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Dissertation
Determining Secondary Structural Elements of Hepatitis B Virus Pregenomic RNA and Preliminary Analysis of the DDB1-HBx Interaction as a Therapeutic Target
Doctor of Philosophy (Ph.D.), Drexel University
Dec 2023
DOI:
https://doi.org/10.17918/00002003
Abstract
Approximately 296 million people worldwide have a chronic hepatitis B virus (CHBV) infection, the leading cause of hepatocellular carcinoma. Despite the availability of an effective vaccine, there are ~1.5 million new infections each year, and there were 820,000 deaths in 2019 due to Hepatitis B virus (HBV)-related liver diseases. Current CHBV therapies are not curative due to the persistence of an HBV replication intermediate, covalently closed circular DNA, an episome localized to the nucleus of infected cells that is the template for HBV RNA transcripts. One of these transcripts, pregenomic (pg) RNA, is translated to generate the viral core and reverse transcriptase/polymerase (RT/Pol) proteins. The pgRNA is also encapsidated and reversed transcribed to generate the HBV DNA genome. Aside from the well-characterized epsilon sequence ([epsilon]) critical for pgRNA encapsidation, little is known about the structure-function relationship of pgRNA and its role in HBV biology. We used selective 2' hydroxylation analyzed by primer extension-mutational profiling (SHAPE-MaP) and Dimethyl sulfate mutational profiling (DMS-MaP) to identify novel secondary structures in in vitro transcribed pgRNA, laying the foundation for studies that will assess the roles of these structures in the viral lifecycle and as targets to block HBV replication. As another method for blocking HBV replication, we developed a protocol for assessing the interaction of the HBV HBx protein with the host-cell protein DNA Damage Binding protein 1 (DDB1), which is required for HBV replication. Previous studies showed that nitazoxanide (NTZ), an antiparasitic, inhibits the interaction of HBx with DDB1 and blocks HBV replication. Using surface plasmon resonance (SPR), we showed that NTZ inhibits the binding of DDB1 to an HBx peptide corresponding to the region of HBx known to interact with and previously co-crystallized with DDB1. This study showed that we have developed a new tool to identify small molecules that may be novel treatments for CHBV.
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Details
- Title
- Determining Secondary Structural Elements of Hepatitis B Virus Pregenomic RNA and Preliminary Analysis of the DDB1-HBx Interaction as a Therapeutic Target
- Creators
- Andrea Rosenkranz
- Contributors
- Michael Bouchard (Advisor)
- Awarding Institution
- Drexel University
- Degree Awarded
- Doctor of Philosophy (Ph.D.)
- Publisher
- Drexel University; Philadelphia, Pennsylvania
- Number of pages
- xv, 317 pages
- Resource Type
- Dissertation
- Language
- English
- Academic Unit
- Biochemistry and Molecular Biology; College of Medicine; Drexel University
- Other Identifier
- 991021837215504721