DNA Polymerase alpha is essential for intracellular amplification of hepatitis B virus covalently closed circular DNA
DNA Polymerase alpha is essential for intracellular amplification of hepatitis B virus covalently closed circular DNA
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DOI：
10.1371/journal.ppat.1007742
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发表时间：
2019-04-01
期刊：
影响因子：
6.7
通讯作者：
Guo, Ju-Tao
中科院分区：
文献类型：
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作者：
Tang, Liudi;Sheraz, Muhammad;Guo, Ju-Tao
持续性B型肝炎病毒（HBV）感染依赖于共价闭合环状（ccc）DNA的建立和维持，所述共价闭合环状（ccc）DNA是作为病毒转录模板的3.2kb附加体，其在感染的肝细胞的细胞核中。虽然有证据表明cccDNA是核衣壳相关松弛环状（rc）DNA的修复产物，但参与修复rcDNA双链不连续性的细胞DNA聚合酶以及潜在机制仍有待充分理解。采用化学遗传学方法，我们发现DNA聚合酶α（Pol）对于cccDNA细胞内扩增是必不可少的，这是一种基因组再循环途径，可在感染的肝细胞中维持稳定的cccDNA库。具体地，通过小分子抑制剂阿非迪霉素或CD 437抑制Pol以及通过siRNA沉默Pol表达导致人肝癌细胞中cccDNA扩增的抑制。将CD 437抗性突变的CRISPR-Cas9敲入Pol基因完全消除了CD 437对cccDNA形成的影响，表明CD 437直接靶向Pol以破坏cccDNA生物合成。从机制上讲，Pol被募集到HBV rcDNA中，并且是产生负链共价闭合环状rcDNA所必需的，这表明Pol参与cccDNA合成中负链DNA切口的修复。因此，我们的研究表明，不同的宿主DNA聚合酶被HBV劫持，以支持从细胞内扩增途径合成cccDNA，而从头病毒感染，这需要Pol和Pol。作者总结CCC DNA是长期抗病毒治疗下最难治的HBV复制中间体，是治疗停止后病毒反弹的原因。因此，了解cccDNA微型染色体的生物合成和维持对于开发治疗慢性HBV感染的新型抗病毒疗法至关重要。虽然已经清楚地证明cccDNA生物合成依赖于宿主细胞DNA修复机制，但将rcDNA转化为cccDNA的分子途径仍有待鉴定。在这里，我们报告了DNA聚合酶α（Pol）以及Pol和通过细胞内cccDNA扩增将rcDNA转化为cccDNA所需的。这一发现为cccDNA生物合成提供了新的分子见解。进一步了解cccDNA合成的机制应该揭示开发治疗剂以根除cccDNA和治愈慢性肝炎B的分子靶点。
Persistent hepatitis B virus (HBV) infection relies on the establishment and maintenance of covalently closed circular (ccc) DNA, a 3.2 kb episome that serves as a viral transcription template, in the nucleus of an infected hepatocyte. Although evidence suggests that cccDNA is the repair product of nucleocapsid associated relaxed circular (rc) DNA, the cellular DNA polymerases involving in repairing the discontinuity in both strands of rcDNA as well as the underlying mechanism remain to be fully understood. Taking a chemical genetics approach, we found that DNA polymerase alpha (Pol ) is essential for cccDNA intracellular amplification, a genome recycling pathway that maintains a stable cccDNA pool in infected hepatocytes. Specifically, inhibition of Pol by small molecule inhibitors aphidicolin or CD437 as well as silencing of Pol expression by siRNA led to suppression of cccDNA amplification in human hepatoma cells. CRISPR-Cas9 knock-in of a CD437-resistant mutation into Pol genes completely abolished the effect of CD437 on cccDNA formation, indicating that CD437 directly targets Pol to disrupt cccDNA biosynthesis. Mechanistically, Pol is recruited to HBV rcDNA and required for the generation of minus strand covalently closed circular rcDNA, suggesting that Pol is involved in the repair of the minus strand DNA nick in cccDNA synthesis. Our study thus reveals that the distinct host DNA polymerases are hijacked by HBV to support the biosynthesis of cccDNA from intracellular amplification pathway compared to that from de novo viral infection, which requires Pol and Pol .Author summary CCC DNA is the most refractory HBV replication intermediate under long-term antiviral therapies and is responsible for the viral rebound after treatment cessation. Therefore, understanding the biosynthesis and maintenance of cccDNA minichromosome is crucial for the development of novel antiviral therapeutics to cure chronic HBV infection. Although it has been clearly demonstrated that cccDNA biosynthesis relies on host cellular DNA repair machinery, the molecular pathways that convert rcDNA into cccDNA remain to be identified. Here we report that DNA polymerase alpha (Pol ) as well as Pol and are required for converting rcDNA into cccDNA through intracellular cccDNA amplification. This finding adds novel molecular insights on cccDNA biosynthesis. Further understanding the mechanism of cccDNA synthesis should reveal molecular targets for developing therapeutic agents to eradicate cccDNA and cure chronic hepatitis B.