Translational Control of Retroviral Unspliced mRNA

逆转录病毒未剪接 mRNA 的翻译控制

• 批准号: 7176238
• 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/7176238
• 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

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.

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未剪接的基因组长度RNA的细胞质表达。我们已经鉴定出与脾坏死病毒 (SNV) 5' 帽相邻的序列，这些序列促进 HIV-1 未剪接报告 RNA 的 Rev/Rev 响应元件 (RRE) 独立表达。 SNV 长末端重复序列的 RU5 区域作为包含内含子的 HIV-1 gag RNA 以及非病毒荧光素酶 (luc) RNA 的独特位置和方向依赖性帽子依赖性翻译增强子。指定为 SNV 转录后控制元件 (PCE)。多核糖体分析表明，尽管 PCE 不是内部核糖体进入序列，但其功能机制是刺激翻译起始。最近，我们在两种不同的逆转录病毒的 5' RNA 末端和细胞原癌基因 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细胞 mRNA 之间共有的一个特征，可在面对有效细胞质表达的多重障碍时实现有效翻译。定点诱变、RNA 亲和层析和 MALDI-TOF 质谱的综合结果确定，冗余茎环基序对于 PCE 活性是必需的，并且 PCE 的结构特征呈现出与 RNA 解旋酶 A (RHA) 相互作用的不配对核苷酸。通过 RNA 沉默敲低内源性 RHA 可消除 PCE 翻译刺激，并表明 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) 逆转录病毒 PCE 共有哪些翻译刺激所需的保守基序？ ii) RHA 中的哪些残基是与 PCE 以及介导 PCE 活性的翻译因子或辅助蛋白相互作用所必需的？ iii) 刺激翻译的哪一步？该提案的总体目标是表征 PCE-RHA 翻译增强的生化机制。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PCE翻译增强所需的RNA解旋酶A的结构域； 3) 评估 PCE-RHA 相互作用在翻译起始中的功能。我们的结果将阐明真核转录后基因表达的独特控制机制，并定义对病毒复制和疾病进展非常重要的病毒与宿主的相互作用。我们关于翻译控制的新基础知识将定义细胞 mRNA 致力于细胞质表达的过程，并产生新的策略来优化不同基因转移应用的载体系统。