Involvement of the host DNA-repair enzyme TDP2 in formation of the covalently closed circular DNA persistence reservoir of hepatitis B viruses
Involvement of the host DNA-repair enzyme TDP2 in formation of the covalently closed circular DNA persistence reservoir of hepatitis B viruses
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DOI：
10.1073/pnas.1409986111
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发表时间：
2014-10-07
影响因子：
11.1
通讯作者：
Nassal, Michael
中科院分区：
文献类型：
--
作者：
Koeniger, Christian;Wingert, Ida;Nassal, Michael
B型肝炎病毒（HBV）是慢性B型肝炎和典型嗜肝DNA病毒的病原体，是通过蛋白质引发的逆转录进行复制的小DNA病毒。产物是一个3-kb松弛环状DNA（RC-DNA），其中一条链通过酪氨酰-DNA磷酸二酯键与病毒聚合酶（P蛋白）连接。感染后，进入的RC-DNA被转化为共价闭合环状（ccc）DNA，其作为病毒持久性储库，其对当前抗HBV治疗是难治的。cccDNA形成的机制尚不清楚，但P蛋白的释放是一个必不可少的步骤。RC-DNA和细胞拓扑异构酶-DNA加合物之间的结构相似性及其已知的酪氨酸-DNA-磷酸二酯酶（TDP）1或TDP 2的修复表明，HBV可能为了自己的目的而篡夺这些酶。在这里，我们证明，人和鸡TDP 2，但只有酵母直向同源物的TDP 1，可以特异性地切割的Tyr-DNA键在病毒适应的模型基板和释放P蛋白从真实的HBV和鸭HBV（DHBV）的RC-DNA在体外，没有事先蛋白水解的大P蛋白。与TPD 2在cccDNA形成中具有生理作用一致，人细胞中RNAi介导的TDP 2消耗显著减缓了RC-DNA向cccDNA的转化。在相同细胞中的异位TDP 2表达恢复了更快的转化动力学。这些数据有力地表明，TDP 2是参与HBV cccDNA生物发生的第一种（尽管可能不是唯一的）宿主DNA修复因子。除了在嗜肝DNA病毒和DNA修复之间建立功能联系外，我们的研究结果还为直接靶向HBV持久性开辟了新的前景。
Hepatitis B virus (HBV), the causative agent of chronic hepatitis B and prototypic hepadnavirus, is a small DNA virus that replicates by protein-primed reverse transcription. The product is a 3-kb relaxed circular DNA (RC-DNA) in which one strand is linked to the viral polymerase (P protein) through a tyrosyl-DNA phosphodiester bond. Upon infection, the incoming RC-DNA is converted into covalently closed circular (ccc) DNA, which serves as a viral persistence reservoir that is refractory to current anti-HBV treatments. The mechanism of cccDNA formation is unknown, but the release of P protein is one mandatory step. Structural similarities between RC-DNA and cellular topoisomerase-DNA adducts and their known repair by tyrosyl-DNA-phosphodiesterase (TDP) 1 or TDP2 suggested that HBV may usurp these enzymes for its own purpose. Here we demonstrate that human and chicken TDP2, but only the yeast ortholog of TDP1, can specifically cleave the Tyr-DNA bond in virus-adapted model substrates and release P protein from authentic HBV and duck HBV (DHBV) RC-DNA in vitro, without prior proteolysis of the large P proteins. Consistent with TPD2's having a physiological role in cccDNA formation, RNAi-mediated TDP2 depletion in human cells significantly slowed the conversion of RC-DNA to cccDNA. Ectopic TDP2 expression in the same cells restored faster conversion kinetics. These data strongly suggest that TDP2 is a first, although likely not the only, host DNA-repair factor involved in HBV cccDNA biogenesis. In addition to establishing a functional link between hepadnaviruses and DNA repair, our results open new prospects for directly targeting HBV persistence.