Molecular Mechanisms in Reovirus mRNA Synthesis

描述(由申请人提供)：分子机制 哺乳动物呼肠孤病毒(呼肠孤病毒)和其他dsRNA病毒 用包装在其中的病毒编码的酶合成它们的mRNA分子 传染性病毒颗粒是积极研究的对象，因为它们承诺 深入了解信使核糖核酸合成的步骤--加链核糖核酸 (转录)、RNA 5‘端封端和RNA运输-发生在分隔的 二十面体病毒颗粒的三维设置。更好的 了解dsRNA病毒的这些机制将有助于 了解相关过程是如何由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、 或者是针对他们的抗细胞药物。我们工作的长期目标 与呼肠孤病毒的关系是为下列角色定义结构功能关系 呼肠孤病毒在病毒复制中的蛋白及其对宿主细胞和动物的影响。 在目前的提案中，重点放在呼肠孤病毒内的蛋白质上。 在体外介导信使核糖核酸合成的核心(亚病毒)颗粒。三个具体的 确定了目标，这些目标反映了最有可能取得进一步进展的地方 有希望的或有必要在这一系统中促进理解的这些目标是 (1)确定呼肠孤病毒转录酶复合体的结构和 它们在核心中的排列，(2)确定呼肠孤病毒的组装途径 核壳，以及(3)剖析呼肠孤病毒核心蛋白的功能。 核糖核酸的合成、封端和运输。重组的呼肠孤病毒颗粒 利用杆状病毒载体表达的重组蛋白起着突出的作用 在拟议的实验中，因为它们广泛适用于 粒子的结构、组装和功能。新近确定的晶体 具有转录和封端功能的呼肠孤病毒核心颗粒的结构 而依赖于呼肠孤病毒rna的rna聚合酶也在这一过程中发挥了重要作用。 通过将注意力集中在结构更多的特定领域来提出建议 需要信息，提出关于信使核糖核酸步骤的具体新假设 合成和组装，并鉴定了核心中的特定氨基酸 为进行结构功能测试而进行突变的蛋白质。结果是 这些研究将加强我们对呼肠孤病毒核心作为一种 设计精美的信使核糖核酸合成分子机器。

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.