PRODUCTS OF THE TRANSFORMING GENES OF POLYOMA VIRUS

该建议涉及多瘤病毒的基因产物， 病毒生长和肿瘤转化。 我们的目标是了解 大T和中T抗原在两个过程中都起作用。 这包括 它们与其他细胞途径的元件的相互作用， 调节细胞生长。 我们强调磷酸化，因为 磷酸化似乎是其功能的核心。我们的方法是 生物化学和遗传学。 大T抗原，这是重要的DNA复制和RNA 转录，具有复杂的磷酸化模式， 分析与功能有关。 我们将继续查明 磷酸化位点。 随着每个地点的确定， 涉及寡核苷酸诱变。 我们将表达和研究 蛋白质C末端结构域的。 我们将探索 大T分子的结构 将进行标记实验， 确定N-和C-末端结构域之间的关键接触点， 在寡聚体中的大T和大T与DNA之间。 二位点 回复突变体将作为一种遗传学方法来确定重要的 交互. 对于生物化学研究，我们将表达重要的 突变体和C-末端结构域。 对于中间T，我们将完成对中间T的分析和诱变。 丝氨酸/苏氨酸磷酸化位点。 磷脂酰肌醇-3-激酶是 中T的靶点与转化关系最为密切。具有广阔 由于其与其他癌基因的关联， 生长因子 我们将继续对这种酶进行分析。 除了 在研究它与中间T的联系的细节时，我们将评估 这种酶是否足以调节细胞生长。 因为 NPXY序列中的突变，该突变与以下定位相关： 包被的小凹使中间T非转化，内体上的实验 将进行中T的瞄准。 中间T对 还将测试内化和内体运输。 多瘤T抗原与其他途径的相互作用， 细胞生长的调节也将被测试。 看来， 中T转化需要功能性ras通路，因此 将审查这一途径的要素。 野生型p53为阴性， SV 40转化灭活的细胞生长调节因子;我们将 确定细胞的多瘤转化是否受p53影响， p53是否又受到多瘤病毒的影响。

This proposal concerns the gene products of polyoma virus required for viral growth and neoplastic transformation. Our goal is to understand how the large T and middle T antigens act in both processes. This includes their interactions with elements of other cellular pathways which are, regulatory for cell growth. We emphasize phosphorylation, because phosphorylation appears central to their function. Our approach is both biochemical and genetic. Large T antigen, which is important for DNA replication and RNA transcription, has a complicated pattern of phosphorylation that mutant analysis relates to function. We will continue identification of the phosphorylation sites. As each site is identified, it will be subjected to oligonucleotide mutagenesis. We will express and study the properties of the C-terminal domain of the protein. We will probe the architecture of the large T molecule. Labeling experiments will be carried out to determine the key contact points between the N- and C-terminal domains, between large T in oligomers and between large T and DNA. Second-site revertants will be sought as a genetic way to determine important interactions. For the biochemical studies, we will express important mutants and the C-terminal domains using baculovirus vectors. For middle T we will complete the analysis and mutagenesis of the serine/threonine phosphorylation sites. Phosphatidylinositol-3-kinase is the target of middle T most closely linked with transformation. It has wide general significance because of its association with other oncogenes and growth factors. Our analysis of this enzyme will continue. Besides working out the details of its association with middle T, we will assess whether this enzyme is sufficient to regulate cell growth. Because a mutation in an NPXY sequence that has been associated with localization to coated pits renders middle T non-transforming, experiments on the endosomal targeting of middle T will be carried out. The effects of middle T on internalization and endosomal trafficking will also be tested. The interactions of polyoma T antigens with other pathways which are regulatory for cell growth win also be tested. It appears that a functioning ras pathway is required for middle T transformation, so elements of that pathway will be examined. Wild type p53 is a negative regulator of cell growth inactivated by SV40 transformation; we will determine whether polyoma transformation of cells is affected by p53 and whether p53 is in turn affected by polyoma virus.