The polymerase L528M mutation cooperates with nucleotide binding-site mutations, increasing hepatitis B virus replication and drug resistance

Abstract

After receiving lamivudine for 3 years to treat chronic hepatitis B, 67-75% of patients develop B-domain L528M, C-domain M552I, or M552V mutations in the HBV polymerase that render hepatitis B virus (HBV) drug-resistant. The aim of this study was to evaluate the influence of these mutations on viral replication and resistance to antiviral agents. We investigated the replication fitness and susceptibility of the wild-type and five mutant HBVs (L528M, M552I, M552V, L528M/M552I, and L528M/M552V) to 11 compounds [lamivudine, adefovir, entecavir (BMS-200475) (+)-BCH-189 (+/-)-FTC (racivir) (-)-FTC (emtricitabine) (+)-FTC, L-D4FC, L-FMAU (clevudine), D-DAPD, and (-)-carbovir] by transfecting HBV DNA into hepatoma cells and monitoring viral products by Southern blotting. The replication competency of the single C-domain mutants M552I and M552V was markedly decreased compared with that of wild-type HBV. However, addition of the B-domain mutation L528M restored replication competence. Only adefovir and entecavir were effective against all five HBV mutants, and higher doses of these compounds were necessary to inhibit the double mutants compared with the single mutants. The B-domain mutation (L528M) of HBV polymerase not only restores the replication competence of C-domain mutants, but also increases resistance to nucleoside analogues.

Figure 1

Southern blot hybridization analysis of replication of wild-type HBV and five mutants. Lanes correspond to DNA extracted from viral core particles derived from HuH-7 cells that were transfected with DNA of wild-type HBV or one of five mutants. Single-stranded bands (SS) were quantified using an LAS1000 image analyzer and then normalized for transfection efficiency based on β-galactosidase activity. The relative ratio of the normalized single-stranded band is shown below each lane, assuming the single-stranded band of wild-type HBV to be 100%. OC, open circular; DS, double-stranded HBV DNA.

Figure 2

Representative Southern blot hybridization used to determine the EC₅₀ value of entecavir against wild-type HBV. (a) Southern blot hybridization analysis of replication of the wild-type treated with entecavir. Lanes correspond to DNA extracted from viral core particles derived from HuH-7 cells that were transfected with wild-type HBV DNA and incubated with increasing concentrations (00001, 0.001, 0.01, and 0.1 μM) of entecavir. OC, open circular; DS, double-stranded; SS, single-stranded HBV DNA. (b) Diagram of replication of wild-type HBV treated with entecavir. Single-stranded bands (SS) were quantified using an LAS1000 image analyzer and then normalized for transfection efficiency based on β-galactosidase activity. The single-stranded band of the wild-type without entecavir was calculated as 100. EC₅₀ was determined to be 0.00036 μM.

Figure 3

Susceptibility of HBV dimer (pSM2) (a) and HBV from 2.2.15 cells (b) to lamivudine and entecavir: drug inhibition curves of wild-type HBV transfected into HuH-7 cells treated with the indicated concentrations of lamivudine and entecavir.