Noncompetitive Inhibition of Hepatitis B Virus Reverse Transcriptase Protein Priming and DNA Synthesis by the Nucleoside Analog Clevudine
Noncompetitive Inhibition of Hepatitis B Virus Reverse Transcriptase Protein Priming and DNA Synthesis by the Nucleoside Analog Clevudine
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DOI：
10.1128/aac.00599-13
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发表时间：
2013-09-01
影响因子：
4.9
通讯作者：
Hu, Jianming
中科院分区：
文献类型：
--
作者：
Jones, Scott A.;Murakami, Eisuke;Hu, Jianming
目前批准用于治疗慢性B型肝炎病毒（HBV）感染的所有抗病毒药物都是核苷（酸）逆转录酶抑制剂（NRTI），其抑制HBV聚合酶的DNA合成活性。聚合酶是一种独特的逆转录酶（RT），具有新的蛋白质引发活性，其中HP使用自身作为蛋白质引物启动病毒DNA合成。我们已经确定了NRTI-三磷酸盐（TP）抑制HBV蛋白引发的能力及其作用机制。恩替卡韦-TP（一种dGTP类似物）抑制了dGTP特异性引发的蛋白质引发，而克来夫定-TP（一种TTP类似物）能够独立于脱氧核苷三磷酸（dNTP）底物抑制蛋白质引发，并且不掺入DNA中。我们接下来研究了NRTI对蛋白质引发的第二阶段的影响，其中将两个dAMP核苷酸添加到初始脱氧鸟苷核苷酸。所获得的结果表明，clevudine-TP以及替诺福韦DF-DP强烈抑制蛋白引发的第二阶段。替诺福韦DF-DP掺入到病毒DNA引物中，而克来夫定-TP抑制引发的第二阶段而不被掺入。最后，使用HBV内源性聚合酶测定的动力学分析表明，clevudine-TP抑制DNA链延长HP在一个非竞争性的方式。因此，clevudine-TP似乎具有独特的能力，通过结合和扭曲的HP活性位点，抑制HBV RT，与NRTI和非核苷RT抑制剂共享的属性。
All currently approved antiviral drugs for the treatment of chronic hepatitis B virus (HBV) infection are nucleos(t)ide reverse transcriptase inhibitors (NRTI), which inhibit the DNA synthesis activity of the HBV polymerase. The polymerase is a unique reverse transcriptase (RT) that has a novel protein priming activity in which HP initiates viral DNA synthesis using itself as a protein primer. We have determined the ability of NRTI-triphosphates (TP) to inhibit HBV protein priming and their mechanisms of action. While entecavir-TP (a dGTP analog) inhibited protein priming initiated specifically with dGTP, clevudine-TP (a TTP analog) was able to inhibit protein priming independently of the deoxynucleoside triphosphate (dNTP) substrate and without being incorporated into DNA. We next investigated the effect of NRTIs on the second stage of protein priming, wherein two dAMP nucleotides are added to the initial deoxyguanosine nucleotide. The obtained results indicated that clevudine-TP as well as tenofovir DF-DP strongly inhibited the second stage of protein priming. Tenofovir DF-DP was incorporated into the viral DNA primer, whereas clevudine-TP inhibited the second stage of priming without being incorporated. Finally, kinetic analyses using the HBV endogenous polymerase assay revealed that clevudine-TP inhibited DNA chain elongation by HP in a noncompetitive manner. Thus, clevudine-TP appears to have the unique ability to inhibit HBV RT via binding to and distorting the HP active site, sharing properties with both NRTIs and nonnucleoside RT inhibitors.