Mechanism of p53 Silencing By Adenovirus E1B 55K Protein

描述（由申请人提供）：腺病毒是了解细胞功能基本方面的重要模型系统。它也被用作基因治疗的载体，并且正在开发腺病毒突变体和重组体，用作治疗剂以在肿瘤细胞中复制并杀死肿瘤细胞，引起肿瘤中的扩散感染，而不通过正常组织扩散。我们的目标是了解腺病毒E1B-55K蛋白在生产性病毒感染过程中的功能，以及如何应用这些知识来制造更有效的治疗剂。我们实验室开发的E1B-55K无效突变体dl1520（又名ONYX-015）已用于治疗人类肿瘤的临床试验，对某些患者有效，而对其他患者无效。这种方法最初的基本原理是，由于E1B-55K的功能是抑制p53，而大多数肿瘤在p53或p53-通路中有缺陷，因此不能抑制p53的突变病毒预计将限制在正常p53+细胞中复制，但能够在p53-细胞中复制。然而，进一步的研究表明，突变体在不同肿瘤细胞系中不同程度复制的能力与包含病毒颗粒的病毒晚期蛋白的表达有关，而与细胞的p53状态无关。我们发现E1B-55K与另一种病毒蛋白E4orf6和几种多聚泛素化p53的细胞蛋白组装泛素蛋白连接酶，标记它被蛋白体降解。其他人发现，这种腺病毒泛素-蛋白连接酶可能也导致参与DNA双链断裂修复的细胞MRN复合物的降解，并且如果MRN复合物未失活，则病毒DNA复制被抑制，并且病毒DNA连接成长的线性DNA分子，其太长而不能包装在病毒体中。最近，我们发现这种泛素连接酶复合物的活性也是刺激病毒晚期基因表达所必需的。对细胞突变体的研究表明，MRN复合物必须失活以允许正常水平的病毒晚期mRNA从细胞核输出到细胞质并在细胞质中翻译。结果表明，MRN？ATM/ATR？下游靶向途径诱导抑制病毒mRNA核输出和晚期病毒mRNA翻译的抗病毒应答。我们将研究ATM激活如何抑制病毒mRNA核输出和翻译。我们的发现可能使我们能够从基因上描述肿瘤的特征，以确定哪些肿瘤可以用dl1520有效治疗。

DESCRIPTION (provided by applicant): Adenovirus is an important model system for understanding basic aspects of cell function. It is also being used as a vector for gene therapy, and adenovirus mutants and recombinants are being developed for use as therapeutic agents to replicate in and kill neoplastic cells, causing a spreading infection in tumor, without spreading through normal tissue. Our goal is to understand the function of the adenovirus E1B-55K protein during a productive viral infection and how this knowledge can be applied to make a more effective therapeutic agent. An E1B-55K null mutant developed in our lab, dl1520 (aka ONYX-015), has been used in clinical trials for the treatment of human tumors, with some efficacy in some patients and not others. The original rationale for this approach was that since E1B-55K functions to inactivate p53, and most tumors are defective in p53 or in the p53-pathway, a mutant virus that cannot inactivate p53 would be expected to be restricted for replication in normal p53+ cells, but able to replicate in p53-minus cells. However, further study revealed that the ability of the mutant to replicate to varying extents in different tumor cell lines was related to the expression of viral late proteins that comprise the virus particle, and not the p53-status of the cells. We found that E1B-55K assembles a ubiquitin-protein ligase with another viral protein, E4orf6, and several cellular proteins that polyubiquitinates p53, marking it for degradation by proteosomes. Others found that this adenovirus ubiquitin-protein ligase probably also causes the degradation of the cellular MRN complex involved in DNA double-strand break repair, and that if the MRN complex is not inactivated, viral DNA replication is inhibited and viral DNA is concatenated into long linear DNA molecules too long to package in the virion. Recently, we found that the activity of this ubiquitin-ligase complex is also required to stimulate viral late gene expression. Studies with cell mutants suggest that it is the MRN complex that must be inactivated to allow the normal level of viral late mRNA export from the nucleus to the cytoplasm and translation in the cytoplasm. The results imply that the MRN ? ATM/ATR ? downstream targets pathway induces an anti-viral response that inhibits viral mRNA nuclear export and translation of late viral mRNAs. We will study how ATM activation inhibits viral mRNA nuclear export and translation. Our findings may allow us to genetically characterize tumors to determine which ones might be effectively treated with dl1520.