Flap endonuclease 1 is involved in cccDNA formation in the hepatitis B virus

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.乙型肝炎病毒（Hepatitis B virus, HBV）是引发肝硬化与肝细胞癌的主要病原之一。慢性HBV感染是上述重症肝病的关键致病因素。感染过程中，HBV会以共价闭合环状DNA（covalently closed circular DNA, cccDNA）的形式在宿主细胞核内形成病毒游离基因。现有治疗手段无法高效清除受感染肝细胞内的cccDNA。cccDNA是病毒复制的核心模板，其由前体松弛环状DNA（relaxed circular DNA, rcDNA）转化生成。然而，对于cccDNA形成至关重要的宿主因子仍有待明确。本研究评估了一种潜在宿主因子——瓣状核酸内切酶1（flap structure-specific endonuclease 1, FEN1）是否参与rcDNA的瓣状结构切割。我们采用细胞培养HBV模型（Hep38.7-Tet细胞系），通过小干扰RNA（small interfering RNA, siRNA）、短发夹RNA（short hairpin RNA, shRNA）、CRISPR/Cas9介导的基因组编辑技术以及FEN1抑制剂，下调FEN1的表达与活性。FEN1表达与活性的下调并未影响核衣壳DNA（nucleocapsid DNA, NC-DNA）的生成，但可降低Hep38.7-Tet细胞内的cccDNA水平。将野生型FEN1进行外源性过表达，可挽救FEN1敲低的Hep38.7-Tet细胞中cccDNA生成减少的表型。抗FEN1免疫沉淀实验证实，FEN1可与HBV DNA结合。体外FEN活性实验表明，该酶可切割人工合成的HBV DNA底物的5’-瓣状结构。进一步实验发现，将纯化的rcDNA与重组FEN1、DNA聚合酶及DNA连接酶共同孵育，可在体外生成cccDNA；且该体外cccDNA生成实验严格依赖FEN1的存在。上述结果证实，在细胞培养体系中FEN1参与HBV cccDNA的形成，且体外实验证明FEN1、DNA聚合酶与DNA连接酶的活性足以将rcDNA转化为cccDNA。