Microbiology and Immunology Graduate Program, Drexel University College of Medicine, Philadelphia, Pennsylvania, USA.
Baruch S. Blumberg Institute, Doylestown, Pennsylvania, USA.
J Virol. 2019 May 15;93(11). doi: 10.1128/JVI.02230-18. Print 2019 Jun 1.
In order to identify host cellular DNA metabolic enzymes that are involved in the biosynthesis of hepatitis B virus (HBV) covalently closed circular (ccc) DNA, we developed a cell-based assay supporting synchronized and rapid cccDNA synthesis from intracellular progeny nucleocapsid DNA. This was achieved by arresting HBV DNA replication in HepAD38 cells with phosphonoformic acid (PFA), a reversible HBV DNA polymerase inhibitor, at the stage of single-stranded DNA and was followed by removal of PFA to allow the synchronized synthesis of relaxed circular DNA (rcDNA) and subsequent conversion into cccDNA within 12 to 24 h. This cccDNA formation assay allows systematic screening of the effects of small molecular inhibitors of DNA metabolic enzymes on cccDNA synthesis but avoids cytotoxic effects upon long-term treatment. Using this assay, we found that all the tested topoisomerase I and II (TOP1 and TOP2, respectively) poisons as well as topoisomerase II DNA binding and ATPase inhibitors significantly reduced the levels of cccDNA. It was further demonstrated that these inhibitors also disrupted cccDNA synthesis during HBV infection of HepG2 cells expressing sodium taurocholate cotransporting polypeptide (NTCP). Mechanistic analyses indicate that whereas TOP1 inhibitor treatment prevented the production of covalently closed negative-strand rcDNA, TOP2 inhibitors reduced the production of this cccDNA synthesis intermediate to a lesser extent. Moreover, small interfering RNA (siRNA) knockdown of topoisomerase II significantly reduced cccDNA amplification. Taking these observations together, our study demonstrates that topoisomerase I and II may catalyze distinct steps of HBV cccDNA synthesis and that pharmacologic targeting of these cellular enzymes may facilitate the cure of chronic hepatitis B. Persistent HBV infection relies on stable maintenance and proper functioning of a nuclear episomal form of the viral genome called cccDNA, the most stable HBV replication intermediate. One of the major reasons for the failure of currently available antiviral therapeutics to cure chronic HBV infection is their inability to eradicate or inactivate cccDNA. We report here a chemical genetics approach to identify host cellular factors essential for the biosynthesis and maintenance of cccDNA and reveal that cellular DNA topoisomerases are required for both synthesis and intracellular amplification of cccDNA. This approach is suitable for systematic screening of compounds targeting cellular DNA metabolic enzymes and chromatin remodelers for their ability to disrupt cccDNA biosynthesis and function. Identification of key host factors required for cccDNA metabolism and function will reveal molecular targets for developing curative therapeutics of chronic HBV infection.
为了鉴定参与乙型肝炎病毒 (HBV) 共价闭合环状 (ccc) DNA 生物合成的宿主细胞 DNA 代谢酶，我们开发了一种基于细胞的测定法，支持从细胞内子代核衣壳 DNA 中同步快速合成 cccDNA。这是通过在 HepAD38 细胞中用膦甲酸 (PFA) 抑制 HBV DNA 聚合酶来实现的，PFA 是一种可逆的 HBV DNA 聚合酶抑制剂，在单链 DNA 阶段，然后去除 PFA 以允许松弛环状 DNA (rcDNA) 的同步合成，并在 12 至 24 小时内随后转化为 cccDNA。这种 cccDNA 形成测定法允许系统筛选 DNA 代谢酶的小分子抑制剂对 cccDNA 合成的影响，但避免了长期治疗时的细胞毒性作用。使用该测定法，我们发现所有测试的拓扑异构酶 I 和 II (TOP1 和 TOP2，分别) 毒物以及拓扑异构酶 II DNA 结合和 ATP 酶抑制剂均显著降低了 cccDNA 的水平。进一步表明，这些抑制剂还在表达牛磺胆酸钠共转运蛋白 (NTCP) 的 HepG2 细胞的 HBV 感染过程中破坏了 cccDNA 的合成。机制分析表明，尽管 TOP1 抑制剂治疗阻止了共价闭合的负链 rcDNA 的产生，但 TOP2 抑制剂对这种 cccDNA 合成中间体的产生抑制作用较小。此外，拓扑异构酶 II 的小干扰 RNA (siRNA) 敲低显著降低了 cccDNA 的扩增。综合这些观察结果，我们的研究表明，拓扑异构酶 I 和 II 可能催化 HBV cccDNA 合成的不同步骤，并且细胞酶的药理学靶向可能有助于慢性乙型肝炎的治愈。持续性 HBV 感染依赖于病毒基因组的核附加体形式的稳定维持和正常功能，称为 cccDNA，是最稳定的 HBV 复制中间体。目前可用的抗病毒治疗无法治愈慢性 HBV 感染的主要原因之一是它们无法根除或失活 cccDNA。我们在这里报告了一种化学遗传学方法来鉴定合成和维持 cccDNA 所必需的宿主细胞因子，并揭示细胞 DNA 拓扑异构酶对于 cccDNA 的合成和细胞内扩增都是必需的。这种方法适用于系统筛选靶向细胞 DNA 代谢酶和染色质重塑剂的化合物，以确定它们破坏 cccDNA 生物合成和功能的能力。鉴定 cccDNA 代谢和功能所必需的关键宿主因子将揭示开发慢性 HBV 感染治愈性治疗的分子靶点。
