Molecular Mechanisms of HBV cccDNA Formation

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ABSTRACT Hepatitis B virus (HBV) covalently closed circular (ccc) DNA plays a central role in the establishment of viral infection and persistence, and is the basis for viral rebound after the cessation of therapy, as well as the elusiveness of a cure even after extended treatment with current approved medications. HBV cccDNA is established upon initial infection through conversion of the partially double stranded relaxed circular (rc) DNA viral genome containing terminal peculiarities, through employment of the host cell’s DNA repair mechanisms in the nucleus. The cccDNA episome levels are maintained through a replication cycle that involves retrotranscription of a cccDNA transcript, termed pregenomic RNA, into progeny rcDNA genomes, some of which are returned to the nucleus for conversion into cccDNA. The conversion of rcDNA into cccDNA requires the removal of a covalently-linked copy of the polymerase from the 5’ end of one of the DNA strands, and this “deproteination” step generates a DNA intermediate, the deproteinated rcDNA (DP-rcDNA), as precursor for cccDNA formation. The rcDNA deproteination is a trigger signal for transportation of HBV nucleocapsid containing mature viral DNA into nucleus, where the rcDNA to cccDNA conversion takes place. We have recently mapped the termini of cytoplasmic DP-rcDNA, which demonstrated that the viral polymerase and RNA primer are completely removed from rcDNA during deproteination, the plus strand DNA is further elongated but the terminal redundant sequence is maintained on DP-rcDNA. In addition, recent studies by us and others have identified a handful of host DNA repair factors involved in cccDNA formation. However, there are many molecular details yet to be elucidated for a better understanding of cccDNA biosynthesis, and the establishment of immunocompetent small animal model for HBV infection is hampered by the inability of cccDNA formation in mouse hepatocyte. In this research application, by making use of a battery of molecular biology, biochemistry, proteomics and genomics technologies, we propose to further elucidate the molecular mechanisms underlying the biogenesis of DP-rcDNA (Aim 1) and cccDNA (Aim 2), and to define the host determinant(s) for the failure of cccDNA formation in mouse hepatocyte (Aim 3). Our ultimate goal is to illustrate a coherent picture of the molecular mechanisms/pathway for HBV cccDNA formation. The accomplishment of this project will reveal new potential antiviral targets for treatment of hepatitis B and aid the development of a mouse model fully susceptible to HBV infection.