Files and links (1)
PDFOpen Access (License Unspecified), Open Access
Dissertation
Identification of host proteins required for hepatitis B virus covalently closed circular DNA biosynthesis by a chemogenetic approach
Doctor of Philosophy (Ph.D.), Drexel University
Apr 2019
DOI:
https://doi.org/10.17918/s5gd-qd64
Abstract
Chronic hepatitis B virus (HBV) infection relies on stable maintenance and proper functioning of a nuclear episomal form of viral genome called covalently closed circular (ccc) DNA, 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. Identification of key host factors required for cccDNA metabolism and function will reveal molecular targets for developing curative therapeutics of chronic HBV infection. In order to identify host cellular DNA metabolic enzymes that are involved in cccDNA biosynthesis, 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 followed by removal of PFA to allow the synchronized synthesis of relaxed circular (rc) DNA and subsequent conversion into cccDNA within 12 to 24 h. This cccDNA formation assay allows for systematic screening of small molecular inhibitors of DNA metabolic enzymes on cccDNA synthesis, but avoids cytotoxic effects upon long term treatment. Using this assay, we screened more than 140 compounds targeting DNA repair enzymes and host cellular proteins involved in chromatin assembly/remodeling and histone modifications and found several groups of compounds that modulate cccDNA synthesis. Particularly, we found that all the tested topoisomerase I and II 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 while 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, siRNA knockdown of topoisomerase II significantly reduced cccDNA amplification. Taken together, our study demonstrates that topoisomerase I and II may catalyze distinct steps of HBV cccDNA synthesis and pharmacologic targeting of these cellular enzymes may facilitate the cure of chronic hepatitis B.
Metrics
46 File views/ downloads
23 Record Views
Details
- Title
- Identification of host proteins required for hepatitis B virus covalently closed circular DNA biosynthesis by a chemogenetic approach
- Creators
- Muhammad Sheraz - DU
- Contributors
- Ju-Tao Guo (Advisor) - Drexel University (1970-)
- Awarding Institution
- Drexel University
- Degree Awarded
- Doctor of Philosophy (Ph.D.)
- Publisher
- Drexel University; Philadelphia, Pennsylvania
- Number of pages
- xiii, 145, 6 pages
- Resource Type
- Dissertation
- Language
- English
- Academic Unit
- Microbiology and Immunology; College of Medicine; Drexel University
- Other Identifier
- 9360; 991014632415104721