Picornavirus Genome Replication

描述(由申请人提供)： 将采用协作、多学科的方法研究脊髓灰质炎病毒基因组复制的第一步--负链RNA合成。鉴于现有的关于脊髓灰质炎病毒分子生物学的丰富信息，很容易假设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合成发生在基因组的中心，而不是在基因组的3‘端，这一发现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，并挑战了这样一种教条，即在感染细胞内积累的复制蛋白的加工形式确实与建立启动复合体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体，该复合体可能具有关闭基因组翻译的功能。前体蛋白结合到起始的内部位置，将基因组的末端招募到中间。然后，蛋白质引物被聚合酶尿苷基化，然后，由于它预先存在于该复合体中，两个核苷酸延伸的引物被有效地转移到3‘端。持续的合成导致了全长负链RNA的产生。我们将通过追求以下特定目标来开始检验这一假说：(1)评估未加工形式的PV复制蛋白与顺式作用复制元件(CRs)：oriL、orIL和oriR的相互作用；(2)确定P3蛋白与各种CRs相互作用的特异性的分子基础；(3)结合反向遗传和经典遗传学方法，定义用于招募聚合酶到起始复合体的蛋白3C的表面；(4)建立体外研究负链RNA合成启动(即VPG启动)的系统，该系统在生物学相关的时间尺度上发挥作用。脊髓灰质炎病毒仍然是皮科纳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。这些研究将对微小核糖核酸病毒基因组复制的分子机制提供更准确的理解。这项研究中确定的对基因组复制至关重要的蛋白质-RNA和蛋白质-蛋白质相互作用是化疗干预的靶点。因此，这些信息可能会被证明有助于制定控制微小核糖核酸病毒感染的战略，而这些病毒感染是无法获得疫苗的。

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.