ADENOVIRUS DEATH PROTEIN

人腺病毒2(AD2)编码一个E311600 mW的蛋白质，命名为 “腺病毒死亡蛋白”(ADP)。ADP是一种核糖蛋白 在感染后期大量合成。ADP 与AD2 E1B-19K共定位，AD2 E1B-19K是维持细胞生长的Bcl2同源物 生存能力。ADP可能取代E1B-19K对细胞的抑制作用 死亡；因此，细胞裂解并释放病毒。结构-功能 分析已经确定了ADP促进细胞死亡所需的结构域 并从ER到ADP起作用的高尔基体和NM 促进细胞死亡。 我们建议确定ADP是否单独促进细胞死亡，它废除了 E1B-19K对肿瘤坏死因子或抗Fas的杀伤作用 影响ICE家族的功能(导致死亡)，而且它 通过细胞凋亡杀死细胞。ADP的“死亡”结构域将突变为 深入了解它们是如何运作的。 蛋白质之间的相互作用将通过免疫共沉淀进行检测。 利用ADP抗血清和标记ADP的表位，亲和结合GST- ADP融合蛋白和酵母双杂交系统。细胞蛋白质 与ADP结合的基因将被克隆和鉴定。我们将重点关注是否 ADP与E1B-19K结合，细胞蛋白与E1B-19K结合，ADP与E1B-19K结合 Bcl2家族的成员。 我们将确定ADP是定位于内NM还是外NM，然后 利用现有突变体识别瞬时转基因中的结构域 在ADP中将ADP从跨高尔基网络传输到 ADP与E1B-19K的相互作用。一名CD8-ADP记者将 用于识别ADP中足以靶向NM的序列。 ADP将通过共聚焦免疫荧光法和平衡法进行监测 膜分离的密度离心法。 ADP是一种新的蛋白质，它代表了病毒中的一个新概念 发病机制。ADP应成为了解细胞凋亡和细胞凋亡的有力工具 E1B-19K癌蛋白的功能，可能是Bcl2的成员 一家人。ADP也可能被用作自杀基因来杀死癌细胞。

Human adenovirus 2 (Ad2) encodes an E3 11,600 MW protein named the "Adenovirus Death Protein" (ADP). ADP is a nuclear (NM) glycoprotein synthesized in large amounts at late stages of infection. ADP colocalizes with Ad2 E1B-19K, a homolog of Bcl-2 which maintains cell viability. ADP may supersede the ability of E1B-19K to inhibit cell death; therefore, cells lyse and release virus. Structure-function analyses have identified domains required for ADP to promote cell death and be sorted from the ER to the Golgi and the NM where ADP functions to promote cell death. We propose to determine if ADP alone promotes cell death, it abrogates the ability of E1B-19K to inhibit cytolysis by TNF or anti-Fas, it affects the functions of the ICE family (which induces death), and it kills cells by apoptosis. The 'death' domains of ADP will be mutated to gain insight into how they function. Protein-protein interactions will be examined by coimmunoprecipitation using antisera to ADP and epitope tagged ADP, affinity binding to a GST- ADP fusion protein, and the yeast two-hybrid system. Cellular proteins that bind ADP will be cloned and characterized. We will focus on whether ADP binds to E1B-19K, cellular proteins that bind to E1B-19K, and to members of the Bcl-2 family. We will determine whether ADP localizes to the inner or outer NM, then use existing mutants to identify in transient transfections the domains in ADP necessary for transport of ADP from the trans-Golgi network to the NM, and for the interaction of ADP with E1B-19K. A CD8-ADP reporter will be used to identify sequences in ADP sufficient for targeting to the NM. ADP will be monitored by confocal immunofluorescence and by equilibrium density centrifugation of membrane fractions. ADP is a novel protein, and it represents a new concept in viral pathogenesis. ADP should be a powerful tool to understand apoptosis and the function of the E1B-19K oncoprotein, and perhaps members of the Bcl-2 family. ADP might also be used as a 'suicide' gene to kill cancer cells.