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Antimicrobial Agents and Chemotherapy, April 2002, p. 1005-1013, Vol. 46, No. 4
0066-4804/02/$04.00+0     DOI: 10.1128/AAC.46.4.1005-1013.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Kinetic Analysis of Wild-Type and YMDD Mutant Hepatitis B Virus Polymerases and Effects of Deoxyribonucleotide Concentrations on Polymerase Activity

Richard K. Gaillard,1* Jennifer Barnard,1 Vincent Lopez,1 Paula Hodges,2 Eric Bourne,1 Lance Johnson,1 Marchelle I. Allen,1 Patrick Condreay,2 Wayne H. Miller,3 and Lynn D. Condreay1

Departments of Virology,1 Protein Sciences,2 Molecular Biochemistry, GlaxoSmithKline, Research Triangle Park, North Carolina3

Received 13 August 2001/ Returned for modification 8 November 2001/ Accepted 15 January 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 Km values for the natural dNTP substrates, indicating decreased affinities for these substrates, as well as increased Ki values for lamivudine triphosphate, indicating decreased affinity for the drug. The effect of the differences in apparent Km values between YMDD-mutant polymerase and wt HBV polymerase could be masked by high levels of dNTP substrates (>20 µM). 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 Km 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.

* Corresponding author. Mailing address: GlaxoSmithKline, Dept. of Virology, RC2 Rm. 3848, P.O. Box 13398, Research Triangle Park, NC 27709. Phone: (919) 483-9265. Fax: (919) 315-5243. E-mail: rkg36200{at}gsk.com.

Antimicrobial Agents and Chemotherapy, April 2002, p. 1005-1013, Vol. 46, No. 4
0066-4804/02/$04.00+0     DOI: 10.1128/AAC.46.4.1005-1013.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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