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制剂构成重大威胁。尽管黄病毒感染对全世界的健康有相当大的影响，但没有抗病毒治疗方法，现有的黄病毒疫苗的效用有限。该项目的长期目标是基于黄病毒复制酶复合体的结构和生化信息开发抗病毒治疗方法。由于复制酶复合体是病毒复制所绝对需要的，因此了解它们的结构和机制应该能够确定治疗的潜在靶点，并提出针对这些传染病威胁的干预策略。黄病毒复制酶复合体由病毒编码的RNA聚合酶和解旋酶以及其他病毒和未识别的细胞蛋白组成，负责复制病毒基因组。在复制过程中，新生链的5'-RNA盖帽与RNA合成同时发生。病毒NS5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capping过程可能是耦合的。由于缺乏全长聚合酶的详细结构信息，在复制过程中这两种活性是否或如何协调尚不清楚。在Aim 1中，登革热病毒NS5中RdRp和MTase结构域的相互作用以及在聚合酶和甲基化反应中发生的构象变化将通过溶液中的小角度x射线散射来确定（Aim 1a）。由此产生的NS5模型将在重组蛋白和感染性病毒中使用定点诱变（Aim 1b）进行测试。在Aim 2中，全长NS5聚合酶与RNA的相互作用将被研究。一种可以同时结合RdRp和MTase结构域的“双重”底物将被设计并测试其与全长NS5的结合。在Aim 3中，将确定全长登革热NS5聚合酶及其RNA复合物在催化途径中不同步骤的x射线晶体结构。结合结构、生化和病毒学研究将有助于阐明RNA合成的机制及其与NS5聚合酶capping反应的潜在协调。

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.