BIOLOGICAL PROPERTIES OF SV40 EARLY PROTEINS

SV40 早期蛋白的生物学特性

• 批准号: 3165844
• 负责人: JANET S BUTEL
• 金额: $ 17.83万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1978
• 资助国家: 美国
• 起止时间: 1978-01-01 至 1988-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3165844
• 关键词: embryo /fetus tissue /cell culture; gene expression; genetic transcription; immunochemistry; membrane activity; membrane structure; mutant; oncogenes; oncogenic virus; radiotracer; simian virus 40; tissue /cell culture; transforming growth factors; transforming virus; tumor antigens; viral leukemogenesis; virus antigen; virus genetics; virus replication

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.

本实验室的长期目标是了解功能 肿瘤病毒与宿主细胞之间的相互作用。这种方法已经 我一直在定义SV40转换所表达的生物学功能 蛋白质，大肿瘤抗原(T-Ag)，在感染和转化的细胞中。 我们在上一个资助期所做的努力集中在 一株病毒SV40T-Ag的质膜相关形式及分析 编码胞质T-Ag(Ct-Ag)核缺陷的突变体 运输。我们发现表面T-Ag与宿主蛋白P53有关 在转化细胞的膜上，这种复合体迅速地 在膜上，膜上的构象暴露出 T-Ag的氨基和羧基末端位于细胞的外部，并且 细胞表面T-Ag的表达与细胞生长密切相关。对中国传统文化的研究 核运输缺陷病毒突变体成功鉴定出 阻止核转运的单一氨基酸变化 T-AG。最后，我们发现T-Ag是通过糖基化修饰的。这些 努力的最终结果是形成了当前 建议，即SV40T-ag的表面伴生形式为 功能上很重要。提议的目标是我们的 以前的研究，在当前思维的背景下是及时的 关于癌症发生的可能的分子机制。 提出了以下具体目标。(1)归类为 表示的变换相关函数的补集 T-AG。该方法将包括共转染/转化分析。 利用突变的Ct-Ag编码质粒和已知的克隆癌基因 原始的和永生化的宿主细胞。(2)进一步检查观察到的 细胞表面T-Ag表达与细胞生长的关系(3)测试 表面相关T-AG可能在生长过程中发挥作用的假设 通过模仿一种“激活的”生长因子受体来实现因子途径。 表面T-Ag的内化及溶酶体酶Will的加工 以及与细胞形成复合体的可能要求 蛋白P53，以产生一个“活性”的T-Ag构象检查。(4)至 进一步表征T-Ag的糖基化修饰，特别是 关于绘制多肽上的糖基化位点。T-AG 总的来说，这可能是研究O-糖蛋白的一个很好的模型。最后，(5)研究 T-ag的胞内转运。我们将特别强调 细胞内T-Ag合成和O-糖基化位点的测定 以及研究将T-AG分子分离出来的分类 用于原子核和表面的定位。这些研究应该有所收获 对病毒原型生物功能的重要见解 转化蛋白质，拓宽我们对分子机制的理解 参与了细胞转化。