MECHANISM OF P53 SILENCING BY ADENOVIRUS E2B 55K PROTEIN

将分析腺病毒E1B-55K在体外表明细胞共抑制物(S)需要抑制基础转录的一个步骤的机制，该步骤特别是从带有p53结合位点的启动子开始的。我们建议纯化这个辅助抑制因子，并分析它抑制转录的机制。在p53阴性的细胞系中，E1B-55K缺失突变体d11520在32度时复制的主要缺陷是未能有效翻译病毒晚期mRNAs。值得注意的是，这一缺陷在很大程度上得到了39度孵化的补充。这一观察结果表明，热休克应激反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摄氏度诱导应激反应的化学试剂是否也解除对E1B-55K的要求来测试。在wtAd5感染的后期，翻译起始因子eIF-4E(帽结合复合体)的去磷酸化抑制了宿主细胞的mRNA翻译。据报道，一个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，而升温在很大程度上减轻了对E1B-55K的需求，我们建议进行研究来检验这一模型，即eIF-4E的去磷酸化是病毒晚期mRNAs有效翻译所必需的。我们还将测试eIF-4E的去磷酸化通过阻止新运输的mRNAs释放穿梭的hnRNP蛋白间接导致细胞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复制E1B-55K突变的有效宿主提供了一种简单的手段，因此，可能成为感染E1B-55K突变的治疗的候选对象。

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.