MECHANISM OF P53 SILENCING BY ADENOVIRUS E1B 55K PROTEIN

将分析腺病毒E1 B-55 K在体外表明需要细胞辅阻遏物来抑制特异性来自具有p53结合位点的启动子的基础转录步骤的机制。我们建议纯化这种共阻遏物，并分析其抑制转录的机制。在p53阴性细胞系中，32度E1 B-55 K无效突变体d11520复制的主要缺陷是未能有效翻译病毒晚期mRNA。值得注意的是，这一缺陷在很大程度上被39度的孵育所弥补。这一观察结果表明，热休克应激反应的诱导可以在很大程度上取代E1 B-55 K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试剂是否也减轻对E1 B-55 K的需求来测试。在wtAd 5感染的晚期，宿主细胞mRNA翻译受到翻译起始因子eIF-4 E（帽结合复合物）去磷酸化的抑制。据报道，E1 B-55 K突变体在诱导这种eIF-4 E去磷酸化方面存在缺陷。由于热休克也诱导eIF-4 E去磷酸化，并且升高的温度在很大程度上减轻了对E1 B-55 K的需求，因此我们提出研究来测试eIF-4 E的去磷酸化是病毒晚期mRNA有效翻译所需的模型。我们还将测试eIF-4 E的去磷酸化通过阻止穿梭hnRNP蛋白从新转运的mRNA中释放而间接导致细胞mRNA核质转运的晚期抑制的模型。这些研究可能提供一种简单的方法来预测哪些肿瘤细胞将是E1 B-55 K突变体复制的有效宿主，因此，可能是E1 B-55 K突变体感染治疗的候选者。

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.