PROJECT SUMMARY Hepatitis B virus chronically infects approximately 300 million people worldwide. All are at significantly increased risk of developing hepatocellular carcinoma. Although virus replication can be blocked by therapy with nucleoside analogs, infected hepatocytes are not cured, because covalently closed circular DNA (cccDNA), the template for transcription of viral RNAs persists in infected cells. So far, therapeutic strategies to prevent cccDNA formation or functionally inactivate or even destroy cccDNA within infected hepatocytes are lacking. The purpose of this application is to investigate four problems pertinent to cccDNA biology: identification of the enzymes required for cccDNA formation, elucidation of the exact mechanism for cccDNA formation, investigating how cccDNA is distributed to daughter cells during regeneration of hepatocytes and determining the physical organization of cccDNA in infected hepatocytes. Research on the mechanism of cccDNA formation could reveal the identity of cellular DNA repair enzymes that in turn, could be become targets for novel antiviral therapies. Investigations on the physical location of cccDNA in nuclei of infected cells could shed light on how cccDNA is maintained and distributed to daughter cells following cell division, and how it is eventually lost during spontaneous recovery from natural HBV infections. A better understanding of these processes is deemed essential for the development of novel strategies to cure chronic hepatitis B (CHB). Aim 1. Mechanism for cccDNA synthesis. We will identify still elusive cellular enzymes responsible for the conversion of rc to cccDNA. Candidates are DNA ligase(s) and endonuclease(s), required for the processing and joining of the 5’ and 3’ ends of the two rcDNA strands. Gene knockout cells will be used to determine how rcDNA is converted into cccDNA. In addition, we propose the development of a novel screening assay for identification of host genes required for HBV infection and cccDNA formation. Aim 2. cccDNA fate during cell division and nuclear localization. This aim builds on our ability to visualize cccDNA in metaphase and interphase nuclei by fluorescence in situ hybridization (FISH). The goal is to determine the distribution of cccDNA in DHBV and HBV producing cells and investigate how cccDNA is distributed to daughter cells under normal conditions and in the presence of cytokines. Experiments are designed to investigate the nuclear localization of functional and transcriptionally silenced cccDNA. To address these aims, we will rely on an experimental approach that builds on our research efforts during the past two years leading to cell culture platforms permitting HBV infections of HepG2 cells in combination with CRISPR/Cas9 gene knockout technology. In addition, the aims build on our recent success in developing methods permitting detection of cccDNA by FISH.