Hong Young Bin, Choi Yongwook, Jung Guhung
Division of Genetic Disease, Korean National Institute of Health, Seoul 122-701, Korea.
J Biochem Mol Biol. 2004 Mar 31;37(2):167-76. doi: 10.5483/bmbrep.2004.37.2.167.
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.
尽管高效抗乙肝病毒药物拉米夫定用于乙肝病毒感染患者，但核苷类似物的长期治疗常导致拉米夫定耐药变异体的出现。在本研究中，我们评估了拉米夫定耐药变异体的保真度。乙肝病毒DNA聚合酶的FLAG标签野生型（FPolE）和Met550变异体（FPolE/M550A、M550V和M550I）在昆虫细胞中表达，然后进行纯化。与许多其他逆转录酶一样，在乙肝病毒DNA聚合酶中未检测到3'→5'外切核酸酶活性。由于没有校对活性，因此使用位点特异性核苷酸错配掺入方法是有益的。从f(ins)值分析可以明显看出，M550I和M550V的保真度值高于野生型乙肝病毒DNA聚合酶，而M550A的保真度值与之相似。因此，有人认为拉米夫定耐药性源于M550V和M550I中对dNTP结合的严格性以及对dCTP和拉米夫定的辨别。
