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性载体的主干。 然而，在所有情况下，逆转录病毒都会招募宿主细胞蛋白以实现其细胞质表达 未剪接的基因组长度的RNA。我们已经鉴定出邻近脾脏坏死 5' 帽的序列 促进 HIV-1 未剪接的 Rev/Rev 反应元件 (RRE) 独立表达的病毒 (SNV) 报告基因RNA。 SNV 长末端重复序列的 RU5 区域作为独特的位置和 含有内含子的 HIV-1 gag RNA 的方向依赖性帽子依赖性翻译增强子以及 非病毒荧光素酶 (luc) RNA。指定为 SNV 转录后控制元件 (PCE)。多核糖体 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，我们在两个RNA的5'端发现了PCE活性 不同的逆转录病毒和细胞原癌基因 mRNA。我们的新假设是 5' PCE 是 不同逆转录病毒和选定的细胞 mRNA 之间共享的特征，以实现高效翻译 面对有效细胞质表达的多重障碍。现场定向的综合结果 诱变、RNA 亲和层析和 MALDI-TOF 质谱已确定冗余 茎环基序是 PCE 活性所必需的，并且 PCE 的结构特征呈不配对状态 与 RNA 解旋酶 A (RHA) 相互作用的核苷酸。通过 RNA 沉默抑制内源性 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 研究结果产生了以下基本问题： i) 翻译所需的保守基序是什么 逆转录病毒 PCE 之间共享刺激吗？ ii) RHA 中的哪些残基对于与 PCE 以及介导 PCE 活性的翻译因子或辅助蛋白？ iii) 哪一步 翻译是刺激吗？该提案的总体目标是表征 PCE-RHA 翻译增强。该提案的三个综合具体目标是：1) 定义 不同逆转录病毒中 PCE 的保守特征； 2) 定义RNA解旋酶A的结构域 PCE 转化增强所必需的； 3) 评估 PCE-RHA 相互作用的功能 翻译启动。我们的结果将阐明真核生物转录后的独特控制机制 基因表达并定义病毒与宿主的相互作用，这对于病毒复制和进展很重要 疾病。我们关于翻译控制的新基础知识将定义细胞的过程 mRNA 致力于细胞质表达并产生优化载体的新策略 用于多种基因转移应用的系统。