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Identification of Host Factors Regulating Hepatitis B Virus Covalently Closed Circular DNA Transcription

Yu, Xiaoyang (2024) Identification of Host Factors Regulating Hepatitis B Virus Covalently Closed Circular DNA Transcription. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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Abstract

Hepatitis B virus (HBV) infection continues to pose a significant public health challenge, with approximately 300 million individuals chronically infected. HBV cccDNA is the persistent form of viral genome and the authentic viral transcription template, which exists in the host cell nucleus as an episomal minichromosome and is resistant to current antivirals. This research aims to identify host factors that regulate HBV cccDNA transcription.
Growing evidence shows that the transcriptional activity of the cccDNA minichromosome undergoes epigenetic regulations, suggesting a new perspective for anti-cccDNA drug development through targeting histone modifications. By screening an epigenetic compound library in the cccDNA reporter cell line HepBHAe82, a bromodomain-containing protein 4 (BRD4) inhibitor MS436 exhibited marked inhibition of cccDNA transcription with noncytotoxic concentrations. Chromatin immunoprecipitation (ChIP) assay demonstrated that MS436 dramatically reduced the enrichment of H3K27ac, an activating histone modification of histone H3, on cccDNA. Interestingly, the MS436-mediated inhibition of cccDNA transcription was accompanied by cccDNA destabilization in in vitro HBV infection systems, indicating that BRD4 activity may also play a role in cccDNA maintenance. Furthermore, depletion of BRD4 resulted in cccDNA level reduction, further validating BRD4 as an antiviral target. Taken together, our study has demonstrated the practicability of HepBHAe82-based anti-HBV drug screening system and provided a proof-of-concept for targeting HBV cccDNA with epigenetic compounds.
IFNα is currently the only FDA approved immunomodulatory drug for treatment of chronic HBV, and it possesses intracellular antiviral activity through the induction of IFN-stimulated genes (ISGs). IFI27 (also known as ISG12) expression is highly induced by IFNα in both HepG2 cells and primary human hepatocytes. Ectopic-expression of IFI27 exhibited significant antiviral effect in HBV-transfected or infected cell cultures by reducing HBV total RNA and core DNA. Furthermore, knocking down IFI27 markedly abrogated the antiviral effect of IFNα in HBV-infected HepG2-NTCP cells. Interestingly, IFI27 exhibits HBV promoter-specific transcriptional inhibition. Immunofluorescence microscopy revealed that IFI27 is predominantly localized in cytoplasm, indicating that IFI27 indirectly inhibits HBV transcription in the nucleus. Our results demonstrate that the anti-HBV mechanism of IFI27 is through downregulation of the level of a host transcription factor, CEBPα, via ubiquitination-mediated proteasome degradation, facilitated by the E3 ubiquitin ligase SKP2. Consequently, the binding of CEBPα to the HBV promoters is impeded, leading to the inhibition of cccDNA transcription. Overall, our study suggests that IFI27 mediates the antiviral activity of IFNα against HBV replication at the transcriptional level by downregulating cellular transcription factor(s) exploited by HBV.

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Details

Item Type: University of Pittsburgh ETD
Status: Unpublished
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Yu, Xiaoyangyux2@upmc.eduxiy103
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairGao, Shou-Jianggaos8@upmc.edu
Committee MemberGuo, Haitaoguoh4@upmc.edu
Committee MemberAmbrose, Zandreazaa4@pitt.edu
Committee MemberBility, Moses Tmtbility@pitt.edu
Committee MemberSarkar, Saumendra Nsaumen@pitt.edu
Date: 16 September 2024
Date Type: Publication
Defense Date: 27 July 2023
Approval Date: 16 September 2024
Submission Date: 8 November 2023
Access Restriction: 2 year -- Restrict access to University of Pittsburgh for a period of 2 years.
Number of Pages: 144
Institution: University of Pittsburgh
Schools and Programs: School of Medicine > Microbiology and Immunology
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: HBV, cccDNA, Epigenetics, BRD4, ISG, IFI27, C/EBPα
Date Deposited: 16 Sep 2024 18:47
Last Modified: 16 Sep 2024 18:47
URI: http://d-scholarship.pitt.edu/id/eprint/45502

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