BIOLOGICAL PROPERTIES OF SV40 EARLY PROTEINS

本项目的长期目标是了解 DNA肿瘤病毒和靶细胞之间的相互作用。 该方法具有 定义了分子间的相互作用和生物学功能 由SV 40转化蛋白、大肿瘤抗原（T-ag）引起。 有 不同的T-ag亚群对 转化细胞，包括质膜相关的可能作用， T-ag（pmT-ag）。 此更新应用程序侧重于基本问题 了解SV 40致癌的分子机制。 这 程序将T-ag结构和功能与转化相关联 事件 提出了以下具体目标。(1)结构和功能 含SV 40 T-ag的膜相关蛋白复合物将 分析了 我们开发了一种丁烷提取方法， 回收含有pmT-ag和p53相关的膜蛋白复合物， 蛋白（p56）。 复合物中的其他细胞组分（60 K，50 K， 45 K，35 K）将被识别;假设他们是一个 受体复合物 化学成分的改变， 将研究含pmT-Ag复合物的活性。 T-ag可能参与生长因子样途径， 考察 (2)新的同相缺失突变体的转化表型 将与突变T-89与细胞的特异性相互作用有关。 蛋白质和质膜。不同的检测将区分T-ag 细胞永生化和表型转化所需的结构域。 互补转化试验可能允许T-ag的分配 根据已知的致癌基因分类。 突变型T-ag 多肽将在结构上和生物化学上表征， 包括与膜蛋白形成复合物， 与突变体的转化有关。 ⑶贩运SV 40 T- 将研究Ag对细胞表面的影响。 缺失突变体将揭示 T-ag中短疏水序列对转运的可能影响 和膜的相互作用。 可能与细胞热量有关- 将阐述pmT-ag转运中休克蛋白p73（结合T-ag）。 O-糖基化在运输或膜结合中的作用 将被考虑。 (4)参与muoti的细胞和分子事件 将定义SV 40转基因小鼠肝脏中的阶段性致癌作用。 我们发现，携带由α-1- 抗胰蛋白酶启动子促进肝肿瘤的发生。 新的转基因小鼠携带 将制备p53或突变体T-ag基因。 各种转基因株系 将进行杂交并确定子代动物的病理学。 的 病毒（pmT-ag）和细胞（癌基因）事件的表达将被 与肿瘤进展的不同阶段相关。 增生性 将培养肝细胞以确定体外生长特性， 对应于体内的癌前病变。

The long-range goal of this project is to understand the transforming interactions between DNA tumor virus and target cells. The approach has been to define molecular interactions and biological functions expressed by the SV40 transforming protein, large tumor antigen (T-ag). There is evidence for multiple effects exerted by distinct T-ag subpopulations on transformed cells, including possible role for plasma-membrane-associated T-ag (pmT-ag). This renewal application focuses on questions fundamental to understanding the molecular mechanisms of SV40 carcinogenesis. This program will correlate T-ag structure and function with transformation events. The following specific aims are present. (1) The structure and function of SV40 T-ag containing, membrane-associated protein complexes will be analyzed. We have developed a butane extraction method that selectively recovers membrane protein complexes that contain pmT-ag and p53-related protein (p56). Other cellular components in the complexes (60K, 50K, 45K, 35K) will be identified; it is hypothesized they are members of a receptor complex. the composition modifications, and biochemical activities of pmT-ag-containing complexes will be investigated . Possible involvement of T-ag in a growth-factor-like pathway will be examined. (2) Transformation phenotypes of new in-phase deletion mutants will be related to specific interactions of mutant T-89 with cellular proteins and the plasma membrane. Different assays will distinguish T-ag domain required for cell immortalization and phenotypic transformation. Complementation transformation tests may permit assignment of T-ag functions to categories defined by known oncogenes. Mutant T-ag polypeptides will be characterized structurally and biochemically, including complex formation with membrane proteins, and those analyses correlated with transformation by the mutants. (3)Trafficking of SV40 T- ag to the cell surface will be studied. Deletion mutants will reveal possible effects of the short hydrophobic sequence in T-ag on transport and membrane interaction. The potential involvement of cellular heat- shock protein p73 (that bind T-ag) in pmT-ag transport will be addressed. A role for O-glycosylation o T-ag in trafficking or membrane association will be considered. (4) Cellular and molecular events involved in muoti- stage carcinogenesis in livers of SV40 transgenic mice will be defined. We have found that mice with the WT T-ag gene controlled by the alpha-1- antitrypsin promoter develop liver tumors. New transgenic mice carrying the p53 or mutants T-ag genes will be made. Various transgenic lines will be crossed and pathology in progeny animal determined. The expression of viral (pmT-ag and cellular (oncogenes) events will be correlated with different stages of neoplastic progression. Hyperplastic liver cells will be cultured to determine in vitro growth properties that correspond to preneoplasia in vivo.