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Nebioglu, Firat

[thumbnail of FiratNebioglu-PhdThesis-Final.pdf] PDF, English
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An estimated 257 million people are chronically infected with hepatitis B virus (HBV), which is a major cause of mortality among viral hepatitis related deaths despite the availability of a safe and effective vaccine. Current antiviral therapies against HBV focus on the suppression of viral replication and although improving the quality of life and long-term survival of patients this therapy only rarely leads to functional cure. To comply with the goals of WHO (summarized in “Global Health Sector Strategy on viral hepatitis”), more potent HBV therapeutics are needed and thus, a better understanding of the basics of HBV biology is essential to explore novel antiviral strategies. Hepatocytes infected with HBV contain highly stable episomal viral genomes, called covalently closed circular DNA (cccDNA), acting as a template for the synthesis of all viral transcripts. In addition, genomic integration of replication-defective, partial HBV genomes is frequently observed early after infection, which is thought to contribute to the chronicity of hepatitis B by providing a stable reservoir for the production HBV antigens. The aim of my thesis was to delineate host cell factors contributing to the HBV replication cycle. To this end, I addressed two specific aspects: First, to identify host factors modulating the secretion of subviral particles (SVPs) that originate from sub-genomic HBV integrates; second, to determine host factors incorporated into HBV virions and subviral particles in the course of particle morphogenesis. With respect to the first aim, I conducted an RNAi-based mini-screen using a custom-made library. I found that the neddylation pathway components Nedd8 and Ube2m are required for hepatitis B surface antigen (HBsAg). Knock-down of these components or pharmacological inhibition with the small molecule neddylation inhibitor MLN4924 inhibited the secretion of HBsAg. Previously, MLN4924 was reported to re-instate an epigenetic cccDNA silencing complex called Smc5/6 (structural maintenance of chromosomes complex 5/6) by preventing its HBx dependent degradation. I found that in addition, neddylation also contributes to the secretion of HBsAg from integrated HBV genome sequences in an HBx-independent manner. To sum up, my siRNA screen identified a previously unknown role of the neddylation pathway on HBsAg secretion that is independent of cccDNA. In the second part of my thesis, I established a protocol combining affinity chromatography, size exclusion chromatography, isopycnic centrifugation, and HBsAg immune-precipitation to enrich HBV particles and SVPs derived from two independent cell lines. Obtained particles were analyzed by mass spectrometry to determine their proteome. In this way, we identified approximately 160 proteins associated with both particle fractions, 6 of them exclusively detected in HBV virions. In addition to the previously published HBV associated proteins, we could identify numerous novel host factors, laying the ground for mechanistic follow-up studies and the possible use of identified host factors as targets for novel antiviral therapy.

Document type: Dissertation
Supervisor: Bartenschlager, Prof. Dr. Ralf
Place of Publication: Heidelberg
Date of thesis defense: 2 December 2021
Date Deposited: 05 Jul 2022 06:36
Date: 2023
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
Controlled Keywords: Biologie, Infektionskrankheit, Virologie
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