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

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 replic ation fitness and susceptibility of the wild-type and five mutant HBVs (L52 8M, 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 (-)-carbov ir] by transfecting HBV DNA into hepatoma cells and monitoring viral produc ts by Southern blotting. The replication competency of the single C-domain mutants M552I and M552V was markedly decreased compared With that of wild-t ype HBV. However, addition of the B-domain mutation L528M restored replicat ion 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.