The Hepatitis B virus (HBV) is a common infectious agent. The HBV protein product, X, has shown to be essential for viral replication, and it is strongly implicated in the development of hepatocellular carcinoma in chronically infected HBV patients, yet its role in these events is not well-understood. Our previous work has comprehensively defined the mechanism for how X transactivated RNA polymerase I and III promoters. We made the interesting discovery that X induces the promoters by activating the Ras signal transduction pathway which then increases the cellular levels of the central transcription factor, TATA-binding protein (TBP). Increases in cellular TBP augment RNA pol I and III transcription and differentially regulate RNA pol II promoters. Our research plan will clearly delineate how X, an oncogenic Ras, increase cellular TBP levels. The proposed aims will rigorously investigate each event in the TBP gene expression process that gives rise to the final TBP product. By examining the individual contribution of each process, and how it differs when either X or oncegenic Ras is expressed in cells, we will obtain a good quantitative picture of how this X- and oncogenic Ras-mediated increase in TBP occurs. We have promising new data indicating that a key step leading to the increase in TBP is due to an increase in TBP promoter activity. Therefore, a major focus of the proposed studies will be to examine how the TBP promoter is regulated by X and by oncogenic Ras. We will comprehensively define the X-mediated signaling events downstream of Ras to the promoter that modulate TBP promoter activity. Since X has been shown to transform cells, and Ras is strongly oncegenic, we will also determine how alterations in the cellular levels of TBP can affect the transformation potential of cells. Focus formation, growth in soft agar, and mouse tumorigenesis assays will be used to assess whether directly overexpressing TBP can enhance transforming activity or whether down-regulating its production in cells can prevent Ras-induced transformation. Using mutant TBP proteins that are specifically defective in RNA pol II or pol III transcription, we will define specific changes in cellular gene expression occurring in TBP-overexpressing cells that contribute to transformation. These studies promise to make unique and important new contributions to our understanding of the function of the HBV X protein and oncegenic Ras, the regulation of TBP, and their consequences on cellular gene activity that leads to cellular transformation.
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