Translational Control of Retroviral Unspliced mRNA

描述(由申请人提供)：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，逆转录病毒被用作构建用于基因转移应用的非致病载体的骨架。然而，在所有情况下，逆转录病毒都会招募宿主细胞蛋白来实现其未剪接基因组长度的RNA的细胞质表达。我们已经确定了靠近脾坏死病毒(SNV)5‘端的序列，它促进了HIV-1非剪接报告RNA的REV/REV反应元件(RRE)的非剪接表达。SNV长末端重复序列的RU5区域作为含内含子的HIV-1 Gag RNA和非病毒荧光素酶(Luc)RNA的不同位置和方向依赖的帽依赖的翻译增强子发挥作用。命名为SNV转录后调控元件(PCE)。多聚体分析表明，尽管PCE不是一个内部核糖体进入序列，但其作用机制是刺激翻译起始。最近，我们在两个不同的逆转录病毒的5‘RNA端和一个细胞原癌基因的mRNA中发现了PCE活性。我们的新假设是，5‘PCE是不同逆转录病毒和选定的细胞mRNAs的共同特征，在面对有效细胞质表达的多个障碍时实现有效翻译。定点突变、RNA亲和层析和MALDI-TOF质谱学的联合结果表明，PCE活性需要多余的茎环基序，并且PCE的结构特征为与RNA解旋酶A(RHA)相互作用的不成对核苷酸。通过RNA沉默抑制内源性RHA可以消除PCE翻译刺激，表明RHA是PCE活性所必需的。我们的发现产生了以下基本问题：i)逆转录病毒PCE之间共享哪些翻译刺激所必需的保守基序？Ii)RHA中的哪些残基是与PCE以及与介导PCE活性的翻译因子或辅助蛋白相互作用所必需的？Iii)促进翻译的哪一步？这项建议的总体目标是表征PCE-RHA翻译增强的生化机制。该建议的三个综合的具体目标是：1)确定不同逆转录病毒之间PCE的保守特征；2)定义PCE翻译增强所必需的RNA解旋酶A的结构域；以及3)评估PCE-RHA相互作用在翻译启动中的功能。我们的结果将阐明真核转录后基因表达的独特控制机制，并确定对病毒复制和疾病进展至关重要的病毒-宿主相互作用。我们关于翻译控制的新的基础知识将定义细胞mRNAs致力于细胞质表达的过程，并产生新的策略来优化载体系统，以适应不同的基因转移应用。

DESCRIPTION (provided by applicant): 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 necessary for 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.