The public health problem caused by hepatitis B virus (HBV) is of worldwide importance since the chronic carrier state in Far East Asia and tropical Africa represents 10% or more of the population and chronic active hepatitis and liver cirrhosis are major causes of mortality. HBV infection can generally be prevented by vaccination. However, reliable treatments for the approximately 200 million HBV chronic carriers worldwide are currently unavailable. The elimination of replicating virus from chronic carriers might be achieved by inhibiting viral transcription, an essential step in the replication cycle of the virus. Therefore, a detailed understanding of the mechanisms regulating the expression of the HBV transcription units may suggest rational approaches to disrupting this step in the viral life cycle. The nucleotide sequence elements in the 3.2kb HBV genome involved in regulating the expression of the four viral transcription units will be examined in a variety of liver and non-liver cell lines using transient transfection assays and in vitro transcription systems. A series of promoter deletions will be constructed for the major surface antigen, large surface antigen, nucleocapsid antigen and X genes and examined for transcriptional activity by measuring RNA levels and/or reporter gene activities directed by these regulatory sequence elements. A detailed examination of the transcriptional regulatory elements of these genes will be performed by characterizing the effects of clustered and single point mutations on the activities of the HBV promoters. The factors present in various cell extracts that bind to the HBV regulatory sequences will be examined using gel retardation and Dnasel footprinting assays. The results of the transcriptional analysis and the DNA binding assays will be compared to determine any correlations between these two activities. This analysis may indicate the presence of regulatory sequence elements which interact with hepatocyte specific transcription factors and therefore may contribute to HBV liver tropism. The sequence elements necessary for transcriptional modulation of the HBV promoter activities by glucocorticoids and transforming growth factor p will also be examined. Defining the HBV promoter regulatory sequence elements and detection of sequence specific DNA binding factors represents an initial step towards the characterization and cloning of the genes encoding these transcription factors. The cloning of the genes for these factors will permit the analysis of the mechanism by which these factors interact with their recognition sequences and influence the transcriptional activity of the HBV promoters.
