Biosynthesis of Adenovirus Early RNA's

这项拟议研究的长期目标是了解腺病毒E1a蛋白是如何激活的 来自病毒启动子的转录，E1A如何修改诱导细胞的细胞基因的转录 增殖，腺病毒E1B-55K蛋白如何抑制P53功能，以及如何识别人类肿瘤 这可能通过瘤内注射E1B-55K缺失突变体来成功治疗。基因调控 表达是几乎所有生物过程的一个基本方面；基因表达的异常控制 有助于许多病理过程，如癌症的发展。了解 详细的转录控制分子机制应该允许治疗干预措施的设计 治疗癌症等人类疾病。我们将使用显微荧光方法来分析相互作用 E1a保守区3和其他带有介体复合体的激活结构域的一般转录 因子，以及活细胞中的其他共激活复合体。我们将使用染色质免疫沉淀(CHIP)来 分析E1a对PIC组装和Pol II在体内对病毒染色质重新启动的影响。我们会 使用芯片上分析来确定E1AN-末端区域与CBP和p300结合的结果 在最近发现的组蛋白H3K18乙酰化的重新分布中，一种与 转录激活。我们将分析E1B-55K通过拴住P53来抑制P53功能的能力。 PML核体依赖E1B-55K苏莫化，并完全抑制P53激活功能 通过新发现的E1B-55K SUM01-P53连接酶活性引起的SUMO1修饰。我们还将 追求E1B-55K通过阻止抗病毒反应刺激病毒晚期基因表达的模型 这抑制了病毒mRNA的核输出和翻译。我们将追求目前的结果，表明 这种抗病毒反应的诱导需要由线性病毒激活的MRN复合体激活ATM DNA这项工作应该会导致对控制分子相互作用的更完整的理解 人类细胞中的转录。了解E1B-55K刺激小鼠体内病毒复制的机制 不同的人类肿瘤细胞系应该可以让临床医生确定哪些人类肿瘤可能更好地治疗 通过瘤内注射E1B-55K缺失突变体有效。

The long term goals of the proposed research are to understand how the adenovirus E1A protein activates transcription from viral promoters, how E1A modifies transcription of cellular genes that induce cell proliferation, how adenovirus E1B-55K protein inhibits p53 function, and how human tumors can be identified that might be successfully treated by intratumoral injection of an E1B-55K deletion mutant. Control of gene expression is a fundamental aspect of nearly all biological processes; abnormal contol of gene expression contributes to many pathological processes such as the development of cancer. Understanding the molecular mechanisms of transcription control in detail should allow the design of therapeutic interventions to treat human disease such as cancer. We will use microscopic fluorescent methods to analyze the interaction of E1A conserved region 3 and other activation domains with mediator complexes, general transcription factors, and other co-activator complexes in living cells. We will use chromatin-immunoprecipitation (ChIP) to analyze the influence of E1A on PIC assembly and re-initiation by Pol II on viral chromatin in vivo. We will use ChIP on chip assays to determine how the binding of the E1A N-terminal region to CBP and p300 results in the recently discovered re-distribution of histone H3K18 acetylation, a modification that correlates with transcriptional activation. We will analyze the ability of E1B-55K to inhibit p53 function by tethering p53 in PML nuclear bodies dependent on E1B-55K sumoylation, and complete inhibition of p53 activation function by SUMO1-modification induced by a newly discovered E1B-55K SUM01-p53 ligase activity. We will also pursue the model that E1B-55K stimulates viral late gene expression by preventing an anti-viral response that inhibits viral mRNA nuclear export and translation. We will pursue current results suggesting that induction of this anti-viral response requires activation of ATM by MRN complexes activated by linear viral DNA. This work should lead to a more complete understanding of molecular interactionsthat control transcription in human cells. Understanding the mechanism by which E1B-55K stimulates viral replication in various human tumor cell lines should allow clinicians to determine which human tumors might betreated effectively by intratumoral injection of an E1B-55K deletion mutant.