Gaillard Richard K, Barnard Jennifer, Lopez Vincent, Hodges Paula, Bourne Eric, Johnson Lance, Allen Marchelle I, Condreay Patrick, Miller Wayne H, Condreay Lynn D
Department of Virology, GlaxoSmithKline, Research Triangle Park, North Carolina 27709, USA.
Antimicrob Agents Chemother. 2002 Apr;46(4):1005-13. doi: 10.1128/AAC.46.4.1005-1013.2002.
Mutations in the YMDD motif of the hepatitis B virus (HBV) DNA polymerase result in reduced susceptibility of HBV to inhibition by lamivudine, at a cost in replication fitness. The mechanisms underlying the effects of YMDD mutations on replication fitness were investigated using both a cell-based viral replication system and an in vitro enzyme assay to examine wild-type (wt) and YMDD-mutant polymerases. We calculated the affinities of wt and YMDD-mutant polymerases for each natural deoxyribonucleoside triphosphate (dNTP) and determined the intracellular concentrations of each dNTP in HepG2 cells under conditions that support HBV replication. In addition, inhibition constants for lamivudine triphosphate were determined for wt and YMDD-mutant polymerases. Relative to wt HBV polymerase, each of the YMDD-mutant polymerases showed increased apparent K(m) values for the natural dNTP substrates, indicating decreased affinities for these substrates, as well as increased K(i) values for lamivudine triphosphate, indicating decreased affinity for the drug. The effect of the differences in apparent K(m) values between YMDD-mutant polymerase and wt HBV polymerase could be masked by high levels of dNTP substrates (>20 microM). However, assays using dNTP concentrations equivalent to those measured in HepG2 cells under physiological conditions showed decreased enzymatic activity of YMDD-mutant polymerases relative to wt polymerase. Therefore, the decrease in replication fitness of YMDD-mutant HBV strains results from the lower affinities (increased K(m) values) of the YMDD-mutant polymerases for the natural dNTP substrates and physiological intracellular concentrations of dNTPs that are limiting for the replication of YMDD-mutant HBV strains.
乙型肝炎病毒（HBV）DNA聚合酶的YMDD基序发生突变，会导致HBV对拉米夫定抑制作用的敏感性降低，代价是复制适应性下降。使用基于细胞的病毒复制系统和体外酶测定法，研究了YMDD突变对复制适应性影响的潜在机制，以检测野生型（wt）和YMDD突变型聚合酶。我们计算了wt和YMDD突变型聚合酶对每种天然脱氧核糖核苷三磷酸（dNTP）的亲和力，并确定了在支持HBV复制的条件下HepG2细胞中每种dNTP的细胞内浓度。此外，还测定了wt和YMDD突变型聚合酶对三磷酸拉米夫定的抑制常数。相对于wt HBV聚合酶，每种YMDD突变型聚合酶对天然dNTP底物的表观K(m)值均增加，表明对这些底物的亲和力降低，同时对三磷酸拉米夫定的K(i)值也增加，表明对该药物的亲和力降低。YMDD突变型聚合酶与wt HBV聚合酶之间表观K(m)值的差异效应可能会被高水平的dNTP底物（>20 microM）掩盖。然而，使用与生理条件下HepG2细胞中测得的dNTP浓度相当的dNTP浓度进行的测定表明，YMDD突变型聚合酶的酶活性相对于wt聚合酶有所降低。因此，YMDD突变型HBV毒株复制适应性的降低是由于YMDD突变型聚合酶对天然dNTP底物的亲和力较低（K(m)值增加）以及生理细胞内dNTP浓度对YMDD突变型HBV毒株的复制具有限制作用。
