Increased DNA polymerase fidelity of the Lamivudine resistant variants of human hepatitis B virus DNA polymerase.
Although efficient antiviral lamivudine is used for HBV-infected patients, a prolonged treatment with nucleoside analogs often results in lamivudine-resistant variants. In this study, we evaluated the fidelity of the lamivudine-resistant variants. The FLAG-tagged wild-type (FPolE) and Met550 variants (FPolE/M550A, M550V, and M550I) of HBV DNA polymerases were expressed in insect cells, then purified. Like many other reverse transcriptases, no 3' --> 5' exonuclease activity was detected in the HBV DNA polymerase. Since there is no proofreading activity, then the use of the site-specific nucleotide misincorporation method is beneficial. From the f(ins) value analysis, it is evident that M550I and M550V exhibit higher fidelity values than the wild-type HBV DNA polymerase, while M550A exhibits similar fidelity values. It is therefore suggested that lamivudine resistance comes from the stringency to dNTP binding and the discrimination of dCTP and lamivudine in M550V and M550I.
Hong
Young Bin
YB
Division of Genetic Disease, Korean National Institute of Health, Seoul 122-701, Korea.
Choi
Yongwook
Y
Jung
Guhung
G
D000818:Animals; D000998:Antiviral Agents / Q000627:therapeutic use*; D016367:Baculoviridae; D020137:Base Pair Mismatch; D001483:Base Sequence; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004352:Drug Resistance, Microbial / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D005092:Exonucleases / Q000235:genetics / Q000378:metabolism; D014644:Genetic Variation; D054303:HIV Reverse Transcriptase / Q000378:metabolism; D006515:Hepatitis B virus / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D019259:Lamivudine / Q000627:therapeutic use*; D016297:Mutagenesis, Site-Directed; D018411:Spodoptera; D013698:Templates, Genetic
D000998:Antiviral Agents; D017931:DNA Primers; D019259:Lamivudine; D054303:HIV Reverse Transcriptase; D004259:DNA-Directed DNA Polymerase; D005092:Exonucleases