POLIOVIRUS USING INFECTIOUS VIRAL CDNA CLONE

包围许多动物病毒的蛋白质衣壳在 在病毒感染中的作用。在遇到目标细胞时，衣壳 必须与细胞表面受体相互作用并释放病毒核 酸性物质进入细胞。在受感染的细胞中，新合成的病毒粒子 蛋白质包裹病毒基因组，并在病毒传播到 新的细胞目标。因此，病毒衣壳必须稳定到 细胞外环境，但足够灵活，可以释放核物质 酸性物质进入细胞。脊髓灰质炎病毒是一个特别好的研究模型。 衣壳是如何调节这些功能的。立体结构 已经通过X射线结晶学确定了脊髓灰质炎病毒的数量，细胞 脊髓灰质炎病毒的受体已经被确定，基因操作 这种病毒可能存在于病毒基因组的感染性cDNA拷贝中。 该方案中的实验使用这些工具来理解 脊髓灰质炎病毒衣壳控制感染的早期事件，包括受体 结合和受体介导的构象转变，两者都在 培养的细胞和动物模型。为了实现这一目标，四个 提出了具体的目标。(1)衣壳序列的鉴定 控制脊髓灰质炎病毒与其细胞受体的相互作用。脊髓灰质炎病毒 抗可溶性受体中和突变体(SRR突变体) 已分离出降低与HeLa细胞结合亲和力的基因。 将对这些突变进行研究，以确定控制病毒的衣壳残基 脊髓灰质炎病毒结合，过渡到改变的颗粒，并揭开涂层。这个 结果可能会对可溶性受体的使用产生影响，如 抗病毒化合物。(2)SRR表型的功能基础。一个 重组的脊髓灰质炎病毒可溶型受体将在 细胞和纯化，以及它与SRR突变体的相互作用将被研究 以了解他们抵抗中和的基础。(3) 脊髓灰质炎病毒宿主范围分析。脊髓灰质炎病毒在小鼠体内的宿主限制 可通过改变VP1B-C环或N末端来克服。 计划进行实验，以解决这样的假设：这些序列 通过控制病毒粒子的能力来确定宿主范围 构象转换，从而调节受体的相互作用。(4) VP1B-C环和N端的功能。两个品种的表型 脊髓灰质炎病毒VP1B-C环氨基酸变化的突变体 N-末端，被认为控制构象转变的结构 将对病毒进行研究，以确定这些序列在 培养细胞中的病毒复制。这些实验的结果 将有助于这项研究的长期目标，即 提供有关病毒复制和发病机制的完整说明 人类病毒病原体。

The protein capsid that surrounds many animal viruses plays an important role in viral infections. Upon encountering a target cell, the capsid must interact with a cell surface receptor and release the viral nucleic acid into the cell. In the infected cell, newly synthesized virion proteins encapsidate the viral genome and protect it as it travels to a new cell target. The viral capsid must therefore be stable to the extracellular environment, yet flexible enough to discharge the nucleic acid into the cell. Poliovirus is a particularly good model for studying how the capsid mediates these functions. The three-dimensional structure of poliovirus has been determined by X-ray crystallography, the cell receptor for poliovirus has been identified, and genetic manipulation of the virus is possible with infectious cDNA copies of the viral genome. Experiments in this proposal use these tools to understand how the poliovirus capsid controls early events in infection, including receptor binding and receptor-mediated conformational transitions, both in cultured cells and in an animal model. To achieve this goal, four specific aims are proposed. (1) Identification of capsid sequences that control the interaction of poliovirus with its cell receptor. Poliovirus mutants resistant to neutralization with soluble receptor (srr mutants) have been isolated which have reduced binding affinity for HeLa cells. These mutants will be studied to identify capsid residues that control poliovirus binding, transition to altered particles, and uncoating. The results may have implications for the use of soluble receptors as antiviral compounds. (2) Functional basis for the srr phenotype. A recombinant soluble form of the poliovirus receptor will be expressed in cells and purified, and its interaction with srr mutants will be studied to understand the basis for their resistance to neutralization. (3) Analysis of poliovirus host range. Poliovirus host restriction in mice can be overcome by changes in the VP1 B-C loop or the N-terminus. Experiments are planned to address the hypothesis that these sequences determine host range by controlling the ability of the virion to undergo conformation transitions, thereby regulating receptor interactions. (4) Functions of the VP1 B-C loop and the N-terminus. The phenotypes of two poliovirus mutants with amino acid changes in the VP1 B-C loop and N-terminus, structures believed to control conformational transitions of the virus, will be studied to determine the role of these sequences in viral replication in cultured cells. The results of these experiments will contribute to the long term goal of this research, which is to provide a complete description of the replication and pathogenesis of a human viral pathogen.