Our overall goal is to study hepatitis B virus (HBV) gene expression in animal cells and to determine its role in chronic liver disease and hepatocellular carcinoma. Studies will be divided into 3 general components 1) descriptive viral pathophysiology, 2) viral molecular biology, and 3) viral cell biology. These studies require a variety of materials and approaches, many of which have been made possible by recent availability of recombinant DNA molecules containing HBV sequences. Our use of labelled cloned HBV-DNA to identify and characterize HBV sequences in a human hepatocellular carcinoma line (PLC/PRF/5 and liver biopsy specimens from chimpanzee HBV carriers indicates the feasibility of this project. In PLC/PRF/5 cells, we observed integrated HBV-DNA and three RNAs with viral sequences by restriction enzyme and molecular hybridization analysis. Experiments will be performed to determine in what percentage of hepatomas in which HBV-sequences are present, are these sequences integrated into the host genome, and when does integration occur? We would also like to identify and characterize mRNAs coding for specific HBV proteins, map these mRNAs on the viral genome, and study the viral transcription mechanism. This will be accomplished by pulse labelling of HBV-RNAs in vivo and by studying in vitro transcription of cloned HBV-DNAs in a DNA-dependent HeLa cellular extract. The latter system will also be used to identfy the promoter site(s) for HBV transription. By using recombinant clones of PLC/PRF/5 HBV-DNA containing host flanking regions, we will characterize HBV and host sequences surrounding the integration sites in the PLC/PRF/5 cell line and in human hepatocellular carcinomas. RNA transcripts containing both host and viral sequences will also be studied in the PLC/PRF/5 cell line. Finally, cellular transformation experiments will be performed to determine whether HBV-DNA is oncogenic in tissue culture and, is so, what portion of the HBV gene confers oncogenicity. These experiments will involve direct transformation of eukaryotic cells by HBV-DNA, restriction fragments of HBV-DNA and cloned integrated HBV-DNA from the PLC/PRF/5 cell line. These studies will, therefore, use multiple approaches to study the influence of HBV-genes on the molecular properties of liver cells and the role of HBV in chronic liver disease and primary liver cancer.
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