GENETICS OF 5' NONCODING REGIONS OF PICORNAVIRUS RNAS

小核病毒 RNA 5 非编码区的遗传学

• 批准号: 2063526
• 负责人: Bert L Semler
• 金额: $ 18.03万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-07-01 至 1997-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2063526
• 关键词: HeLa cells; Picornaviridae; RNA binding protein; RNA biosynthesis; gel filtration chromatography; gel mobility shift assay; gene mutation; genetic manipulation; genetic regulatory element; genetic translation; host organism interaction; ion exchange chromatography; northern blottings; nucleic acid sequence; nucleic acid structure; poliovirus; protein biosynthesis; protein purification; protein sequence; recombinant DNA; site directed mutagenesis; temperature sensitive mutant; tissue /cell culture; viral rescue; virus RNA; virus genetics; virus replication; western blottings

The proposed project will extend our molecular genetic analysis of the 5' noncoding regions (5'NCRs) of picornavirus RNAs to include biochemical assays as functional tests for RNA-protein interactions. We will utilize point mutations, deletions, and linker scanning mutations within predicted stem-loop structures in the poliovirus 5' NCR to address the role of sequence versus structure in the formation of functional complexes required for cap-independent translation. Our functional analysis for viable viruses containing 5' NCR lesions will include assays for viral-specific protein and RNA synthesis, complementation with genetically-marked viruses, determination of growth characteristics in human cells of neuronal origin, and RNA sequence analysis of pseudo-revertants. The need by picornavirus RNAs for specific complexes of cellular proteins distinct from those employed by cap-dependent translation mechanisms will be explored by binding and translation assays in the presence of fractionated cell extracts enriched in known and perhaps novel translation components. We propose a series of experiments employing U.V. crosslinking, primer extension footprinting, and chemical modification to define the specific nucleotide sequences in the 5'NCR of poliovirus RNA that are involved in the formation RNA-protein complexes. In addition, we will identify and isolate cellular proteins that bind to specific stem-loop structures in the poliovirus 5' NCR. The overall goal of the proposed studies is to determine the viral genetic and cellular polypeptide determinants in RNA-proteins interactions within the 5' NCR of genomic RNAs, information that ultimately will help to unravel the molecular mechanisms of cap-independent translation initiation utilized by picornaviruses. The health relatedness of this project stems from uncovering genetic and biochemical mechanisms employed by viral pathogens to subvert the cellular translation apparatus for dedicated synthesis of their own viral gene products. On a more general level, our studies will contribute to a basic understanding of how RNA-protein interactions are involved in translational regulation in eukaryotic cells.

拟议的项目将扩大我们对5‘的分子遗传分析。 小核糖核酸的非编码区(5‘NCR)，包括生化 作为RNA-蛋白质相互作用的功能测试。我们将利用 点突变、缺失和链接器扫描突变 预测脊髓灰质炎病毒5‘NCR中的茎环结构以解决 序列与结构在功能形成中的作用 不区分大小写的翻译所需的复合体。我们的职能部门 对含有5‘NCR损伤的活病毒的分析将包括化验 对于病毒特异的蛋白质和RNA合成，与 基因标记病毒生长特性的测定 神经细胞来源的人类细胞及其RNA序列分析 伪回复变种。小核糖核酸对特定复合体的需要 与依赖帽子的蛋白质不同的细胞蛋白质 翻译机制将通过约束性分析和翻译分析来探索 在分离的细胞提取物存在的情况下 也许是新奇的翻译成分。我们提出了一系列实验 采用UV交联剂、底漆延伸印迹和化学品 修改以定义5‘NCR中的特定核苷酸序列 脊髓灰质炎病毒的RNA参与了RNA-蛋白质复合体的形成。 此外，我们还将鉴定和分离结合到 脊髓灰质炎病毒5‘NCR中的特定茎环结构。总目标 建议的研究之一是确定病毒的遗传和细胞 5‘NCR内RNA-蛋白质相互作用中的多肽决定因素 基因组RNA中，最终将有助于解开 帽非依赖性翻译起始的分子机制 被小核糖核酸病毒感染。这个项目与健康相关的原因是 揭示病毒病原体的遗传和生化机制 颠覆细胞翻译设备，用于专门合成 他们自己的病毒基因产物。在更广泛的层面上，我们的研究将 有助于基本理解RNA-蛋白质相互作用是如何 参与真核细胞的翻译调控。