MECHANISM OF P53 SILENCING BY ADENOVIRUS E2B 55K PROTEIN

腺病毒 E2B 55K 蛋白沉默 P53 的机制

• 批准号: 6046158
• 负责人: ARNOLD J BERK
• 金额: $ 20.95万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-02-01 至 2004-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6046158
• 关键词: Adenoviridae; DNA footprinting; HeLa cells; affinity chromatography; gel mobility shift assay; gene induction /repression; genetic promoter element; genetic transcription; heat shock proteins; host organism interaction; intermolecular interaction; mutant; oncogenes; p53 gene /protein; phosphoproteins; protein purification; ribonucleoproteins; transcription factor; virus protein; western blottings

Mechanisms will be analyzed by which adenovirus E1B-55K in vitro indicated that a cellular co-repressor(s) is required to inhibit a step in basal transcription specifically from promoters with p53-binding sites. We propose to purify this co-repressor and analyze the mechanism by which it represses transcription. In a p53-minus cell line, the principal defect in the replication of the E1B- 55K null mutant d11520 at 32 degrees is failure to efficiently translate viral late mRNAs. Remarkably, this defect is largely complemented by incubation at 39 degrees. This observation suggests that induction of the heat-shock stress response can largely substitute for the E1B-55K late function. This possibility will be tested by determining if chemical agents that induce the stress response at 32 degrees also relieve the requirement for E1B-55K. During the late phase of infection by wtAd5, host cell mRNA translation is inhibited by the dephosphorylation of the translation initiation factor eIF-4E, the cap binding complex. An E1B- 55K mutant has been reported to be defective in inducing this eIF-4E dephosphorylation. Since heat shock also indues eIF-4E dephosphorylation and elevated temperature largely relieves the requirement for E1B-55K, we propose studies to test the model that the dephosphorylation of eIF-4E is required for the efficient translation of viral late mRNAs. We will also test the model that dephosphorylation of eIF-4E indirectly causes the late phase inhibition of cellular mRNA nucleocytoplasmic transport by preventing the release of shuttling hnRNP proteins from newly transported mRNAs. These studies may provide a simple means for predicting which tumor cells would be effective hosts for the replication of E1B-55K mutants and, therefore, might be candidates for therapy by infection with an E1B-55K mutant.

体外研究表明，E1B-55K腺病毒需要一种细胞共抑制因子来抑制具有p53结合位点的启动子的基础转录的一个步骤。我们建议纯化该共抑制因子，并分析其抑制转录的机制。在p53-阴性细胞系中，E1B- 55K零突变体d11520在32度下复制的主要缺陷是不能有效地翻译病毒晚期mrna。值得注意的是，这一缺陷在很大程度上可以通过39度的孵化来弥补。这一观察结果表明，热休克应力响应的诱导在很大程度上可以替代E1B-55K的晚期功能。这种可能性将通过确定在32度下诱导应激反应的化学剂是否也减轻对E1B-55K的需求来测试。在wtAd5感染的后期，宿主细胞mRNA翻译被翻译起始因子eIF-4E（帽结合复合体）的去磷酸化所抑制。据报道，E1B- 55K突变体在诱导eIF-4E去磷酸化方面存在缺陷。由于热休克也会诱导eIF-4E去磷酸化，而升高的温度在很大程度上减轻了对E1B-55K的需求，因此我们提出研究来验证eIF-4E去磷酸化是有效翻译病毒晚期mrna所必需的模型。我们还将测试eIF-4E的去磷酸化通过阻止新运输mRNA的穿梭hnRNP蛋白的释放间接导致细胞mRNA核质运输的后期抑制的模型。这些研究可能为预测哪些肿瘤细胞是E1B-55K突变体复制的有效宿主提供了一种简单的方法，因此，可能是通过感染E1B-55K突变体进行治疗的候选细胞。