Mechanism of RNA synthesis and 5'-capping by dengue virus NS5 polymerase

描述（由申请人提供）：黄病毒，包括登革热、西尼罗河、黄热病和日本脑炎病毒，作为新兴疾病和潜在的生物恐怖剂构成重大威胁。尽管黄病毒感染对世界范围的健康有相当大的影响，但没有抗病毒疗法可用，并且现有的黄病毒疫苗的效用有限。该项目的长期目标是根据黄病毒复制酶复合物的结构和生化信息开发抗病毒治疗药物。由于复制酶复合物是病毒复制所必需的，因此对其结构和机制的了解应确定潜在的治疗靶点，并提出针对这些传染病威胁的干预策略。黄病毒复制酶复合物由病毒编码的RNA聚合酶和解旋酶以及其他病毒和未鉴定的细胞蛋白组成，负责复制病毒基因组。在复制过程中，新生链的5 '-RNA加帽沿着RNA合成。病毒NS 5聚合酶在单个多肽内具有RNA依赖性RNA聚合酶（RdRp）和5 '-RNA甲基转移酶（MTase）活性，表明RNA合成和加帽过程可能是偶联的。很少有人知道这两种活动是否或如何在复制过程中协调，主要是由于缺乏有关全长聚合酶的详细结构信息。在目标1中，登革热病毒NS 5内的RdRp和MTase结构域的相互作用以及在聚合酶和甲基化反应期间在其中发生的构象变化将通过溶液中的小角X射线散射来确定（目标1a）。将使用定点诱变在重组蛋白和感染性病毒中检测所得NS 5模型（Aim 1b）。在目标2中，将研究全长NS 5聚合酶与RNA的相互作用。将设计可同时结合RdRp和MTase结构域的“双重”底物并测试其与全长NS 5的结合。在目标3中，将确定全长登革NS 5聚合酶及其RNA复合物在沿着催化途径的沿着不同步骤的X射线晶体结构。结合结构，生物化学和病毒学研究将有助于阐明RNA合成的机制及其与NS 5聚合酶中的加帽反应的潜在协调。 公共卫生相关性：黄病毒，如登革热病毒，作为新兴疾病和潜在的生物恐怖制剂构成重大威胁。我们将对进行病毒基因组复制过程的登革病毒聚合酶进行综合结构、生物化学和病毒学研究。我们的研究结果将为开发抗病毒疗法奠定基础，以治疗这些目前无法治疗且往往致命的传染病。

DESCRIPTION (provided by applicant): Flaviviruses including dengue, West Nile, yellow fever, and Japanese encephalitis viruses, pose significant threats as emerging diseases and potential bioterror agents. Despite the considerable impact of flavivirus infection on world-wide health, no antiviral therapies are available, and existing flavivirus vaccines are of limited utility. The long-term goal of this project is to develop antiviral therapeutics based on structural and biochemical information regarding the flavivirus replicase complex. Since replicase complexes are absolutely required for virus replication, knowledge of their structures and mechanisms should identify potential targets for therapeutics and suggest strategies for intervention against threats from these infectious diseases. The flavivirus replicase complex, consisting of a virally-encoded RNA polymerase and helicase as well as other viral and unidentified cellular proteins, is responsible for copying the viral genome. During replication, 5'-RNA capping of the nascent strand occurs along with RNA synthesis. The viral NS5 polymerase has both RNA-dependent RNA polymerase (RdRp) and 5'-RNA methyltransferase (MTase) activities within a single polypeptide, indicating that the RNA synthesis and capping processes may be coupled. Little is known as to whether or how the two activities are coordinated during replication, largely due to a lack of detailed structural information about the full-length polymerase. In Aim 1, the interactions of the RdRp and MTase domains within dengue virus NS5 and the conformational changes that occur therein during the polymerase and methylation reactions, will be determined by small-angle X-ray scattering in solution (Aim 1a). The resulting NS5 model will be tested in recombinant protein and in the infectious virus using site-directed mutagenesis (Aim 1b). In Aim 2, interactions of the full-length NS5 polymerase with RNA will be investigated. A 'dual' substrate that can bind simultaneously to both the RdRp and MTase domains will be designed and tested for its binding to the full- length NS5. In Aim 3, the X-ray crystal structures of the full-length dengue NS5 polymerase and its RNA complexes at different steps along the catalytic pathway will be determined. The combined structural, biochemical, and virological studies will help elucidate the mechanisms for RNA synthesis and its potential coordination with capping reactions in the NS5 polymerase. PUBLIC HEALTH RELEVANCE: Flaviviruses such as dengue virus pose significant threats as emerging diseases and potential bioterror agents. We will perform integrated structural, biochemical, and virological studies on the dengue virus polymerase that carries out viral genome replication processes. The results of our studies will lay the foundation for developing antiviral therapeutics to treat these currently untreatable and often deadly infectious diseases.