Hepadnaviruses can cause chronic, productive, infections, associated with both progressive liver damage and the accumulation of aberrant, integrated forms of viral DNA. Both events appear to be co-factors in the development of hepatocellular carcinoma, which occurs with high frequency in chronically-infected individuals. The productive phase of hepadnavirus infections is apparently maintained solely by the transcriptional activity of a covalently-closed, circular (CCC) viral DNA found at low copy number in the nucleus of infected hepatocytes. Effective antiviral chemotherapies to eliminate this species, by ending virus production, should not only stop further liver damage but, also, substantially reduce the life-time risk of liver cancer. The possibility of devising such virus-specific therapies is encouraged by a recent report that, in duck hepatocyte tissue culture, hepadnavirus CCC DNA is synthesized via a virus-specified mechanism, involving reverse transcription of a viral RNA, and not by host-specific, semi-conservation replication. A detailed understanding of this process, including its role and significance in in vivo virus replication will, therefore, be a major goal of our investigations. The process of formation and of maintenance of CCC viral DNA in chronic, hepadnavirus infections will be studied, primary, in the context of both in vivo and in vitro infections by duck hepatits virus. Questions to be addressed will include: (1) What are the roles of viral and cellular proteins in the conversion of virion, relaxed circular, partially double- stranded DNA to CCC DNA? (2) How is CCC DNA maintained during chronic, hepatocellular infections? (3) What is the detailed molecular mechanism of viral DNA synthesis during chronic infections? Towards these goals, we have identified major events in hepadnaviral DNA synthesis and developed the first detailed model for this process, obtained preliminary evidence that CCC DNA can accumulate, in vitro, via an intracellular route, identified a number of drugs that appear to be useful for characterization of the hepadnavirus life cycle, and established a system for in vitro infection of primary woodchuck hepatocytes with woodchuck hepatitis virus.
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