DNA聚合酶α对于乙型肝炎病毒共价闭合环状DNA的细胞内扩增

StephenW

DNA Polymerase alpha is essential for intracellular amplification of hepatitis B virus covalently closed circular DNA

    Liudi Tang,
    Muhammad Sheraz,
    Michael McGrane,
    Jinhong Chang,
    Ju-Tao Guo


PLOS x

    Published: April 26, 2019
    Https://doi.org/10.1371/journal.ppat.1007742

   
Abstract

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. The product of nucleocapsid associated relaxing 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 α) is essential for cccDNA intracellular amplification, a genome recycling pathway that maintains a stable cccDNA pool in infected hepatocytes. Specifically, inhibition of Pol α by small molecule inhibitors aphidicolin or CD437 as well as silencing of Pol α expression by siRNA led to Singer of cccDNA amplification in human hepatoma cells. CRISPR-Cas9 knock-in of a CD437-resistant mutation into Pol The singularly abolished the effect of CD437 on cccDNA formation, indicating that CD437 directly targets Pol α to disrupt cccDNA biosynthesis. Mechanistically, Pol α is recruited to HBV rcDNA and required for the generation of minus strand covalently closed circular rcDNA, suggesting that Pol α is 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 κ and Pol λ.
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., 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 α) as well as Pol δ and ɛ are The cc cDNA synthesis should be revealed to the 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.

StephenW

DNA聚合酶α对于乙型肝炎病毒共价闭合环状DNA的细胞内扩增至关重要

Liudi Tang，
穆罕默德·谢拉兹
Michael McGrane，
Jinhong Chang，
郭菊涛


PLOS x

发布时间：2019年4月26日
Https://doi.org/10.1371/journal.ppat.1007742


抽象

持续性乙型肝炎病毒（HBV）感染依赖于在感染的肝细胞的细胞核中建立和维持共价闭合环状（ccc）DNA，一种3.2kb的附加体，其作为病毒转录模板。核衣壳相关的松弛环状（rc）DNA的产物，涉及修复rcDNA两条链中的不连续性的细胞DNA聚合酶以及潜在的机制仍有待完全理解。采用化学遗传学方法，我们发现DNA聚合酶α（Polα）是cccDNA细胞内扩增所必需的，这是一种基因组循环途径，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aphidicolin或CD437抑制Polα以及通过siRNA沉默Polα表达导致Singer在人肝细胞瘤细胞中扩增cccDNA。 CRISPR-Cas9将CD437抗性突变敲入Pol中单独消除CD437对cccDNA形成的影响，表明CD437直接靶向Polα以破坏cccDNA生物合成。机械地，Polα被募集到HBV rcDNA并且需要产生负链共价闭合的环状rcDNA，表明Polα在cccDNA合成中修复负链DNA缺口。因此，我们的研究表明，与需要Polκ和Polλ的新生病毒感染相比，HBV劫持了不同的宿主DNA聚合酶，以支持细胞内扩增途径中cccDNA的生物合成。
作者摘要

CCC DNA是长期抗病毒治疗中最难治的HBV复制中间体，是治疗停止后病毒反弹的原因。了解cccDNA微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已经清楚地证明cccDNA生物合成依赖于宿主细胞DNA修复机制，将rcDNA转化为cccDNA的分子途径仍有待鉴定。在这里，我们报告DNA聚合酶α（Polα）以及Polδ和ɛ是cc cDNA合成应该揭示cccDNA生物合成。进一步了解cccDNA合成的机制应揭示开发治疗剂以根除cccDNA和治疗慢性乙型肝炎的分子靶点。

StephenW

https://journals.plos.org/plospa ... ournal.ppat.1007742