VIRAL PROTEINS THAT INHIBIT TNF CYTOLYSIS

我们最近确定并开始描述一组 由人腺病毒E3区编码的独特蛋白质。这些 蛋白质，称为14.7K和10.4K/14.5K，当在病毒中表达时， 受感染的细胞保护细胞免受肿瘤坏死因子的裂解 （TNF）。 肿瘤坏死因子是一种多功能的细胞因子，具有多种效应 从体外转化和感染细胞的细胞溶解， 体内炎症反应的协调。TNF细胞溶解 通过凋亡发生，类似于在细胞凋亡过程中观察到的程序性细胞死亡。 胚胎发生，并表明，了解细胞内的 这种现象所涉及的信号可能是一种广泛的 各种重要的发展信号。TNF的作用机制 行动已经非常难以剖析，因为 它所产生的影响的多样性。因此，识别特定 抑制剂，抵消一些，但不是全部，TNF诱导的反应 介绍了一种新的途径，研究复杂的系列， TNF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TNF的作用机制 抑制蛋白具体目标是：（l）评估 E3蛋白在转染细胞中的功能， 其他病毒蛋白质，并产生一小组人类和小鼠 表达E3蛋白的细胞系用于进一步研究;（2） 阐明E3蛋白的结构/功能关系， 分析了一组23个缺失突变体和点突变体， l0.4K基因。这一目标也将决定 10.4K/14.5K的两种功能，即抑制TNF裂解和降低- EGF受体的调节;（3）分析E3的能力， 抑制与TNF细胞溶解相关的选定功能的蛋白质 重点是TNF受体的表达与I0.4KI 14.5K和 14.7K对细胞基因表达的调控;（4）利用cDNA 克隆以鉴定与E3蛋白 相互作用介导TNF抗性。这一目标利用了我们的 先前鉴定的变异细胞系，其中E3蛋白 表达但不阻止TNF裂解。我们将引入cDNA 通过转染这些变体从正常细胞克隆 表达E3蛋白，并利用TNF的强大选择性 细胞裂解以选择抗性克隆。

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.