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

Date Published: June 21, 2018

Publisher: Public Library of Science

Author(s): Kouichi Kitamura, Lusheng Que, Miyuki Shimadu, Miki Koura, Yuuki Ishihara, Kousho Wakae, Takashi Nakamura, Koichi Watashi, Takaji Wakita, Masamichi Muramatsu, Aleem Siddiqui.


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.

Partial Text

Hepatitis B virus (HBV) is a major pathogenic cause of human cirrhosis and hepatocellular carcinoma [1]. Infectious HBV particles contain relaxed circular DNA (rcDNA) encapsidated by core proteins [2]. After entering the host hepatocyte, rcDNA is converted into covalently closed circular DNA (cccDNA), which is stably maintained as an episome in the nucleus. cccDNA serves as the template for all HBV transcripts, including pregenomic RNA (pgRNA), a viral replicative intermediate [2–4]. pgRNA, viral reverse transcriptase P protein, and core proteins assemble into a nucleocapsid, where pgRNA undergoes reverse transcription by the P protein to produce rcDNA. The mature nucleocapsid is further assembled with surface proteins to allow secretion as an infectious virion. Alternatively, the rcDNA containing nucleocapsid is recycled back to the nucleus to maintain the pool of cccDNA [5].

Host DNA repair factors are expected to be involved in cccDNA conversion because the virus genome does not encode the responsible DNA modifiers [2, 3, 6, 27]. The TDP2 enzyme has been proposed to remove P protein [28], although another study reported that TDP2 is not required for cccDNA formation in vivo [29]. We previously showed that the host DNA repair enzyme UNG removes uracil residues from deaminated duck HBV (DHBV) cccDNA (or its precursor), thus changing its mutation frequency [30]. FEN1 plays a role in various DNA metabolic pathways, including Okazaki fragment maturation and LP-BER. During lagging strand DNA synthesis, Polδ/Polε use the strand exchange activity to produce the 5′-flap structure, and then FEN1 cleaves the 5′-flap and generates a ligatable end to facilitate lagging strand synthesis. In LP-BER, FEN1 removes the 5′-flap structure containing a damaged sugar and generates a ligatable 5′ end to facilitate its repair process [9, 10]. However, it remains unknown whether other back-up systems can substitute for absence of FEN1 activity.