Novel methods for recombinantly producing hepadnaviral relaxed circular and covalently closed circular DNA

Abstract

Worldwide, an estimated 296 million people are chronically infected with hepatitis B virus (HBV). Chronic hepatitis B (CHB) is a known cause of life-threatening liver diseases including cirrhosis, fibrosis, and hepatocellular carcinoma (HCC). Central to the establishment and maintenance of CHB is the formation of covalently closed circular (ccc) DNA within the nucleus of infected hepatocytes, which is notoriously difficult to eliminate. cccDNA is formed from the repair of a relaxed circular (rc) DNA molecule present within viral particles upon infection. This repair process requires a set of host factors that has yet to be completely defined, but could offer new targets for antiviral treatments that aim to eliminate cccDNA, potentially providing solutions to chronic infection. An efficient pathway for the generation of rcDNA and cccDNA in vitro is, therefore, desirable to allow for the study of rcDNA to cccDNA repair. Previously, methods have been defined for recombinantly producing HBV rcDNA and cccDNA, but we strived to define more efficient protocols for their generation. Thus, we developed novel techniques for the production of both rcDNA and cccDNA, outlined in this thesis. We used our rcDNA production method to generate five HBV rcDNA constructs with different length plus strands and showed that all species are competent for expressing viral proteins upon transfection into permissive cells. We used a previously defined technique for producing circular DNAs known as ligase assisted minicircle accumulation (LAMA) to create wild type and mutant cccDNA that led to robust protein expression when transfected in cell culture. Several other hepadnaviruses have been important tools in understanding HBV and may have differences in repair that can be investigated. For this purpose, we also generated four additional hepadnaviral cccDNAs. We were able to test some of these and found them, also, to be competent for protein expression. Altogether, these results highlight the promise our techniques hold for allowing the study of rcDNA, cccDNA, and the repair process of HBV and other hepadnaviruses to define remaining unknown roles of host factors and present new targets for CHB therapeutics.