GENETICS OF FIVE NONCODING REGIONS OF PICORNAVIRUS RNAS

拟议的项目将利用小核糖核酸病毒分子 重组体和突变体的功能结构域进行测定， 脊髓灰质炎病毒RNA的5 '-非编码区。 康贝特人将以 基因5 '-非翻译区的遗传改变的影响 脊髓灰质炎基因组对RNA复制和核糖体结合/病毒- 特异性蛋白质合成 我们将进行体外操作 克隆的cDNA构建了许多不同的位点特异性的 5 '-非编码区内的重组和突变基因组 脊髓灰质炎病毒和柯萨奇病毒的RNA。 5 '-非编码cDNA 然后将嵌合体和突变体连接到编码区 脊髓灰质炎病毒RNA的感染性cDNA克隆。 质粒 包含完整的重组/突变基因组的基因组将被 通过转染培养的灵长类动物细胞测定感染性。 将回收感染性重组和突变病毒， 分析（i）的改变的或温度敏感的表达， RNA复制功能，（ii）病毒蛋白质合成功能， 和（iii）宿主细胞关闭功能。 最后，任何 重组质粒不产生感染性病毒， 将分析转染测定的体外翻译 使用来自噬菌体的体外转录物的效率 T7启动子和RNA聚合酶系统。 我们的总体目标是 这个项目是为了了解特定块的作用， 病毒基因组内的非编码核苷酸在调节 脊髓灰质炎基因产物的单个翻译单位的表达。 拟议项目的健康相关性基于 先前的研究表明， 脊髓灰质炎病毒RNA在病毒的复制适应性和 病毒神经毒力决定簇的完全表达。 的 从拟议项目中获得的信息应提供 疫苗株遗传修饰的合理方案 通过重组DNA方法获得小核糖核酸病毒。 这种非编码 区域修饰具有降低 疫苗株细胞致病性和不稳定性， 扰乱病毒体结构，这对引发完全的 保护性免疫反应

The proposed project will utilize picornavirus molecular recombinants and mutants to assay the functional domains of the 5'-non-coding region of poliovirus RNA. We will determine the effects of genetic alteration of the 5'-untranslated region of the polio genome on RNA replication and ribosome binding/viral- specific protein synthesis. We will carry out in vitro manipulation of cloned cDNAs to construct a number of different site-specific recombinant and mutant genomes within the 5'-noncoding regions of polio-virus and coxsackievirus RNAs. The 5'-noncoding cDNA chimeras and mutants will then be ligated to the coding region from an infectious cDNA clone of poliovirus RNA. Plasmids containing the complete recombinant/mutant genomes will be assayed for infectivity by transfection of cultured primate cells. Infectious recombinant and mutant viruses will be recovered and analyzed for altered or temperature-sensitive expression of (i) RNA replication functions,(ii) viral protein synthesis functions, and (iii) host cell shut off functions. Finally, any of the recombinant plasmids that do not produce infectious virus in the transfection assay will be analyzed for in vitro translation efficiency using in vitro transcripts derived from a bacteriophage T7 promoter and RNA polymerase system. Our overall goal in this project is to understand the role of specific blocks of noncoding nucleotides within a viral genome in the regulation of expression of the single translational unit of polio gene products. The health-relatedness of the proposed project is based upon previous studies that implicate the 5'-noncoding region of poliovirus RNA in both the replicative fitness of the virus and the full expression of viral neurovirulence determinants. The information gained from the proposed project should provide a rational scheme for the genetic modification of vaccine strains of picornaviruses by recombinant DNA methodology. Such noncoding region modifications have the potential advantage of reducing vaccine strain cytopathogenicity and instability without perturbing the virion structure that is critical for eliciting a fully- protective immune response.