BIOLOGICAL PROPERTIES OF SV40 EARLY PROTEINS

该项目的长期目标是了解 DNA肿瘤病毒与靶细胞的相互作用。这种方法已经 一直在定义分子相互作用和表达的生物功能 由SV40转化蛋白、大肿瘤抗原(T-Ag)组成。的确有 不同T-Ag亚群对人的多重影响的证据 转化细胞，包括与质膜相关的可能作用 T-AG(PMT-AG)。此续订申请侧重于基本问题 了解SV40致癌的分子机制。这 计划将T-ag的结构和功能与转换相关联 事件。 目前存在以下具体目标。(一)结构和功能 含有SV40T-Ag的膜相关蛋白复合体将 分析过了。我们开发了一种丁烷提取方法，可以选择性地 恢复含有PMT-Ag和P53相关的膜蛋白复合体 蛋白质(P56)。配合物中的其他细胞成分(60K，50K， 45K、35K)将被识别；假设他们是 受体复合体。成分修饰和生化 含PMT-Ag的络合物的活性将被研究。 T-AG可能参与生长因子样通路 检查过了。(2)新的同相缺失突变体的转化表型 将与突变体T-与细胞的特异性相互作用有关 蛋白质和质膜。不同的检测方法会区分T-Ag 细胞永生化和表型转化所需的结构域。 互补转换测试可能允许分配T-ag 对已知癌基因所定义的类别的功能。突变型T-Ag 多肽将在结构和生物化学上进行表征， 包括与膜蛋白形成复合体，以及这些分析 与突变体的转化相关。(3)贩运SV40T- 银对细胞表面的影响将被研究。缺失突变体将揭示 T-Ag中短疏水序列对转运的可能影响 和膜相互作用。细胞热的潜在参与-- 休克蛋白p73(结合T-Ag)在PMT-Ag转运中将被解决。 T-Ag的O-糖基化在转运或膜结合中的作用 将被考虑。(4)参与突变的细胞和分子事件-- SV40转基因小鼠肝脏的分期癌变将被定义。 我们发现携带WTT-Ag基因的小鼠受α-1-g基因控制。 抗胰蛋白酶启动子会发展成肝肿瘤。新的转基因小鼠携带 P53或突变的T-Ag基因将被制造出来。各种转基因品系 将进行杂交，并在后代动物中确定病理。这个 病毒(PMT-Ag)和细胞(癌基因)事件的表达将是 与肿瘤进展的不同阶段相关。增生性 肝细胞将进行培养，以确定其体外生长特性 相当于体内的创业成熟症。

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.