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Dissertation
Role and mechanism of host cellular DNA repair proteins in hepatitis B virus cccDNA biosynthesis
Doctor of Philosophy (Ph.D.), Drexel University
Apr 2019
DOI:
https://doi.org/10.17918/pkxt-se38
Abstract
Persistent hepatitis B virus (HBV) infection relies on the establishment and maintenance of covalently closed circular (ccc) DNA, a 3.2-kb episome that serves as a viral transcription template, in the nucleus of an infected hepatocyte. Although evidence suggests that cccDNA is converted from its precursor molecule, relaxed circular (rc) DNA, by exploitation of host cellular DNA repair machineries, the host DNA polymerases involved in repairing the discontinuity in both strands of rcDNA remain to be fully understood. Taking a chemical genetics approach, DNA polymerase alpha (Pol [alpha]) was identified as a novel host factor essential for cccDNA biosynthesis from progeny rcDNA. Specifically, inhibition of Pol [alpha] by the small molecule inhibitors aphidicolin or CD437, as well as silencing of Pol [alpha] expression by small interfering (si)RNA, led to suppression of cccDNA formation in human hepatoma cells. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 knock-in of a CD437-resistant mutation into Pol [alpha] genes completely abolished the effect of CD437 on cccDNA formation, indicating that CD437 directly targets Pol [alpha] to disrupt cccDNA biosynthesis. Mechanistically, Pol [alpha] is recruited to HBV rcDNA and involved in the repair of the rcDNA minus-strand nick during cccDNA synthesis. Our study also identified a small molecule compound that enhances the conversion of rcDNA into cccDNA. The enhanced synthesis of cccDNA molecules requires the induction of cellular responses to assemble more productive DNA repair complexes onto rcDNA. Further investigation of the mechanism and molecular regulation of cccDNA biosynthesis should reveal molecular targets for developing therapeutic agents to eradicate cccDNA and cure chronic hepatitis B.
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Details
- Title
- Role and mechanism of host cellular DNA repair proteins in hepatitis B virus cccDNA biosynthesis
- Creators
- Liudi Tang - 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
- xvi, 153 pages
- Resource Type
- Dissertation
- Language
- English
- Academic Unit
- Microbiology and Immunology; College of Medicine; Drexel University
- Other Identifier
- 9358; 991014632334504721