Translational Control of Retroviral Unspliced mRNA

逆转录病毒未剪接 mRNA 的翻译控制

• 批准号: 7339629
• 负责人: Kathleen A. Boris-Lawrie
• 金额: $ 19.31万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-02-03 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7339629
• 关键词: ATP phosphohydrolase; Address; Affect; Affinity Chromatography; Be++ element; Beryllium; Binding; Binding Proteins; Binding Sites; Biochemical; Biological Assay; Biological Models; Categories; Cell Nucleus; Cells; Commit; Complex; Development; Disease; Double-Stranded RNA Binding Domain; EMSA; Electrophoretic Mobility Shift Assay; Elements; Encephalomyocarditis virus; Enhancers; Face; Feline Leukemia Virus; Gagging; Gene Expression; Gene Transfer; Genome; Goals; HIV; HIV-1; Human; Human Spumavirus; Immunologic Deficiency Syndromes; Immunoprecipitation; Internal Ribosome Entry Site; Introns; Knowledge; Length; Long Terminal Repeats; Luciferases; MALDI-TOF Mass Spectrometry; Mason-Pfizer monkey virus; Mass Spectrum Analysis; Mediating; Medical Surveillance; Messenger RNA; Modeling; Necrosis; Neoplastic Cell Transformation; Nuclear; Nuclear Export; Nuclear Localization Signal; Nucleotides; Oryctolagus cuniculus; Outcome; Polyribosomes; Positioning Attribute; Post-Transcriptional Regulation; Post-Translational Protein Processing; Process; Property; Proteins; Proto-Oncogenes; Purpose; RNA; RNA Binding; RNA Interference; RNA Recognition Motif; RNA helicase A; RNA-Protein Interaction; RNase protection assay; Recruitment Activity; Reporter; Research Personnel; Resistance; Resolution; Response Elements; Reticulocytes; Retroviridae; Ribonucleoproteins; Ribosomes; Role; Rous sarcoma virus; Signal Transduction; Site; Site-Directed Mutagenesis; Small Interfering RNA; Spleen; Structure; Sucrose; System; Testing; Translation Initiation; Translations; Untranslated Regions; Vertebral column; Viral; Viral Structural Proteins; Virus; Work; cofactor; experience; gain of function; gene transfer vector; helicase; leukemia virus; mutant; novel; nucleocytoplasmic transport; prevent; programs; research study; rev-Responsive Elements; stem; translation factor; vector; virus host interaction

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.

逆转录病毒在引起肿瘤转化或免疫缺陷的能力方面差异很大。此外， 逆转录病毒被用作构造基因转移应用的非致病矢量的骨干。 但是，在所有情况下，逆转录病毒募集宿主细胞蛋白以实现其细胞质表达 未绘制的基因组长度RNA。我们已经确定了与脾脏坏死5'帽相邻的序列 促进Rev/Rev响应元件（RRE）无综合表达的病毒（SNV） 记者RNA。 SNV长末端重复的RU5区域作为独特的位置 - 和 依赖于含有内含子内含子HIV-1的HIV-1插入RNA的方向依赖性帽依赖性翻译增强剂以及 非病毒荧光素酶（LUC）RNA。指定了SNV转录后控制元件（PCE）。多层 分析表明其功能机制是刺激翻译起始，尽管PCE不是 内部核糖体进入序列。最近，我们确定了两个RNA末端的PCE活性 发散逆转录病毒和细胞原子元mRNA中。我们的新颖假设是5'PCE是一个 发散逆转录病毒和选定的细胞mRNA中共享的特征，以实现有效的翻译 有效细胞质表达的多个障碍的面孔。站点定向的结合结果 诱变，RNA亲和色谱和MALDI-TOF质谱已确定冗余 茎环基序是PCE活性所必需的，并且PCE的结构特征呈现未配对 核苷酸与RNA解旋酶A（RHA）相互作用。通过RNA沉默敲除内源性RHA 消除了PCE翻译刺激，并证明RHA对于PCE活性是必需的。我们的 调查结果产生了以下基本问题：i）翻译必要的必要主题 逆转录病毒PCE共享刺激？ ii）RHA中的残基与与 PCE和翻译因子或辅助蛋白介导PCE活性？ iii）什么步骤 翻译被刺激？该提议的总体目标是表征 PCE-RHA转化增强。该提案的三个集成具体目的是：1）定义 发散逆转录病毒中PCE的保守特征； 2）定义RNA解旋酶A的域 PCE转化增强所需的必要； 3）评估PCE-RHA相互作用的功能 翻译启动。我们的结果将阐明转录后真核生物的独特控制机制 基因表达并定义病毒宿主相互作用，这对于病毒复制和发展至关重要 疾病。我们对翻译控制的新基本知识将定义蜂窝的过程 mRNA致力于细胞质表达，并产生新的策略以优化向量 用于多种基因转移应用的系统。