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 用重组体和突变体分析细胞的功能结构域 脊髓灰质炎病毒RNA的5‘-非编码区。我们将确定 人乳头瘤病毒5‘-非翻译区基因改变的影响 脊髓灰质炎基因组的RNA复制和核糖体结合/病毒- 特定的蛋白质合成。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 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‘端非编码的cDNA 然后将嵌合体和突变体连接到编码区 来自脊髓灰质炎病毒RNA的感染性cDNA克隆。质粒 包含完整的重组/突变基因组的将是 通过对培养的灵长类细胞进行转基因来检测传染性。 传染性重组和突变病毒将被回收和 分析(I)的改变或温度敏感表达 RNA复制功能，(Ii)病毒蛋白质合成功能， 以及(Iii)主单元关闭功能。最后，任何一个 在体内不产生传染性病毒的重组质粒 将分析体外翻译的转染法。 使用来自噬菌体的体外转录本的效率 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 病毒基因组中的非编码核苷酸在调节 表达脊髓灰质炎基因产物的单一翻译单位。 拟议项目与健康相关的基础是 先前的研究表明，该基因的5‘-非编码区 脊髓灰质炎病毒RNA在病毒复制适合性和 病毒神经毒力决定因素的全面表达。这个 从拟议项目中获得的信息应提供 猪瘟疫苗株基因改造的合理方案 用重组DNA方法检测小核糖核酸病毒。这样的非编码 区域修改的潜在优势是减少 无菌疫苗株细胞致病性和不稳定性 扰乱病毒粒子的结构，而病毒粒子结构对于引发完全- 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.