Cellular DNA repair in cccDNA formation during hepatitits B virus infection - addressing functionally redundant repair pathways

乙型肝炎病毒感染期间 cccDNA 形成中的细胞 DNA 修复 - 解决功能冗余修复途径

• 批准号: 134164308
• 负责人: Professor Dr. Michael Nassal
• 金额: --
• 依托单位: Klinik für Innere Medizin II - Gastroenterologie, Hepatologie, Endokrinologie und Infektiologie
• 依托单位国家: 德国
• 项目类别: Research Units
• 财政年份: 2009
• 资助国家: 德国
• 起止时间: 2008-12-31 至 2015-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/134164308?language=en
• 关键词: Cellular; DNA; repair; cccDNA; formation

Acting as template for all viral transcripts, the covalently closed circular (ccc) DNA form of the hepatitis B virus (HBV) genome is crucial for persistence. None of the current drugs directly targets or even eradicates cccDNA; a few persisting cccDNA molecules can reactivate infection. Improving this situation requires cccDNA biogenesis to be understood. Currently, little is known on how the P protein- and RNA-linked relaxed circular (RC) DNA genome from virions is converted into cccDNA, except that this requires several distinct enzymatic steps. Shared features between RC-DNA and damaged cellular DNA (strand-breaks, non- DNA adducts) suggested that these activities are provided by the cell´s DNA repair machinery. Indeed, results obtained so far strongly imply tyrosyl-DNA-phosphodiesterase 2 (TDP2) as one cellular repair factor in cccDNA formation but they also suggest an involvement of mechanistically distinct alternative pathways. Such redundancy is a hallmark of cellular DNA repair. The overall aim of the project is to elucidate the molecular pathway of cccDNA biogenesis. Focusing on P protein removal from RC-DNA we obtained compelling biochemical evidence that TDP2, involved in cellular protein-DNA adduct repair, can perform this function. However, in-cell TDP2 knockdown by conventional RNAi techniques revealed only modest reductions in cccDNA. Such phenotypes are well known from cellular DNA repair where endonucleolytic pathways can substitute for TDP. Beyond final proof for a physiological role of TDP2, the proposal therefore aims at defining whether, and if so which, of these alternative pathways operate in cccDNA formation. Furthermore, nucleolysis is the only way to remove the RNA primer from RC-DNA, hence this step will be also be analyzed. As before, we will combine in vitro plus in-cell approaches to identify additional repair factors, in particular structure-specific endonucleases, that can act on RC-DNA and then evaluate the consequences of modulating their cellular levels on cccDNA formation. Numerous new tools including in vitro assays for repair factor activity and human cell lines producing well detectable cccDNA are now available. However, we also realized that the limited silencing efficiency of the RNAi techniques used so far hampered definite conclusions on whether a cell factor is decisive for, or contributing to, cccDNA biogenesis. Hence we will exploit advanced technologies, including inducibly shRNA expressing lentivirus vectors and genomeediting by designer nucleases, to increase knockdown efficiency or completely knockout individual repair factors. In case of factors where this approach is not applicable, small compounds and adenovirus early proteins will be used for inhibition. In combination, this repertoire of methods will enable inactivation of more than one repair factor at a time, and thus identify at least some of the key cell factors involved in hepadnaviral cccDNA biogenesis.

作为所有病毒转录物的模板，B肝炎病毒（HBV）基因组的共价闭合环状（ccc）DNA形式对于持久性至关重要。目前没有一种药物直接靶向甚至根除cccDNA;一些持续存在的cccDNA分子可以重新激活感染。改善这种情况需要理解cccDNA的生物发生。目前，关于来自病毒体的P蛋白和RNA连接的松弛环状（RC）DNA基因组如何转化为cccDNA知之甚少，除了这需要几个不同的酶促步骤。RC-DNA和受损细胞DNA之间的共同特征（链断裂、非DNA加合物）表明这些活动是由细胞的DNA修复机制提供的。事实上，迄今为止获得的结果强烈暗示酪氨酰-DNA-磷酸二酯酶2（TDP 2）作为cccDNA形成中的一种细胞修复因子，但它们也表明涉及机械上不同的替代途径。这种冗余是细胞DNA修复的标志。该项目的总体目标是阐明cccDNA生物发生的分子途径。通过研究P蛋白从RC-DNA中的去除，我们获得了令人信服的生物化学证据，表明参与细胞蛋白-DNA加合物修复的TDP 2可以执行此功能。然而，通过常规RNAi技术进行的细胞内TDP 2敲低显示cccDNA仅适度减少。这些表型是细胞DNA修复中众所周知的，其中核酸内切途径可以取代TDP。除了最终证明TDP 2的生理作用之外，该提案还旨在定义这些替代途径中是否以及如果是，则哪些在cccDNA形成中起作用。此外，核溶解是从RC-DNA中去除RNA引物的唯一方法，因此也将分析该步骤。如前所述，我们将联合收割机体外加细胞内方法相结合，以鉴定可作用于RC-DNA的其他修复因子，特别是结构特异性核酸内切酶，然后评估调节其细胞水平对cccDNA形成的影响。许多新的工具，包括在体外检测修复因子的活性和人类细胞系产生良好的可检测的cccDNA，现在是可用的。然而，我们也意识到，迄今为止使用的RNAi技术的有限沉默效率阻碍了关于细胞因子是否决定或有助于cccDNA生物发生的明确结论。因此，我们将利用先进的技术，包括可诱导的shRNA表达慢病毒载体和设计核酸酶的基因组编辑，以提高敲除效率或完全敲除单个修复因子。在这种方法不适用的因素的情况下，小化合物和腺病毒早期蛋白将用于抑制。组合起来，这种方法库将能够一次灭活超过一种修复因子，从而鉴定参与嗜肝DNA病毒cccDNA生物发生的至少一些关键细胞因子。

项目成果

Involvement of the host DNA-repair enzyme TDP2 in formation of the covalently closed circular DNA persistence reservoir of hepatitis B viruses

• DOI: 10.1073/pnas.1409986111
• 发表时间: 2014-10-07
• 期刊: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• 影响因子: 11.1
• 作者: Koeniger, Christian;Wingert, Ida;Nassal, Michael
• 通讯作者: Nassal, Michael

Hepatitis B Virus-Infected HepG2hNTCP Cells Serve as a Novel Immunological Tool To Analyze the Antiviral Efficacy of CD8+ T Cells In Vitro

• DOI: 10.1128/jvi.00605-15
• 发表时间: 2015-07-01
• 期刊: JOURNAL OF VIROLOGY
• 影响因子: 5.4
• 作者: Hoh, Alexander;Heeg, Maximilian;Thimme, Robert
• 通讯作者: Thimme, Robert

HBV cccDNA: viral persistence reservoir and key obstacle for a cure of chronic hepatitis B

• DOI: 10.1136/gutjnl-2015-309809
• 发表时间: 2015-12-01
• 期刊: GUT
• 影响因子: 24.5
• 作者: Nassal, Michael