The hepatitis B virus (HBV) is an infectious agent affecting over 300 million people world-wide. Chronic infection can lead to liver necrosis and hepatocellular carcinoma. One of the HBV gene products, the X protein, transactivates a large number of cellular and viral genes, yet the mechanism by which this occurs is still not understood. Compelling evidence supports the view that the X protein has a critical role in establishing infection and a direct role in the development of hepatocellular carcinoma. Therefore, uncovering the function of this protein in the transactivation of cellular genes is critical to our understanding the role of X in pathogenesis. The investigator has recently demonstrated that X mediates an increase in the cellular levels of the TATA-binding protein (TBP), a factor involved in the transcription of all cellular genes, and that this protein is limiting for the expression of RNA pol III genes. Both X-mediated increases in RNA pol III gene expression and TBP are dependent upon X-activation of cellular protein kinases. The investigator's aim is to define the signaling pathway activated by X and the consequence of this event on gene activity and TBP levels. The proposed studies will determine: (1) at what level X regulates the cellular increase in TBP; (2) whether both the X-mediated events are dependent upon the activation of the Ras signal transduction pathway; (3) whether X activates Sos-Grb2 complexes and whether this is necessary for gene induction; and (4) whether RNA pol III gene induction and the increase in cellular TBP is mediated exclusively by activating signaling proteins. The proposed studies will comprehensively assess the relationship between X-activation of RNA pol III genes, X-mediated increases in TBP, and the activation of Ras. The investigator will also examine whether X can activate RNA pol I gene expression. These results could demonstrate, for the first time, a whole new class of cellular genes that are induced by X. If so, the investigator will also determine whether induction by X is also dependent upon Ras activation and increased TBP levels. The proposed experiments will be carried out using rat 1 cells and/or Drosopholia S-2 cells that are transiently transfected or genetically altered to express expression vectors for X or other genes. The goal of the studies will potentially be to define a key function of the X protein that allows HBV to intrude into the cellular transcription machinery and ultimately alter the growth properties of the cell. These basic studies will directly impact treatment of HBV-infected patients. With the many new anti-Ras therapeutic agents under investigation, these studies could lead the way for a potential new use of these compounds: by inhibiting X mediated Ras activation to prevent pathogenesis in individuals chronically infected with HBV.
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