Translational Control of Retroviral Unspliced mRNA

逆转录病毒引起肿瘤转化或免疫缺陷的能力差异很大。此外，本发明还提供了一种方法， 逆转录病毒被用作构建用于基因转移应用的非致病性载体的骨架。 然而，在所有情况下，逆转录病毒募集宿主细胞蛋白质以实现它们的细胞质表达。 未剪接的基因组长度RNA。我们已经确定了邻近脾坏死5'帽的序列 促进HIV-1未剪接的Rev/Rev应答元件（RRE）非依赖性表达的病毒（SNV） 报告RNA。SNV长末端重复序列的RU 5区作为不同的位置发挥作用， 含有内含子的HIV-1 gag RNA的方向依赖性帽依赖性翻译增强子，以及 非病毒荧光素酶（luc）RNA。命名为SNV转录后控制元件（PCE）。多核糖 分析表明，其功能机制是刺激翻译起始，而PCE不是 内部核糖体进入序列。最近，我们鉴定了两个人的5' RNA末端的PCE活性， 趋异逆转录病毒和细胞原癌基因mRNA。我们的新假设是，5'PCE是一个 不同的逆转录病毒和选定的细胞mRNA之间共享的特征，以实现在不同的逆转录病毒中的有效翻译。 面对有效细胞质表达的多重障碍。站点定向的组合结果 诱变，RNA亲和层析和MALDI-TOF质谱已经确定了冗余的 茎-环基序是PCE活性所必需的，PCE的结构特征呈现不配对 与RNA解旋酶A（RHA）相互作用的核苷酸。通过RNA沉默敲低内源性RHA 消除了PCE翻译刺激，并证明RHA是PCE活性所必需的。我们 研究结果产生了以下基本问题：i）翻译所必需的保守基序是什么 刺激在逆转录病毒PCE之间共享？ii）RHA中的哪些残基是与 PCE与翻译因子或介导PCE活性的辅助蛋白的结合？（三）什么步骤 翻译是刺激的吗？本提案的总体目标是描述 PCE-RHA翻译增强。该提案的三个综合具体目标是：1）确定 不同逆转录病毒PCE中的保守特征; 2）定义RNA解旋酶A的结构域 PCE翻译增强所必需的;和3）评估PCE-RHA相互作用在 翻译起始我们的研究结果将阐明真核生物转录后调控的独特机制 基因表达，并确定病毒-宿主相互作用，这对病毒复制和进展很重要， 疾病我们对翻译控制的新的基础知识将定义细胞 mRNA致力于细胞质表达并产生新的策略来优化载体 用于多种基因转移应用的系统。

Retroviruses vary widely in their ability to cause neoplastic transformation or immunodeficiency. In addition, retroviruses are used as a backbone for constructing nonpathogenic vectors for gene transfer applications. However, in all cases, retroviruses recruit host cell proteins to achieve cytoplasmic expression of their unspliced genome-length RNA. We have identified sequences adjacent to the 5' cap of spleen necrosis virus (SNV) that facilitate Rev/Rev responsive element (RRE)-independent expression of HIV-1 unspliced reporter RNA. The RU5 region of the SNV long terminal repeat functions as a distinct position- and orientation-dependent cap-dependent translational enhancer of intron-containing HIV-1 gag RNA as well as nonviral luciferase (luc) RNA. Designated the SNV post-transcriptional control element (PCE). polysome analyses indicate that its functional mechanism is to stimulate translation initiation, although the PCE is not an internal ribosome entry sequence. Recently, we identified PCE activity in the 5' RNA terminus of two divergent retroviruses and in a cellular protooncogene mRNA. Our novel hypothesis is that 5' PCEs are a feature shared among divergent retroviruses and selected cellular mRNAs to achieve efficient translation in the face of multiple barriers to efficient cytoplasmic expression. Combined results of site-directed mutagenesis, RNA affinity chromatography and MALDI-TOF mass spectroscopy have determined redundant stem-loop motifs are necessary for PCE activity and that the structural features of the PCE present unpaired nucleotides for interaction with RNA helicase A (RHA). Knockdown of endogenous RHA by RNA silencing eliminates PCE translation stimulation and demonstrates that RHA is necessary for PCE activity. Our findings have generated the following essential questions: i) What conserved motifs necessaryfor translation stimulation are shared among retroviral PCEs? ii) What residues in RHA are necessary for interaction with the PCE and with translation factors or auxiliary proteins that mediate PCE activity? iii)What step of translation is stimulated? The overall goal of this proposal is to characterize the biochemical mechanism of PCE-RHA translational enhancement. Three integrated Specific Aims for this proposal are:1) to define conserved features in PCEs among divergent retroviruses; 2) to define the domains of RNA helicase A necessary for PCE translational enhancement; and 3) to evaluate the function of the PCE-RHA interaction in translation initiation. Our results will illuminate a unique control mechanism of eukaryotic post-transcriptional gene expression and define virus-host interactions that are important for viral replication and progression to disease. Our new fundamental knowledge of translational control will define the process by which cellular mRNAs become committed to cytoplasmic expression and produce new strategies to optimize vector systems for diverse gene transfer applications.