Abstract
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.
Highlights
Hepatitis B virus (HBV) is a major pathogenic cause of human cirrhosis and hepatocellular carcinoma [1]
After entering the host hepatocyte, relaxed circular DNA (rcDNA) is converted into covalently closed circular DNA, which is stably maintained as an episome in the nucleus. cccDNA serves as the template for all HBV transcripts, including pregenomic RNA, a viral replicative intermediate [2,3,4]. 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
flap structure-specific endonuclease 1 (FEN1) is involved in HBV cccDNA formation of the r sequence was determined by fluorescence intensity (S1C Fig) and polyacrylamide gel electrophoresis (PAGE) (S1D Fig)
Summary
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. 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. Reverse-transcriptase inhibitors are the major medical intervention for controlling HBV infection. These inhibitors can effectively shut down viral replication, but are unable to eliminate cccDNA from infected hepatocytes; this inability often leads to viral rebound upon therapy withdrawal [2, 3, 6]. A lack of comprehensive knowledge on the molecular mechanisms of cccDNA formation and maintenance has hampered the effective development of such approaches
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