The role of host DNA ligases in hepadnavirus covalently closed circular DNA formation.
The role of host DNA ligases in hepadnavirus covalently closed circular DNA formation.
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DOI：
10.1371/journal.ppat.1006784
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发表时间：
2017-12
期刊：
影响因子：
6.7
通讯作者：
Guo H
中科院分区：
文献类型：
--
作者：
Long Q;Yan R;Hu J;Cai D;Mitra B;Kim ES;Marchetti A;Zhang H;Wang S;Liu Y;Huang A;Guo H
嗜肝DNA病毒共价闭合环（ccc）DNA是病毒真正的转录模板，在病毒的感染和持久性中起着关键作用。在感染后，非复制性cccDNA从进入的和从头合成的病毒基因组松弛环状（rc）DNA转化，推测是通过在细胞核中利用宿主细胞的DNA修复机制。将rcDNA转化为cccDNA需要在rcDNA的切口/缺口区域制备末端用于链连接。在筛选了107个细胞DNA修复基因后，我们在本文中报告了细胞DNA连接酶（LIG）1和3在cccDNA形成中起关键作用。在体外cccDNA形成测定中，连接酶抑制剂或LIG 1/3的功能性敲除/敲除显著降低了cccDNA的产生，并且在cccDNA产生细胞中对病毒核心DNA复制没有直接影响。此外，在相应的敲除或敲低细胞中LIG 1/3的反式互补能够恢复cccDNA形成。此外，发现LIG 4（非同源末端连接DNA修复装置中的组分）负责从病毒双链线性（cDNA）DNA形成cccDNA，而不是rcDNA。总之，我们证明嗜肝DNA病毒利用整个宿主DNA连接酶形成cccDNA，这揭示了cccDNA生物合成的连贯分子途径，以及治疗B型肝炎的新型抗病毒策略的发展。嗜肝DNA病毒cccDNA是病毒基因组的持久形式，就人B肝炎病毒（HBV）而言，cccDNA是治疗停止后病毒反弹的基础，也是目前药物治疗难以治愈的基础。因此，阐明cccDNA形成的分子机制将有助于HBV基础和医学水平的研究。在这项研究中，我们筛选了总共107个细胞DNA修复基因，并确定DNA连接酶1和3是从病毒松弛（开放）环状DNA形成cccDNA的关键因素。此外，我们发现细胞DNA连接酶4负责将病毒双链线性DNA转化为cccDNA。我们的研究进一步证实了宿主DNA修复机制参与cccDNA的形成，并可能为未来治疗B肝炎揭示新的抗病毒靶点。
Hepadnavirus covalently closed circular (ccc) DNA is the bona fide viral transcription template, which plays a pivotal role in viral infection and persistence. Upon infection, the non-replicative cccDNA is converted from the incoming and de novo synthesized viral genomic relaxed circular (rc) DNA, presumably through employment of the host cell’s DNA repair mechanisms in the nucleus. The conversion of rcDNA into cccDNA requires preparation of the extremities at the nick/gap regions of rcDNA for strand ligation. After screening 107 cellular DNA repair genes, we herein report that the cellular DNA ligase (LIG) 1 and 3 play a critical role in cccDNA formation. Ligase inhibitors or functional knock down/out of LIG1/3 significantly reduced cccDNA production in an in vitro cccDNA formation assay, and in cccDNA-producing cells without direct effect on viral core DNA replication. In addition, transcomplementation of LIG1/3 in the corresponding knock-out or knock-down cells was able to restore cccDNA formation. Furthermore, LIG4, a component in non-homologous end joining DNA repair apparatus, was found to be responsible for cccDNA formation from the viral double stranded linear (dsl) DNA, but not rcDNA. In conclusion, we demonstrate that hepadnaviruses utilize the whole spectrum of host DNA ligases for cccDNA formation, which sheds light on a coherent molecular pathway of cccDNA biosynthesis, as well as the development of novel antiviral strategies for treatment of hepatitis B. Hepadnavirus cccDNA is the persistent form of viral genome, and in terms of human hepatitis B virus (HBV), cccDNA is the basis for viral rebound after the cessation of therapy, as well as the elusiveness of a cure with current medications. Therefore, the elucidation of molecular mechanism of cccDNA formation will aid HBV research at both basic and medical levels. In this study, we screened a total of 107 cellular DNA repair genes and identified DNA ligase 1 and 3 as key factors for cccDNA formation from viral relaxed (open) circular DNA. In addition, we found that the cellular DNA ligase 4 is responsible for converting viral double-stranded linear DNA into cccDNA. Our study further confirmed the involvement of host DNA repair machinery in cccDNA formation, and may reveal new antiviral targets for treatment of hepatitis B in future.
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DOI：
10.1007/978-1-62703-484-5_13
发表时间：
2013
期刊：
Methods in molecular biology (Clifton, N.J.)
影响因子：
--
作者：
Cai, Dawei;Nie, Hui;Yan, Ran;Guo, Ju-Tao;Block, Timothy M;Guo, Haitao
通讯作者：
Guo, Haitao
影响因子：
7.6
作者：
Cai, Dawei;Wang, Xiaohe;Yan, Ran;Mao, Richeng;Liu, Yuanjie;Ji, Changhua;Cuconati, Andrea;Guo, Haitao
通讯作者：
Guo, Haitao
影响因子：
7.6
作者：
Liu C;Cai D;Zhang L;Tang W;Yan R;Guo H;Chen X
通讯作者：
Chen X
DOI：
10.1073/pnas.1409986111
发表时间：
2014-10-07
影响因子：
11.1
作者：
Koeniger, Christian;Wingert, Ida;Nassal, Michael
通讯作者：
Nassal, Michael
CRISPR-CAS9介导的基因失活的高度活性SGRNA的合理设计。
影响因子：
46.9
作者：
Doench, John G.;Hartenian, Ella;Graham, Daniel B.;Tothova, Zuzana;Hegde, Mudra;Smith, Ian;Sullender, Meagan;Ebert, Benjamin L.;Xavier, Ramnik J.;Root, David E.
通讯作者：
Root, David E.