UNIVERSITY OF ILLINOIS
This fellowship will support the study of the metabolic control of hepatitis B virus (HBV) biosynthesis and more specifically bile acid regulation of HBV both in cell culture and in vivo. The long-term objectives are to understand the in vivo mechanisms regulating HBV transcription and how alterations in viral RNA synthesis modulate HBV replication. It is proposed to determine the role of the nuclear hormone receptor farnesoid X receptor (FXR) in the modulation of HBV biosynthesis, and the importance of the corepressor, small heterodimer partner (SHP) in regulating FXR-mediated HBV transcription and replication under normal and altered physiological conditions. Previous experiments suggested that bile acids should inhibit HBV biosynthesis by activating SHP expression because SHP negatively regulates HBV biosynthesis in vitro. Subsequently, FXR was examined because FXR is the major bile acid receptor and FXR activates SHP. Retinoid X receptor (RXR) plus FXR was shown to support replication in nonhepatoma cells. Therefore, the mechanism of action of RXR/FXR will be characterized to determine its recognition sequence within the HBV genome and its relative responsiveness to various coactivators and corepressors, with a particular focus on the importance of SHP. This functional mapping of the RXR/FXR recognition element in the nucleocapsid promoter will be done by mutational analysis, and to analyze further the relative importance of the recognition elements for RXR/FXR binding, EMSA and DNaseI footprinting analysis utilizing nucleocapsid promoter sequences will be performed. Also chromatin immunoprecipitation will be done to determine which nuclear receptors bind to the HBV genome both in hepatoma cells and in HBV transgenic mice. In addition, the role of SHP in maintaining tight regulation of HBV transcription and replication will be examined by chracterization of SHP-null HBV transgenic mice for viral RNA and DNA synthesis under normal physiological conditions and in the presence of bile acids.