STUDIES ON THE REPLICATION AND ONCOGENICITY OF HBV

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There are an estimated 176 million chronic carriers of hepatitis B virus (HBV) world wide and strong epidiomological evidence linking the carrier state with development of hepatocellular carcinoma. Thus, it is of utmost importance to have in vitro systems available for studying viral replication and for elucidating the mechanism(s) by which HBV infected cells become malignantly transformed. Although it has been possible to obtain partial expression of viral functions by transfecting rodent cells with cloned HBV DNA, these cells do not support efficient viral replication nor do they become malignantly transformed. Nevertheless, cloned HBV DNA is capable of causing hepatitis in chimpanzees. The working hypothesis for the experiments proposed here is that the relatively weak promoters associated with HBV genes other than the gene coding for the surface antigen of the virus cannot overcome host and/or tissue specific restrictions imposed by rodent cells or cells of non-hepatic origin. Thus, to meet our long-term goal of establishing in vitro systems for studying HBV, we propose 1) to obtain increased expression of viral genes in mouse lines that already carry integrated HBV genes by a) treating them with compounds known to enhance expression of integrated viral genomes; b) exposing them to tumor promoters and subeffective doses of physical and chemical carcinogens and c) by "superinfecting" them with additional recircularized HBV genomes or with new hybrid plasmids in which the viral genome is linked to strong promoters or enhancing sequences; 2) to develop hybrid plasmids designed to replicate episomally in simian cells or to give efficient expression of viral functions in cultured hepatocytes; 3) to create transgenic mice carrying HBV sequences by introducing recircularized HBV genomes or hybrid plasmids with HBV sequences linked to strong promoters into mouse embryo cells; 4) to develop methods of in situ hybridization suitable for use with biopsy material which will allow association of HBV gene expression with various clinical stages of chronic liver disease. These studies should elucidate at least some of the factors that regulate expression of HBV genes in cultured cells, in transgenic mice and in the liver of infected individuals.