Identifying Host Factors Governing Hepatitis B Virus Covalently Closed Circular DNA Biogenesis

Abstract

Hepatitis B Virus (HBV), the major etiologic agent of acute and chronic hepatitis and end-stage liver disease, constitutes a major global health crisis, with over 296 million chronic carriers worldwide. HBV infection can be prevented through prophylactic vaccination, but currently, there is no cure for the disease due to the persistence of the highly stable covalently closed circular DNA (cccDNA). cccDNA serves as the template for the majority of HBV transcripts and its persistence is the root cause for HBV chronicity. By hijacking host DNA repair machinery, cccDNA is formed from lesion-bearing viral relaxed circular DNA (rcDNA), which enters the cell as part of the HBV virion. Our lab has previously identified 5 host factors sufficient for cccDNA biogenesis in vitro. However, in cellulo, it is conceivable that other host factors and redundant pathways may function in this process. In this thesis, we aimed to define all critical host factors essential for rcDNA to cccDNA conversion in cellulo. We first established a CRISPRi platform to downregulate host factors of interest and study its impact on HBV infection. We then validated this platform by silencing DNA Damage Binding Protein 1 (DDB1), a host factor necessary for robust HBV infection, and observed a robust decrease in HBV infection. We utilized this platform to study the impact of silencing 4 out of the 5 factors previously demonstrated to be sufficient for cccDNA biogenesis in vitro as well as several host cellular polymerases. Finally, we developed two novel reporters: one that monitors HBV infected cells by taking advantage of a key viral mechanism, and the other that monitors rcDNA repair by expression of a fluorescent reporter when repaired. Ultimately, the results from this thesis suggest the presence of redundant pathways and factors that function in rcDNA repair in cellulo, and the methodologies established will be utilized in future studies to define a comprehensive set of host factors key for rcDNA repair. These results thus will aid towards a better understanding of key steps in HBV infection and a curative treatment for HBV.