Molecular Mechanisms in Reovirus mRNA Synthesis

描述（由申请人提供）： 哺乳动物正呼肠孤病毒（呼肠孤病毒）和其它dsRNA病毒介导 使用包装在细胞内的病毒编码的酶合成它们的mRNA分子， 传染性病毒颗粒是一个积极研究的主题，因为它们承诺 深入了解mRNA合成的步骤-正链RNA合成 （转录）、RNA 5'加帽和RNA转运-发生在限定的区域内。 二十面体病毒颗粒的三维设置。更好的 对dsRNA病毒中这些机制的理解应该有助于 了解相关过程是如何通过类似的其他病毒介导的， 微生物或细胞酶，并用于设计新的抗病毒，抗微生物， 或针对它们的抗细胞剂。我们工作的长期目标 与呼肠孤病毒是定义结构功能关系的作用， 呼肠孤病毒蛋白在病毒复制中的作用以及对宿主细胞和动物的影响。 在目前的建议中，重点放在呼肠孤病毒内的蛋白质上 在体外介导mRNA合成的核心（亚病毒）颗粒。三个具体 确定了目标，这些目标反映了最有可能取得进一步进展的领域 有希望或有必要在这个系统中促进理解。这些目的是 (1)以确定呼肠孤病毒转录酶复合物的结构， 它们在核心中的排列，（2）定义呼肠孤病毒的组装途径 核心壳，和（3）剖析呼肠孤病毒核心蛋白在 RNA合成、加帽和转运。呼肠孤病毒颗粒复溶自 使用杆状病毒载体表达的重组蛋白质发挥显著作用 在拟议的实验中，由于其广泛的适用性， 粒子结构、组装和功能。最近确定的晶体 具有转录和加帽能力的呼肠孤病毒核心颗粒的结构 呼肠孤病毒RNA依赖性RNA聚合酶也在这一过程中起重要作用。 通过将重点放在需要更多结构的特定领域， 信息是必要的，提出了关于mRNA步骤的具体新假设。 合成和组装，并确定了核心中的特定氨基酸 对蛋白质进行诱变以进行结构-功能测试。结果 这些研究将增强我们对呼肠孤病毒核心的理解， 设计精巧的mRNA合成分子机器。

DESCRIPTION (provided by applicant): The molecular mechanisms by which mammalian orthoreoviruses (reoviruses) and other dsRNA viruses mediate synthesis of their mRNA molecules using virally encoded enzymes packaged within infectious virus particles is a subject of active inquiry because they promise insight into how the steps in mRNA synthesis - plus-strand RNA synthesis (transcription), RNA 5' capping, and RNA transport - occur within the delimited three-dimensional setting of the icosahedral virus particle. A better understanding of these mechanisms in dsRNA viruses should be useful for understanding how related processes are mediated by analogous other viral, microbial, or cellular enzymes and for designing new antiviral, antimicrobial, or anticellular agents directed at them. The long-term objective of our work with reoviruses is to define structure-function relationships for the roles of reovirus proteins in viral replication and effects on host cells and animals. In the current proposal, emphasis is placed on the proteins within the reovirus core (subviral) particle that mediates mRNA synthesis in vitro. Three specific aims are identified, which reflect where further progress appears most promising or necessary to advance understanding in this system. These aims are (1) to determine the structure of the reovirus transcriptase complexes and their arrangement in cores, (2) to define the assembly pathway of the reovirus core shell, and (3) to dissect the functions of the reovirus core proteins in RNA synthesis, capping, and transport. Reovirus particles reconstituted from recombinant proteins expressed using baculovirus vectors play a prominent role in the proposed experiments because of their broad applicability to studies of particle structure, assembly, and functions. Recently determined crystal structures of the transcription- and capping-competent reovirus core particle and the reovirus RNA-dependent RNA polymerase also figure heavily in this proposal by having focused attention on particular areas where more structure information is needed, suggested specific new hypotheses about steps in mRNA synthesis and assembly, and identified specific amino acids in the core proteins to subject to mutagenesis for structure-function testing. The results of these studies will enhance our understanding of the reovirus core as an elegantly designed molecular machine for mRNA synthesis.