Susceptibility of lamivudine-resistant hepatitis B virus to other reverse transcriptase inhibitors

Abstract

The emergence of resistant hepatitis B virus (HBV), with mutations in the YMDD motif of the polymerase gene after treatment with lamivudine, is becoming an important clinical problem. In this study, susceptibility of wild-type and lamivudine-resistant HBV M552I, M552V, and L528M/M552V mutants to other reverse transcriptase inhibitors was investigated by transient transfection of full-length HBV DNA into human hepatoma cells. HBV DNA replication was monitored by Southern blot hybridization, which showed the presence of a single-stranded band (representative of the HBV replicative intermediates) in the drug-free, wild-type HBV-transfected cells. This band was diminished in the samples of wild-type HBV DNA treated with either lamivudine, adefovir, or lobucavir. The band intensities from the lamivudine-resistant mutants were not decreased by treatment with lamivudine, but were decreased by the treatments with adefovir or lobucavir. In contrast, penciclovir and nevirapine did not diminish the intensity of the single-stranded band of wild-type HBV or the lamivudine-resistant mutants. These results demonstrate that lamivudine-resistant HBV is susceptible to adefovir and lobucavir. Lamivudine-resistant HBV should be treated with adefovir or lobucavir, and combination therapy with lamivudine and adefovir/lobucavir may prevent the emergence of lamivudine-resistant HBV.

Figure 1

Southern blotting analysis of HBV DNA extracted from HuH-7 cells transfected with wild-type, M552I, M552V, and L528M/M552V mutants of HBV treated with antivirals at a concentration of 8.7 μM. Lane a: control; lane b: lamivudine; lane c: nevirapine; lane d: adefovir; lane e: lobucavir; and lane f: penciclovir. OC, open circular; DS, double-stranded; SS, single-stranded.

Figure 2

Diagram comparing the mean ± SD susceptibility of wild-type, M552I, M552V, and L528M/M552V mutants of HBV transfected into HuH-7 cells to lamivudine, lobucavir, adefovir, penciclovir, and nevirapine at a concentration of 8.7 μM. Single-stranded bands were quantified using a BAS 2000 image analyzer and normalized for transfection efficiency based on β-galactosidase activity. The single-stranded band of control was calculated as 100.

Figure 3

Drug inhibition curves of wild-type and lamivudine-resistant HBV transfected into HuH-7 cells treated with indicated concentrations of lamivudine (a), adefovir (b), and lobucavir (c). Values represent the mean of 3 experiments.

Figure 4

Southern blotting hybridization analysis of HBV transfected into HuH-7 cells treated with lamivudine and penciclovir. Lanes correspond to DNA extracted from viral core particles derived from HuH-7 cells. The arrow indicates the single-stranded band, representative of HBV replication intermediates. OC, open circular; DS, double-stranded; SS, single-stranded.

Figure 5

Lack of susceptibility of HBV to penciclovir and nevirapine. Drug inhibition curves of wild-type HBV transfected into HuH-7 cells treated with indicated concentrations of penciclovir and nevirapine. Values represent the mean of 3 experiments.