Cellular DNA Topoisomerases Are Required for the Synthesis of Hepatitis B Virus Covalently Closed Circular DNA

Abstract

In order to identify host cellular DNA metabolic enzymes that are involved in the biosynthesis of hepatitis B virus (HBV) covalently closed circular (ccc) DNA, we developed a cell-based assay supporting synchronized and rapid cccDNA synthesis from intracellular progeny nucleocapsid DNA. This was achieved by arresting HBV DNA replication in HepAD38 cells with phosphonoformic acid (PFA), a reversible HBV DNA polymerase inhibitor, at the stage of single-stranded DNA and was followed by removal of PFA to allow the synchronized synthesis of relaxed circular DNA (rcDNA) and subsequent conversion into cccDNA within 12 to 24 h. This cccDNA formation assay allows systematic screening of the effects of small molecular inhibitors of DNA metabolic enzymes on cccDNA synthesis but avoids cytotoxic effects upon long-term treatment. Using this assay, we found that all the tested topoisomerase I and II (TOP1 and TOP2, respectively) poisons as well as topoisomerase II DNA binding and ATPase inhibitors significantly reduced the levels of cccDNA. It was further demonstrated that these inhibitors also disrupted cccDNA synthesis during de novo HBV infection of HepG2 cells expressing sodium taurocholate cotransporting polypeptide (NTCP). Mechanistic analyses indicate that whereas TOP1 inhibitor treatment prevented the production of covalently closed negative-strand rcDNA, TOP2 inhibitors reduced the production of this cccDNA synthesis intermediate to a lesser extent. Moreover, small interfering RNA (siRNA) knockdown of topoisomerase II significantly reduced cccDNA amplification. Taking these observations together, our study demonstrates that topoisomerase I and II may catalyze distinct steps of HBV cccDNA synthesis and that pharmacologic targeting of these cellular enzymes may facilitate the cure of chronic hepatitis B. IMPORTANCE Persistent HBV infection relies on stable maintenance and proper functioning of a nuclear episomal form of the viral genome called cccDNA, the most stable HBV replication intermediate. One of the major reasons for the failure of currently available antiviral therapeutics to cure chronic HBV infection is their inability to eradicate or inactivate cccDNA. We report here a chemical genetics approach to identify host cellular factors essential for the biosynthesis and maintenance of cccDNA and reveal that cellular DNA topoisomerases are required for both de novo synthesis and intracellular amplification of cccDNA. This approach is suitable for systematic screening of compounds targeting cellular DNA metabolic enzymes and chromatin remodelers for their ability to disrupt cccDNA biosynthesis and function. Identification of key host factors required for cccDNA metabolism and function will reveal molecular targets for developing curative therapeutics of chronic HBV infection.

Effect of static magnetic field on DNA synthesis: The interplay between DNA chirality and magnetic field left‐right asymmetry

“…Since Topoisomerases could alter the supercoiling of double‐stranded DNA, we chose topotecan, a topoisomerase I inhibitor that can interfere with supercoil relaxation, in combination with SMFs. Two colon cancer cell lines HCT116 and LoVo were plated first day and treated with corresponding concentrations of topotecan or DMSO as control for another 2 days before they were harvested for analysis.…”

“…It should be noted that different types of cells exhibit different responses to SMF because this response depends on many factors, such as cell type, age, differentiation state, cell rigidity, cell polarity, and other external factors influencing the cell machinery. 39 Since Topoisomerases could alter the supercoiling of double-stranded DNA, we chose topotecan, a topoisomerase I inhibitor 40,41 that can interfere with supercoil relaxation, in combination with SMFs. Two colon cancer cell lines HCT116 and LoVo were plated first day and treated with corresponding concentrations of topotecan or DMSO as control for another 2 days before they were harvested for analysis.…”

“…Topotecan Hydrochloride (HY‐13768A) was purchased from MedChem Express, and stock solution was made by dissolving topotecan in DMSO at 20 mmol/L. 4 × 10 5 /mL cells were plated on the 96 plate first day, and 16 hours later the cells were treated with topotecan at a specific concentration or DMSO as control, then exposed to 1 T SMF or not for another 2 days before they were harvested for cell counting. At the end of experiment, the cells were trypsinized by 100 μL trypsin and terminated by adding 100 μL medium, then analyzed with flow cytometry (CytoFLEX, Beckman Coulter).…”

Involvement of Host ATR-CHK1 Pathway in Hepatitis B Virus Covalently Closed Circular DNA Formation

“…1) from the 5= end of the minus strand. Topo I and II have also very recently been reported to play a role in HBV CCC DNA formation (28).…”

“…This appears to be the best candidate to date for an authentic intermediate in CCC DNA formation, which would further suggest that minus strand closing occurs before the plus strand during CCC DNA formation. Interestingly, Topo I was reported to be more important than Topo II for this early step of cM-RC DNA formation (28).…”