STUDIES ON POLIOVIRUS USING INFECTIOUS VIRAL CDNA CLONES

本提案中实验的目的是使用一种 遗传学和分子生物学相结合的方法 研究脊髓灰质炎病毒复制和发病机制。这位将军 方法是使用具有感染性的脊髓灰质炎病毒cdna克隆。 基因组，以分离确定的病毒突变体和重组体，并 研究这些病毒在培养细胞和 动物。其中一个部分的实验旨在确定 基因组5‘-上游区域的功能，特别是 对RNA合成很重要的茎和环结构，如 以及七个上游的Aug密码子，它们在病毒复制中发挥作用 都是未知的。计划进行实验来解释这种表型。 一种病毒突变体，在受感染的细胞中，导致排列紊乱 翻译控制，例如可能关联的 与eIF2α的磷酸化有关。另一系列 实验采用了脊髓灰质炎的小鼠模型来识别 并研究病毒对疾病的产生至关重要的功能。我们的 以往的研究表明，P2/Lansing感染的能力 小鼠映射到病毒衣壳蛋白和病毒重组体 将在该菌株和老鼠无毒菌株之间构建 为了定义鼠标所需衣壳的精确区域- 适应的表型。该属性的功能基础将是 还将探讨：对小鼠的适应是否涉及选择 识别小鼠中枢神经系统受体的病毒变体 神经系统？的三维结构 P2/Lansing Capsid将确定为 病毒结构与病毒结构关系的研究进展 神经毒性。有关衣壳区域的其他信息 对于小鼠适应的表型来说，重要的是来自 致病基因突变的鉴定 耐中和的P2/Lansing变异体 抗体。最后，我们的研究表明，单一的碱基 病毒RNA 5‘-上游区域的变化，已知是 P3/Sabin表型减弱的部分原因 疫苗，也大大降低了P2/Lansing的神经毒力 老鼠。已经鉴定出一种细胞系，它复制了 这种突变的影响，并将被用于阐明病毒 因突变而改变的功能。这些研究的结果 将为我们的长远目标作出贡献，提供一个完整 对一种病毒的复制和致病机制的描述 会导致人类疾病。

The aim of experiments in this proposal is to employ a combination of genetic and molecular biological approaches for studying poliovirus replication and pathogenesis. The general approach is to use infectious cDNA clones of the poliovirus genome to isolate defined viral mutants and recombinants, and to study the replication of these viruses in cultured cells and in animals. Experiments in one section are aimed at determining the function of the 5'-upstream region of the genome, in particular a stem and loop structure that is important for RNA synthesis as well as seven upstream AUG codons whose role in viral replication are not known. Experiments are planned to explain the phenotype of a viral mutant which, in infected cells, causes a derangement of translational control such as that which might be associated with phosphorylation of eIF2 alpha. Another series of experiments employ a mouse model for poliomyelitis to identify and study viral functions essential for production of disease. Our previous studies showed that the ability of P2/Lansing to infect mice maps to the viral capsid proteins, and viral recombinants between this strain and mouse-avirulent strain will be constructed to define the precise regions of the capsid required for the mouse- adapted phenotype. The functional basis for this property will also be explored: does adaptation to mice involve selection of viral variants that recognize receptors in the mouse central nervous system? The three-dimensional structure of the P2/Lansing capsid will be determined to provide a framework for studies on the relationship between viral structure and neurovirulence. Additional information on regions of the capsid important for the mouse-adapted phenotype will come from identification of the mutations responsible for the attenuation of P2/Lansing variants resistant to neutralization with monoclonal antibodies. Finally, our studies have shown that a single base change in the 5'-up-stream region of the viral RNA, known to be partly responsible for the attenuated phenotype of the P3/Sabin vaccine, also drastically reduces P2/Lansing neurovirulence in mice. A cell line has been identified that the duplicates the effect of this mutation and will be used to elucidate the viral functions altered by the mutation. The results of these studies will contribute to our long term objective, to provide a complete description of the replication and pathogenesis of a virus that causes human disease.