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Core components of DNA lagging strand synthesis machinery are essential for hepatitis B virus cccDNA formation.
Nature Microbiology ( IF 28.3 ) Pub Date : 2020-03-09 , DOI: 10.1038/s41564-020-0678-0 Lei Wei 1 , Alexander Ploss 1
Nature Microbiology ( IF 28.3 ) Pub Date : 2020-03-09 , DOI: 10.1038/s41564-020-0678-0 Lei Wei 1 , Alexander Ploss 1
Affiliation
Chronic hepatitis B virus (HBV) infections result in 887,000 deaths annually. The central challenge in curing HBV is eradication of the stable covalently closed circular DNA (cccDNA) form of the viral genome, which is formed by the repair of lesion-bearing HBV relaxed circular DNA delivered by the virions to hepatocytes. The complete and minimal set of host factors involved in cccDNA formation is unknown, largely due to the lack of a biochemical system that fully reconstitutes cccDNA formation. Here, we have developed experimental systems where various HBV relaxed-circular-DNA substrates are repaired to form cccDNA by both cell extracts and purified human proteins. Using yeast- and human-extract screenings, we identified five core components of lagging-strand synthesis as essential for cccDNA formation: proliferating cell nuclear antigen, the replication factor C complex, DNA polymerase δ, flap endonuclease 1 and DNA ligase 1. We reconstituted cccDNA formation with purified human homologues, establishing these as a minimal set of factors for cccDNA formation. We further demonstrated that treatment with the DNA-polymerase inhibitor aphidicolin diminishes cccDNA formation both in biochemical assays and in HBV-infected human cells. Together, our findings define key components in HBV cccDNA formation.
中文翻译:
DNA落后链合成机器的核心组件对于乙型肝炎病毒cccDNA的形成至关重要。
慢性乙型肝炎病毒(HBV)感染每年导致887,000人死亡。治愈HBV的主要挑战是根除病毒基因组的稳定的共价闭合环状DNA(cccDNA)形式,这种形式是由病毒体传递给肝细胞的带损伤的HBV松弛环状DNA的修复形成的。cccDNA形成所涉及的宿主因子的完整和最少集合是未知的,主要是由于缺乏能够完全重构cccDNA形成的生化系统。在这里,我们开发了实验系统,其中各种HBV松弛圆形DNA底物都被细胞提取物和纯化的人类蛋白质修复形成cccDNA。通过酵母和人提取物筛选,我们确定了滞链合成的五个核心成分对于cccDNA的形成至关重要:增殖细胞核抗原,复制因子C复合体,DNA聚合酶δ,襟翼内切核酸酶1和DNA连接酶1。我们用纯化的人类同源物重建cccDNA的形成,将它们作为cccDNA形成的最小因素。我们进一步证明了用DNA聚合酶抑制剂Aphidicolin的治疗在生化测定和HBV感染的人类细胞中均减少了cccDNA的形成。总之,我们的发现定义了HBV cccDNA形成的关键成分。
更新日期:2020-03-09
中文翻译:
DNA落后链合成机器的核心组件对于乙型肝炎病毒cccDNA的形成至关重要。
慢性乙型肝炎病毒(HBV)感染每年导致887,000人死亡。治愈HBV的主要挑战是根除病毒基因组的稳定的共价闭合环状DNA(cccDNA)形式,这种形式是由病毒体传递给肝细胞的带损伤的HBV松弛环状DNA的修复形成的。cccDNA形成所涉及的宿主因子的完整和最少集合是未知的,主要是由于缺乏能够完全重构cccDNA形成的生化系统。在这里,我们开发了实验系统,其中各种HBV松弛圆形DNA底物都被细胞提取物和纯化的人类蛋白质修复形成cccDNA。通过酵母和人提取物筛选,我们确定了滞链合成的五个核心成分对于cccDNA的形成至关重要:增殖细胞核抗原,复制因子C复合体,DNA聚合酶δ,襟翼内切核酸酶1和DNA连接酶1。我们用纯化的人类同源物重建cccDNA的形成,将它们作为cccDNA形成的最小因素。我们进一步证明了用DNA聚合酶抑制剂Aphidicolin的治疗在生化测定和HBV感染的人类细胞中均减少了cccDNA的形成。总之,我们的发现定义了HBV cccDNA形成的关键成分。