Flap endonuclease 1 is involved in cccDNA formation in the hepatitis B virus.
Flap endonuclease 1 is involved in cccDNA formation in the hepatitis B virus.
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DOI：
10.1371/journal.ppat.1007124
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发表时间：
2018-06
期刊：
影响因子：
6.7
通讯作者：
Muramatsu M
中科院分区：
文献类型：
--
作者：
Kitamura K;Que L;Shimadu M;Koura M;Ishihara Y;Wakae K;Nakamura T;Watashi K;Wakita T;Muramatsu M
乙型肝炎病毒（HBV）是肝硬化和肝细胞癌的主要病原体之一。慢性乙型肝炎病毒感染是这些严重肝脏疾病的关键因素。在感染过程中，HBV 以共价闭合环状 DNA (cccDNA) 的形式形成核病毒附加体。目前的疗法无法有效地消除受感染肝细胞中的 cccDNA。 cccDNA 是病毒复制的主模板，由其前体松弛环状 DNA (rcDNA) 转化而成。然而，对于 cccDNA 形成至关重要的宿主因素仍有待确定。在这里，我们评估了一种潜在的宿主因子，瓣结构特异性核酸内切酶 1 (FEN1)，是否参与 rcDNA 中瓣状结构的切割。在细胞培养 HBV 模型 (Hep38.7-Tet) 中，siRNA、shRNA、CRISPR/Cas9 介导的基因组编辑和 FEN1 抑制剂降低了 FEN1 的表达和活性。 FEN1 表达和活性的降低并不影响核衣壳 DNA (NC-DNA) 的产生，但确实降低了 Hep38.7-Tet 细胞中的 cccDNA 水平。野生型 FEN1 的外源过表达挽救了 FEN1 耗尽的 Hep38.7-Tet 细胞中 cccDNA 产量的减少。抗 FEN1 免疫沉淀揭示了 FEN1 与 HBV DNA 的结合。体外 FEN 活性测定表明 5'-flap 从合成的 HBV DNA 底物上裂解。此外，当纯化的 rcDNA 与重组 FEN1、DNA 聚合酶和 DNA 连接酶一起孵育时，在体外生成 cccDNA。重要的是，体外 cccDNA 形成测定需要 FEN1。这些结果表明FEN1参与细胞培养系统中HBV cccDNA的形成，并且FEN1、DNA聚合酶和连接酶活性足以在体外将rcDNA转化为cccDNA。乙型肝炎病毒 (HBV) 感染仍然是影响超过 3.5 亿人的全球健康问题。 HBV是肝硬化和肝细胞癌的主要病原体之一。 HBV 共价闭合环状 DNA (cccDNA) 是持续感染的关键病毒中间体。然而，cccDNA形成的分子机制尚未阐明。在这里，我们发现宿主因子flap-核酸内切酶1 (FEN1) 在cccDNA 形成中至关重要。我们通过将纯化的病毒 DNA 与重组 FEN1、DNA 聚合酶和 DNA 连接酶一起孵育，开发了一种新型 cccDNA 形成测定。这项研究为 cccDNA 形成的分子机制提供了新的见解，并提出 FEN1 作为潜在的抗 HBV 药物靶点。
Hepatitis B virus (HBV) is one of the major etiological pathogens for liver cirrhosis and hepatocellular carcinoma. Chronic HBV infection is a key factor in these severe liver diseases. During infection, HBV forms a nuclear viral episome in the form of covalently closed circular DNA (cccDNA). Current therapies are not able to efficiently eliminate cccDNA from infected hepatocytes. cccDNA is a master template for viral replication that is formed by the conversion of its precursor, relaxed circular DNA (rcDNA). However, the host factors critical for cccDNA formation remain to be determined. Here, we assessed whether one potential host factor, flap structure-specific endonuclease 1 (FEN1), is involved in cleavage of the flap-like structure in rcDNA. In a cell culture HBV model (Hep38.7-Tet), expression and activity of FEN1 were reduced by siRNA, shRNA, CRISPR/Cas9-mediated genome editing, and a FEN1 inhibitor. These reductions in FEN1 expression and activity did not affect nucleocapsid DNA (NC-DNA) production, but did reduce cccDNA levels in Hep38.7-Tet cells. Exogenous overexpression of wild-type FEN1 rescued the reduced cccDNA production in FEN1-depleted Hep38.7-Tet cells. Anti-FEN1 immunoprecipitation revealed the binding of FEN1 to HBV DNA. An in vitro FEN activity assay demonstrated cleavage of 5′-flap from a synthesized HBV DNA substrate. Furthermore, cccDNA was generated in vitro when purified rcDNA was incubated with recombinant FEN1, DNA polymerase, and DNA ligase. Importantly, FEN1 was required for the in vitro cccDNA formation assay. These results demonstrate that FEN1 is involved in HBV cccDNA formation in cell culture system, and that FEN1, DNA polymerase, and ligase activities are sufficient to convert rcDNA into cccDNA in vitro. Hepatitis B virus (HBV) infection remains a worldwide health problem that affects more than 350 million people. HBV is one of the major etiological pathogens for liver cirrhosis and hepatocellular carcinoma. HBV covalently closed circular DNA (cccDNA) is a key viral intermediate for persistent infection. However, the molecular mechanism of cccDNA formation has not been clarified. Here, we found that the host factor flap-endonuclease 1 (FEN1) is pivotal in cccDNA formation. We developed a novel cccDNA formation assay by the incubation of purified viral DNA with recombinant FEN1, DNA polymerase, and DNA ligase. This study provides new insights into the molecular mechanisms of cccDNA formation and proposes FEN1 as a potential anti-HBV drug target.
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影响因子：
3.7
作者：
Seeger, Christoph;Mason, William S.
通讯作者：
Mason, William S.
影响因子：
6.7
作者：
Liang G;Liu G;Kitamura K;Wang Z;Chowdhury S;Monjurul AM;Wakae K;Koura M;Shimadu M;Kinoshita K;Muramatsu M
通讯作者：
Muramatsu M
影响因子：
4.9
作者：
Ladner, SK;Otto, MJ;King, RW
通讯作者：
King, RW
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
影响因子：
9.2
作者：
Saharia, Abhishek;Guittat, Lionel;Stewart, Sheila A.
通讯作者：
Stewart, Sheila A.