Mechanism of p53 Silencing By Adenovirus E1B 55K Protein

描述(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。它还被用作基因治疗的载体，正在开发腺病毒突变体和重组体作为治疗剂，用于在肿瘤细胞中复制和杀死肿瘤细胞，导致肿瘤的传播性感染，而不是通过正常组织传播。我们的目标是了解腺病毒E1B-55K蛋白在高效病毒感染期间的功能，以及如何将这一知识应用于制造更有效的治疗剂。我们实验室开发的E1B-55K零突变体dl1520(又名Onyx-015)已用于治疗人类肿瘤的临床试验，对一些患者有一定的疗效，但对其他患者没有效果。这种方法最初的基本原理是，由于E1B-55K的功能是灭活P53，而大多数肿瘤在P53或P53途径中存在缺陷，因此不能灭活P53的突变病毒将被限制在正常P53+细胞中复制，但能够在P53阴性细胞中复制。然而，进一步的研究发现，突变体在不同肿瘤细胞系中不同程度复制的能力与构成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和几种使P53泛素化的细胞蛋白组装成泛素蛋白连接酶，标志着P53被蛋白小体降解。其他人发现，这种腺病毒泛素蛋白连接酶可能还会导致参与DNA双链断裂修复的细胞MRN复合体的降解，如果MRN复合体没有失活，病毒DNA的复制就会受到抑制，病毒DNA被连接成太长的线性DNA分子，无法包装在病毒粒子中。最近，我们发现这种泛素-连接酶复合体的活性也需要刺激病毒晚期基因的表达。对细胞突变体的研究表明，只有MRN复合体被灭活，才能使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？自动取款机/自动取款机？下游靶通路诱导抗病毒反应，抑制病毒mRNAs的核输出和晚期病毒mRNAs的翻译。我们将研究ATM激活如何抑制病毒mRNA的核输出和翻译。我们的发现可能使我们能够确定肿瘤的基因特征，以确定哪些肿瘤可能被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.