The long-term objective is to determine how persistent WHV infection of the liver leads to hepatocellular carcinoma (HCC) and study factors regulating persistent infections in woodchucks and in hepatocyte cultures.
The specific aims will be: (1) To determine the function of nuclear WHV DNA by isolating nucleoprotein (NP) complexes containing MHV DNA from the nuclei of chronically infected hepatocytes and: (a) Determine the structure of the complexes and the molecular forms of viral DNA in them. (b) Determine if replicative intermediates can be identified in the DNA from NP complexes, paying special attention to complexes containing CCC forms of the virus. If such molecules are identified, determine their origin and mechanism of replication. (c) Determine the endogenous DNA and RNA polymerase activities of the NP complexes and identify the transcriptional templete. (2) Study the mechanism of integration of WHV DNA by cloning integrations and homologous cell sequences and sequencing the viral-cell junctions and search for """"""""novel"""""""" forms of WHV using molecular cloning. (3) Attempt to establish persistent infections in woodchucks by perturbing the liver during acute infection in ways that will stimulate regeneration and/or suppress the immune response. (4) Utilize bio-matrix system to establish tissue cultures of chronically infected woodchuck hepatocytes. WHV is the only animal model which mimics the human disease with regard to the progression from chronic active hepatitis to HCC. The presence of viral integrations in human and woodchuck tumors has implicated their role in HCC. An understanding of how free WHV DNA is maintained in the nucleus, how it integrates in cellular DNA and how experimental treatments affect it may enable treatments for the prevention or cure of chronic infections to be devised. The methods used will include (1) isolation of NP complexes containing WHV DNA from hepatocyte nuclei by testing and modifying the selective nuclear extraction method for isolating SV40, polyoma and adenovirus NP complexes. Molecular cloning of viral integrations will be carried out by constructing libraries of recombinant DNA in lambda phage vectors and screening libraries with cloned WHV probe. Experimentally infected animals will be treated by partial hepatectomy and immunosuppresants in attempts to establish chronic infections.
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