BIOLOGICAL PROPERTIES OF SV40 EARLY PROTEINS

这个实验室的长期目标是了解 肿瘤病毒和宿主细胞之间的相互作用。 该方法具有 定义SV40转化表达的生物学功能 蛋白质，大肿瘤抗原（T-ag），在感染和转化细胞。 我们在上一个供资期间的工作重点是描述 SV40 T-ag的质膜结合形式和分析病毒 一种编码细胞质T-ag（cT-ag）的突变体， 运输 我们发现表面T-ag与宿主蛋白p53相关， 在转化细胞的膜中，复合物迅速转变为 在膜上，膜上的构象暴露了 氨基和羧基末端的T-ag的细胞的外部， 表面T-ag的表达与细胞生长相关。 的研究 核转运缺陷型病毒突变体成功地鉴定了 单个氨基酸的变化，负责阻断核转运 T-ag 最后，我们发现T-ag被糖基化修饰。 这些 这些努力最终形成了一种观点， 建议，即SV40 T-ag的表面结合形式是 功能重要。 拟议的目标是我们的逻辑延伸， 以前的研究，并及时在当前的思维背景下， 关于致癌的可能分子机制。 提出了以下具体目标。 (1)归入 互补组的转换相关的功能表示， T-ag 该方法将包括共转染/转化测定 使用突变的cT-ag编码质粒和已知的克隆癌基因， 原代和永生化宿主细胞。 (2)为了进一步研究观察到的 表面T-ag表达与细胞生长的关系。 (3)测试 假设表面相关的T-ag可能在生长中起作用， 通过模拟"活化的"生长因子受体来激活生长因子途径。 表面T-ag的内化和溶酶体蛋白酶的加工将 可能需要与细胞形成复合物， 蛋白p53产生"活性"的T-ag构象检查。 (4)到 进一步表征T-ag的糖基化修饰，特别是 关于多肽上糖基化位点的作图。 t-AG 可能是O-糖蛋白的一个很好的模型。 （5）研究 T-ag的细胞内运输。 将特别强调 确定细胞中T-ag合成和O-糖基化的位点， 以及研究T-ag分子的分类， 用于核和表面定位。 这些研究应该会产生 对原型病毒的生物学功能的重要见解 转化蛋白，拓宽我们对分子机制的理解 参与细胞转化。

The long-range goal of this laboratory is to understand the functional interactions between a tumor virus and the host cell. The approach has been to define the biological functions expressed by the SV40 transforming protein, large tumor antigen (T-ag), in infected and transformed cells. Our efforts during the previous funding period focused on characterizing the plasma membrane-associated form of SV40 T-ag and analyzing a viral mutant that encoded a cytoplasmic T-ag (cT-ag) defective for nuclear transport. We showed that surface T-ag is associated with host protein p53 in the membrane of transformed cells, that the complex is rapidly turned over in the membrane, that the conformation in the membrane exposes both amino and carboxy termini of T-ag on the exterior of the cell, and that the expression of surface T-ag correlates with cell growth. Studies of the nuclear-transport-defective viral mutant succeeded in identifying the single amino acid change responsible for blocking nuclear transport of T-ag. Finally, we found that T-ag is modified by glycosylation. These efforts culminated in a perspective that forms the basis of the current proposal, namely that the surface-associated form of SV40 T-ag is functionally important. Proposed objectives are logical extensions of our previous studies and are timely in the context of current thinking regarding possible molecular mechanisms in carcinogenesis. The following specific aims are proposed. (1) To categorize into complementation groups the transformation-related functions expressed by T-ag. The approach will include co-transfection/transformation assays using the mutant cT-ag-encoding plasmid and known cloned oncogenes in primary and immortalized host cells. (2) To further examine the observed relationship between surface T-ag expression and cell growth. (3) To test the hypothesis that surface-associated T-ag may be functioning in a growth factor pathway by mimicking an "activated" growth factor receptor. Internalization of surface T-ag and processing by lysosomal proteases will be sought, and the possible requirement of complex formation with cellular protein p53 to generate an "active" T-ag conformation examined. (4) To further characterize the glycosylation modification of T-ag, especially with respect to mapping the glycosylation sites on the polypeptide. T-ag may be a good model for O-glycoproteins in general. Finally, (5) To study the intracellular trafficking of T-ag. Special emphasis will be placed on determining the sites of T-ag synthesis and O-glycosylation in the cell, as well as investigating the sorting that separates T-ag molecules destined for nuclear and surface localizations. These studies should yeild important insights into the biological functioning of a prototype viral transforming protein and broaden our understanding of molecular mechanisms involved in cellular transformation.