YMDD motif in hepatitis B virus DNA polymerase influences on replication and lamivudine resistance: A study by in vitro full-length viral DNA transfection

Recently lamivudine used to treat patients with hepatitis B virus (HBV) inf ection was revealed to have potent antiviral activity. However, HBV resista nce to lamivudine has been reported and shown to have amino acid substituti ons in the methionine residue of the conserved tyrosine (Y), methionine (M) , aspartate (D), aspartate (D) motif of RNA-dependent DNA polymerase. To ex plore the consequences of substitutions in this motif (YMDD), we made 7 var iants by substituting the methionine of the YMDD motif with isoleucine (I), valine (V), alanine (A), leucine (L), lysine (K), arginine (R), and threon ine (T). Replication ability of these variants was evaluated by transfectio n into human hepatoma cells. Sensitivity to lamivudine was tested for repli cation-competent variants. Four variants with hydrophobic substitutions (I, V; A, and L) remained replication competent, whereas 3 others with hydroph ilic substitutions (K, R, and T) exhibited impaired replication. Of the 4 r eplication-competent variants, 2 (I and V) were resistant, and 2 (A and L) were sensitive to lamivudine. Because the polymerase and the surface gene o verlap, the introduction of these mutations affected the secretion of hepat itis B surface antigen (HBsAg), namely 4 variants (I, V, L, and R) secreted HBsAg, whereas 3 variants (A, K, and T) did not. Our study elucidated that only one amino acid substitution in the YMDD motif was sufficient to cause lamivudine resistance in vitro. As a result of replication competence and lamivudine sensitivity, only viruses having YIDD or YVDD sequences may appe ar during treatment with lamivudine. This in vitro system could be used to study HBV mutations, replication competence, and their susceptibility to an tivirals.