In vitro evaluation of hepatitis B virus polymerase mutations associated with famciclovir resistance

Several mutations (V521L, P525L, L528M, T532S, and V555I) in the gene for hepatitis B virus (HBV) polymerase have been identified in HBV isolated from patients that displayed break-through viremia during famciclovir treatment. To determine whether these mutations cause phenotypic resistance to famciclovir, we compared the inhibition constants (K_i ) of penciclovir triphosphate (PCVTP, the active metabolite of famciclovir) for recombinant wild-type and mutant HBV polymerases containing these mutations. In in vitro enzymatic assays, the V555I mutation displayed the most resistance (with K_i increased by 6.2-fold) to PCVTP. The V521L and L528M mutations showed moderately decreased sensitivity to PCVTP (K_i increased by >3-fold). We also analyzed the cross-resistance profiles of these variants for adefovir and lamivudine, two other antiviral agents that also inhibit DNA replication by HBV polymerase. All 5 famciclovir-associated mutations were sensitive to adefovir diphosphate (ADVDP) in in vitro enzymatic assays (<2.3-fold decreased sensitivity). The V521L, L528M, and T532S mutations were also sensitive to lamivudine triphosphate (LAMTP); however, the P525L and V555I mutations displayed moderately decreased sensitivity to LAMTP in enzymatic assays (3.6-fold decreased sensitivity). The lamivudine-resistant mutations M552I, M552V, and L528M+M552V, which were previously shown to display 8- to 25-fold resistance to LAMTP, were less resistant (≤3.1-fold) to PCVTP.