Picornavirus Genome Replication

描述（由申请人提供）： 将采用多学科协作方法研究脊髓灰质炎病毒基因组复制的第一步，即负链RNA合成。鉴于已经有大量关于脊髓灰质炎病毒分子生物学的信息，很容易认为关于脊髓灰质炎病毒基因组复制的一切都是已知的。事实上，这一过程的生动、丰富多彩、详细的模型出现在每一本病毒学教科书中。然而，最近发现脊髓灰质炎病毒负链RNA合成的起始发生在基因组的中心而不是3'端，这对以前的模型提出了质疑。此外，我们对脊髓灰质炎病毒基因组复制的研究揭示了关键复制蛋白之间存在一种新的相互作用，这种相互作用对于启动负链RNA合成至关重要，并挑战了以下教条：在感染细胞内积累的复制蛋白的加工形式确实与建立起始复合物所需的蛋白质形式相同。推动这些研究的中心假设如下。未加工形式的复制蛋白首先与位于基因组末端5'和3'端的顺式作用复制元件结合，折叠成核糖核蛋白复合物，其可以起到关闭基因组翻译的作用。前体蛋白与起始位点的结合将基因组的末端募集到中间。然后通过聚合酶将蛋白质引物尿苷酰化，然后由于其在该复合物中的预先存在，将两个核苷酸延伸的引物有效地转移到3'端。持续的合成导致全长负链RNA的产生。我们将开始通过追求以下具体目标来检验这一假设：（1）评价PV复制蛋白的未加工形式与顺式作用复制元件（克雷斯）：oriL、oril和oriR的相互作用;（2）确定P3蛋白与各种克雷斯之间相互作用的特异性的分子基础;（3）通过使用反向遗传学和经典遗传学方法的组合来定义用于将聚合酶募集到起始复合物的蛋白3C的表面;（4）开发用于研究负链RNA合成的起始的系统（即，VPg引发），其在生物学相关的时间尺度上起作用。脊髓灰质炎病毒仍然是小核糖核酸病毒家族中研究最深入和最广泛的成员之一。这个家族包括导致无数人类疾病的病毒，包括普通感冒，肝炎和慢性炎症性心脏病。这些研究将有助于我们更准确地了解小核糖核酸病毒基因组复制的分子机制。本研究中鉴定的蛋白质-RNA和蛋白质-蛋白质相互作用对基因组复制至关重要，代表了化疗干预的靶点。因此，这一信息可能被证明是有用的战略，以控制小核糖核酸病毒感染的疫苗是不可用的发展。

DESCRIPTION (provided by applicant): A collaborative, multidisciplinary approach will be applied to the study of the first step in poliovirus genome replication, negative-strand RNA synthesis. Given the wealth of information already available on the molecular biology of poliovirus, it is easy to assume that everything is known about poliovirus genome replication. Indeed, vivid, colorful, detailed models of this process appear in every virology textbook. However, the recent discovery that initiation of poliovirus negative-strand RNA synthesis occurs in the center of the genome rather than at the 3' end calls into question previous models. Moreover, our studies of poliovirus genome replication have uncovered the existence of a novel interaction between key replication proteins that is essential for initiation of negative-strand RNA synthesis and challenge the dogma that the processed forms of the replication proteins which accumulate within infected cells are, indeed, the same forms of the proteins required for establishment of the initiation complex. The central hypothesis driving these studies is as follows. Unprocessed forms of the replication proteins bind first to the cis-acting replication elements located at the extreme 5' and 3' ends of the genome, folding into a ribonucleoprotein complex that may function to turn off translation of the genome. Binding of precursor proteins to the internal site of initiation recruits the ends of the genome to the middle. The protein primer is then uridylylated by polymerase, and the two-nucleotides-extended primer is then efficiently transferred to the 3' end owing to its preexistence in this complex. Continued synthesis results in the production of full-length, negative-strand RNA. We will begin testing this hypothesis by pursuing the following specific aims: (1) Evaluate the interaction of unprocessed forms of PV replication proteins with the cis-acting replication elements (CREs): oriL, oril and oriR; (2) Define the molecular basis for the specificity of the interaction between P3 proteins and the various CREs; (3) Define the surface of protein 3C used to recruit polymerase to the initiation complex by using a combination of reverse-genetic and classical genetic approaches; (4) Develop a system to study initiation of negative-strand RNA synthesis (i.e., VPg priming) in vitro that functions on a biologically relevant time scale. Poliovirus remains one of the most intensively and extensively studied members of the Picornavirus family. This family includes viruses that cause myriad human diseases, including the common cold, hepatitis and chronic, inflammatory heart disease. These studies will provide a more precise understanding of the molecular mechanism of picornavirus genome replication. Protein-RNA and protein-protein interactions identified in this study that are critical for genome replication represent targets for chemotherapeutic intervention. Therefore, this information may prove useful in the development of strategies to control picornavirus infections for which vaccines are not available.