GENETICS OF 5' NONCODING REGIONS OF PICORNAVIRUS RNAS

该项目将扩展我们对5'端的分子遗传学分析。 小核糖核酸病毒RNA的非编码区（5 'NCR），包括生物化学 作为RNA-蛋白质相互作用的功能测试的测定。 我们将利用 点突变、缺失和接头扫描突变， 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 帽独立翻译所需的复合物。 我们的功能 对含有5' NCR病变的活病毒的分析将包括测定 用于病毒特异性蛋白质和RNA合成，与 遗传标记病毒生长特性的测定 神经元来源的人类细胞，以及 假回复突变体 小核糖核酸病毒RNA对特定复合物的需求 细胞蛋白质不同于那些采用帽依赖 翻译机制将通过结合和翻译测定来探索 在存在富含已知和 也许是新的翻译组件。 我们提出了一系列的实验 采用紫外线交联、引物延伸足迹法和化学方法， 修饰以限定在5 'NCR中的特定核苷酸序列， 参与RNA-蛋白质复合物形成的脊髓灰质炎病毒RNA。 此外，我们还将鉴定和分离出与 脊髓灰质炎病毒5' NCR中的特定茎环结构。 总目标 一项拟议的研究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簇 基因组RNA的信息，这些信息最终将有助于解开 利用帽非依赖性翻译起始的分子机制 被微小核糖核酸病毒感染 该项目的健康相关性源于 揭示病毒病原体的遗传和生化机制 为了颠覆细胞翻译装置， 他们自己的病毒基因产物 在更一般的层面上，我们的研究将 有助于基本了解RNA-蛋白质相互作用是如何 参与真核细胞的翻译调控。

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.