VIRAL PROTEINS THAT INHIBIT TNF CYTOLYSIS

抑制 TNF 细胞溶解的病毒蛋白

• 批准号: 2099422
• 负责人: Linda R Gooding
• 金额: $ 22.42万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-01-01 至 1997-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2099422
• 关键词: Adenoviridae; T lymphocyte; apoptosis; cytolysis; epidermal growth factor; gene deletion mutation; gene expression; growth factor receptors; intermolecular interaction; molecular cloning; point mutation; protein structure function; receptor expression; tissue /cell culture; transfection; tumor necrosis factor alpha; virus protein

We have recently identified and begun to characterize a group of unique proteins, encoded by-the E3 region of human adenoviruses. These proteins, called 14.7K and 10.4K/14.5K, when expressed in virus- infected cells protect the cells from lysis by tumor necrosis factor (TNF). TNF is a multifunctional cytokine with pleiotropic effects ranging from cytolysis of transformed and infected cells in vitro to orchestration of the inflammatory response in vivo. Cytolysis by TNF occurs by apoptosis, resembling the programmed cell death seen during embryogenesis, and suggests that understanding of the intracellular signalling involved in the phenomenon may hold the key to a wide variety of important developmental signals. The mechanisms of TNF action have been extraordinarily difficult to dissect because of the diversity of effects it mediates. Thus, the identification of specific inhibitors that counteract some, but not all, TNF-induced responses introduces a new avenue of approach to studying the complex series of events that occurs following TNF binding to its cell surface receptor. We will continue our studies on the mechanism of action of these TNF inhibitory proteins. Specific aims are: (l) to evaluate the ability of the E3 proteins to function in transfected cells in the absence of other viral proteins and to produce a small panel of human and mouse cells lines expressing the E3 proteins for further study; (2) to elucidate structure/function relationships of the E3 proteins by analyzing a panel of 23 deletion and point mutants produced in the l0.4K gene. This aim will also determine the relationship between the two functions of 10.4K/14.5K, namely inhibition of TNF lysis and down- regulation of the EGF receptor; (3) to analyze the ability of the E3 proteins to inhibit selected functions associated with TNF cytolysis with emphasis on TNF receptor expression with I0.4KI14.5K and on modulation of cellular gene expression for 14.7K; (4) to use cDNA cloning to identify cellular proteins with which the E3 proteins interact in mediating TNF resistance. This aim takes advantage of our previous identification of variant cell lines in which the E3 proteins are expressed but do not prevent TNF lysis. We will introduce cDNA clones from normal cells by transfection into these variants expressing the E3 protein and use the powerful selection of TNF cytolysis to select resistant clones.

我们最近已经确定并开始描述一组 独特的蛋白质，由人腺病毒的E3区编码。这些 蛋白质，称为14.7K和10.4K/14.5K，当在病毒中表达时- 感染细胞保护细胞免受肿瘤坏死因子的裂解 (肿瘤坏死因子)。肿瘤坏死因子是一种具有多效性的多功能细胞因子 从体外转化和感染细胞的细胞溶解到 体内炎症反应的协调。肿瘤坏死因子的细胞溶解作用 通过细胞凋亡发生，类似于在 胚胎发生，并表明对细胞内 参与这一现象的信号可能掌握着广泛的 各种重要的发育信号。肿瘤坏死因子的作用机制 行动一直非常难以剖析，因为 它所调节的影响的多样性。因此，识别特定的 抗肿瘤坏死因子诱导的部分但不是全部反应的抑制剂 介绍了一种研究复数列的新途径 肿瘤坏死因子与其细胞表面受体结合后发生的事件。 我们将继续研究这些肿瘤坏死因子的作用机制。 抑制蛋白。具体目标是：(L)评估能力 在缺乏E3蛋白的情况下，在转基因细胞中发挥作用 其他病毒蛋白，并产生一小部分人和小鼠 表达E3蛋白的细胞系，以供进一步研究； 阐明E3蛋白的结构/功能关系 分析由23个缺失和点突变组成的小组 10.4K基因。这个目标也将决定两国之间的关系 10.4K/14.5K的两个功能，即抑制肿瘤坏死因子的裂解和下调。 EGF受体的调节；(3)分析E3的能力 抑制与肿瘤坏死因子细胞溶解相关的特定功能的蛋白质 重点研究了I0.4KI14.5K和On的肿瘤坏死因子受体的表达 14.7K对细胞基因表达的调控；(4)利用cDNAs 克隆鉴定E3蛋白与之结合的细胞蛋白 在调节肿瘤坏死因子抵抗中相互作用。这一目标利用了我们的 E3蛋白变异细胞系的初步鉴定 都有表达，但不能阻止肿瘤坏死因子的裂解。我们将介绍cDNA 通过将正常细胞的克隆导入这些变异体 表达E3蛋白并利用肿瘤坏死因子的强大选择性 细胞溶解以选择抗性克隆。