POLIOVIRUS 3D--COOPERATIVE RNA BINDING & POLYMERIZATION

描述：除逆转录病毒外，所有RNA病毒都编码一种RNA依赖性 RNA聚合酶来扩增它们的基因组。 脊髓灰质炎病毒的研究 RNA依赖的RNA聚合酶最近已成为一个基于结构的问题 3Dpol的三维结构的解决方案。 的 脊髓灰质炎病毒聚合酶与来自脊髓灰质炎病毒的RNA依赖性RNA聚合酶同源。 其他小核糖核酸病毒，如鼻病毒和埃可病毒， 没有疫苗。 脊髓灰质炎病毒的几个有趣特征 聚合酶生物化学需要进一步研究， 详细的结构和功能的了解机制， RNA依赖的RNA聚合和开发药物靶点 破坏 申请人已经表明，RNA结合和RNA 聚合是相对于聚合酶浓度协同的。 到 定义结合的RNA底物的路径，聚合酶的同源性， HIV逆转录酶和U1 snRNP结合蛋白将用于指导 定点诱变实验。 突变体的结合亲和力 将测试RNA聚合酶以区分对内在RNA的影响 结合亲和力来自对蛋白质-蛋白质相互作用的影响， 产生协同RNA结合。 聚合酶-聚合酶相互作用， 可能参与合作的RNA结合被认为是在三维 结构，其显示聚合酶分子的头-尾寡聚化 在晶体内的长纤维中。 凝胶移位RNA结合实验， 野生型和突变型聚合酶，潜在的定点诱变 蛋白质-蛋白质界面和原子力显微镜将用于 确定与RNA底物结合的RNA聚合酶的化学计量。 特异性影响内在RNA结合和蛋白质-蛋白质的突变 将测试相互作用对脊髓灰质炎病毒复制的影响。 在感染的细胞和无细胞提取物中都有。 肽抑制剂 聚合酶-聚合酶的相互作用将寻求使用酵母 双杂交系统，并测试它们对协同RNA结合的影响， 脊髓灰质炎病毒在无细胞提取物中的复制周期。 的抑制剂 多蛋白复合物内的相互作用具有理论优势 对这些抑制剂产生抗性的突变应该是隐性的， 或仅部分占优势。 他们应该是无效的选择，即使在 RNA病毒，以其众所周知的高突变率。

DESCRIPTION: All RNA viruses besides retroviruses encode an RNA-dependent RNA polymerase to amplify their genomes. Study of the poliovirus RNA-dependent RNA polymerase has recently become a structure-based problem with the solution of the three-dimensional structure of 3Dpol. The poliovirus polymerase is homologous to RNA-dependent RNA polymerases from other picornaviruses such as rhinoviruses and echoviruses for which good vaccines are not available. Several interesting features of poliovirus polymerase biochemistry demand further investigation, both to acquire detailed structural and functional understanding of the mechanism of RNA-dependent RNA polymerization and to develop targets for pharmaceutical disruption. The applicant has shown that both RNA binding and RNA polymerization are cooperative with respect to polymerase concentration. To define the path of bound RNA substrates, the homology of the polymerase with HIV reverse transcriptase and U1 snRNP binding protein will be used to guide site-directed mutagenesis experiments. Binding affinities of mutant polymerases for RNA will be tested to distinguish effects on intrinsic RNA binding affinity from effects on the protein-protein interactions that give rise to cooperative RNA binding. Polymerase-polymerase interactions that may be involved in cooperative RNA binding are seen in the three-dimensional structure, which shows head-to-tail oligomerization of polymerase molecules in long fibers within the crystal. Gel-shift RNA binding experiments with wild type and mutant polymerases, site-directed mutagenesis of potential protein-protein interfaces and atomic force microscopy will be used to determine the stoichiometry of RNA polymerase bound to RNA substrates. Mutations that specifically affect intrinsic RNA binding and protein-protein interactions will be tested for their effects on the poliovirus replicative cycle both in infected cells and in cell-free extracts. Peptide inhibitors of polymerase-polymerase interactions will be sought using the yeast two-hybid system and tested for their effects on cooperative RNA binding and the poliovirus replicative cycle in cell-free extracts. Inhibitors of interactions within multiprotein complexes have the theoretical advantage that mutations that confer resistance to such inhibitors should be recessive or only partially dominant. They should be inefficiently selected even in RNA viruses, with their notoriously high mutation rate.