Kinetic analysis of wild-type and YMDD mutant hepatitis B virus polymerases and effects of deoxyribonucleotide concentrations on polymerase activity

Abstract

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.

FIG. 1.

Schematic of HBV genome replication. Pregenomic RNA is shown as a linear molecule, with the locations of direct repeats 1 (DR1) and 2 (DR2) indicated. After initiation of replication at the 5′ end of the pregenomic RNA and translocation to the direct repeat 1 (DR1) at the 3′ end, elongation continues and produces the minus-strand DNA. Second-strand synthesis [producing the DNA plus strand] occurs after circularization of the minus strand. Probes 8F and 4RA are indicated by the heavy black lines.