Regulation of amplification of cccDNA in hepatitis B virus: Involvement of the viral envelope proteins

Hepatitis B virus (HBV) is a human pathogen and affects an estimated 2 billion people worldwide. Chronic infection with HBV can significantly increase an individual's risk of developing liver cancer. In order to establish an infection, HBV must synthesize and maintain a covalently closed circular DNA (cccDNA) form of its genome in the nucleus of host cells. The work presented in this dissertation advances our understanding of how cccDNA is synthesized and a mechanism HBV uses to regulate this process.;To investigate the synthesis of cccDNA, a cell culture system was developed, which has several features that facilitate study of this and other aspects of HBV replication. Cell lines were created from hepatoma cells Huh7 and HepG2 that express Epstein-Barr virus (EBV) nuclear antigen-1 and a TetR-KRAB fusion protein stably. When transfected with a plasmid that contains the origin of plasmid replication of EBV these cell lines express HBV to high levels. Synthesis of cccDNA was increased to levels readily detectable by Southern blotting in Huh7-derived cell lines. Additionally, these cell lines maintained the HBV expression plasmid upon selection and conditionally expressed HBV. Thus, these cell cultures will allow tractable genetic approaches for studying the synthesis of cccDNA and other aspects of HBV replication.;Using these cell cultures, the mechanism through which HBV regulates synthesis of cccDNA was investigated and a contribution of the L and M envelope proteins was demonstrated. Ablation of expression of the three viral envelope proteins, L, M and S, led to an increase in the level of cccDNA. Subsequent restoration of expression of the envelope proteins led to a decrease in the level of cccDNA, the magnitude of which correlated with the level of envelope protein. Complementation with the different combinations of envelope proteins suggested that L and M proteins are necessary, but not sufficient for regulation of synthesis of cccDNA.;A role of the viral envelope proteins in plus-strand DNA synthesis was also reported. It was found that elongation of the plus strand was inhibited by expression of the viral envelope proteins. A model in which envelopment arrests further DNA synthesis is proposed.