Picornavirus Genome Replication

描述(申请人提供)：这是一项研究微小核糖核酸病毒基因组复制的拨款续期申请。小核糖核酸病毒对美国公共卫生构成现有的和新出现的威胁。虽然微小核糖核酸病毒基因组复制所需的蛋白质因子和遗传元件是已知的，并且似乎是保守的，但对产生微小核糖核酸病毒的机制缺乏清楚的了解。该计划的长期目标是在体外从纯化的成分中重建微小核糖核酸病毒的基因组复制。我们已经实现了前一个供资期间的所有主要目标。此外，我们还解决了微小核糖核酸病毒3CD蛋白的第一个晶体结构，发展了利用核磁共振光谱研究3C-RNA相互作用的技术，并发现3CD蛋白具有复制前和复制后的功能。在接下来的资助期间，我们将继续我们对微小核糖核酸病毒基因组复制的研究，并探索我们新发现的3CD的功能，具体目标如下：(1)利用分子遗传学、生化和生物物理方法确定微小核糖核酸病毒VPG尿苷基化复合体的组装和结构组织机制；(2)利用核磁共振光谱确定3C识别序列和结构特异性RNA的分子基础；以及(3)阐明3CD在复制复合体形成中的作用。 公共卫生相关性：微小核糖核酸病毒对美国公共卫生构成现有的和新出现的威胁。该应用的目标的实现将为开发治疗微小核糖核酸病毒感染的抑制剂提供新的靶点和机制，特别是那些没有疫苗可用的感染。

DESCRIPTION (provided by applicant): This is an application for renewal of a grant to study picornavirus genome replication. Picornaviruses represent an existing and emerging threat to US public health. Although protein factors and genetic elements required for picornavirus genome replication are known and appear to be conserved, a clear understanding of the mechanisms employed to produce picornaviral RNA is lacking. The long-term objective of this program is to reconstitute picornavirus genome replication in vitro from purified components. We have achieved all of the major objectives of the previous funding period. In addition, we have solved the first crystal structure for a picornaviral 3CD protein, developed the technology to study 3C-RNA interactions by using nuclear magnetic resonance spectroscopy, and discovered that the 3CD protein has both pre- and post-replication functions. During the next funding period, we will continue our studies of picornavirus genome replication as well as explore our newly discovered function for 3CD by pursuing the following specific aims: (1) Define the mechanism of assembly and structural organization of the picornavirus VPg uridylylation complex by using molecular genetic, biochemical and biophysical approaches; (2) Define the molecular basis for sequence- and structure-specific RNA recognition by 3C by using nuclear magnetic resonance spectroscopy; and (3) Elucidate the function of 3CD in formation of replication complexes. PUBLIC HEALTH RELEVANCE: Picornaviruses represent an existing and emerging threat to US public health. Achievement of the goals of the application will provide novel targets and mechanisms for development of inhibitors to treat infections by picornaviruses, especially those for which vaccines are not available.