Murine polyomavirus provides an important model for neoplastic transformation. The virus causes a broad spectrum of tumors. Conclusions reached in the laboratory can be readily tested in animals. Studies on polyoma have repeatedly provided insight into basic mechanisms of cellular growth regulation. Tyrosine kinase and phosphatidylinositol 3-kinase (PI3K) signaling are two fundamental mechanisms uncovered from studies on polyoma. Transformation results from the action of three viral gene products: large T (LT), middle T (MT) and small T (ST) antigens. There are four specific aims that will provide new insight into how they work: 1) the first specific aim concerns large T. Patterns of host cellular RNA expression that result from different large T pathways will be determined. The mechanism will be established by which LT regulates genes containing CREB/ATF sites. Because acetylation has been connected to transcriptional regulation, the sites of acetylation on LT will be established and their function tested by site-directed mutagenesis. Recent results show that LT causes G2/M arrest. Experiments will be carried out to determine the mechanism by which LT causes this arrest. We have identified two new LT-binding partners, Pinl and Bubl, which are known regulators of G2/M. Their role in LT function will be probed. 2) The second specific aim concerns small T. Important differences in function have been detected between polyoma and SV40 small T. Expression profiling will be used to identify the extent of, and the basis for, the differences between them. New polyoma small T partners will be sought by tandem affinity purification and mass spec. Protein phosphatase 2A is a critical small T target. Experiments will be performed to distinguish between different mechanisms by which small T targets PP2A. 3) Human mammary epithelial cells provide a useful model of human cancer. The third specific aim will use the model to probe the function of middle T and small T. The MT signaling pathways required for transformation will be determined. Small T mutant in binding novel partners will be tested in these cells. 4) Our fourth specific aim involves structural studies using NMR. The structure of the N-terminal domain (NT) of LT will be determined. This domain is sufficient to promote cell growth, to cause apoptosis and to regulate cellular RNA transcription. We will also initiate studies to carry out structure determination of polyoma small T.
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