mutants contained amino acid substitutions in the polymerase protein. We found that the M550I and M550V plus L526M substitutions, which confer lamivudine resistance, did not confer cross-resistance to adefovir or DAPD, but conferred cross-resistance to L-FMAU. The M550V substitution in isolation conferred a similar phenotype to M550I, except that it did not confer significant resistance to L-FMAU. The L526M substitution, which is associated with famciclovir resistance, conferred cross-resistance to L-FMAU but not to adefovir or DAPD. Inhibition of HBV secretion by DAPD, L-FMAU, and adefovir did not always correlate with inhibition of the generation of intracellular HBV replicative intermediates, suggesting that these analogs may preferentially inhibit specific stages of the viral replication cycle.

FIG. 1

Southern hybridization of HBV RI extracted from intracellular core particles extracted from HepG2 cells transiently transfected with plasmid vectors which contained either wild-type (wt) HBV or L526M, M5501, or L526M + M550V mutants following treatment with (a) lamivudine, (b) adefovir, (c) DAPD, or (d) l-FMAU. Bands which correspond to the rcDNA (RC) and ssDNA (SS) species are indicated.

FIG. 2

Graphical analysis of data obtained from autoradiographs shown in Fig. 1. Dose-response plots for inhibition of wild-type HBV replication by lamivudine (LMV), adefovir (ADV), l-FMAU, and DAPD are shown. The finer lines on either side of each plot represent 95% confidence intervals for each curve fit.