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

描述(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，作为新出现的疾病和潜在的生物恐怖因子，构成重大威胁。尽管黄病毒感染对全球健康有相当大的影响，但没有抗病毒疗法可用，现有的黄病毒疫苗效用有限。该项目的长期目标是基于有关黄病毒复制酶复合体的结构和生化信息开发抗病毒疗法。由于复制酶复合体对于病毒复制是绝对必要的，对其结构和机制的了解应该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。黄病毒复制酶复合体由病毒编码的RNA聚合酶和解旋酶以及其他病毒和不明原因的细胞蛋白组成，负责复制病毒基因组。在复制过程中，新生链的5‘-RNA封顶伴随着RNA的合成。病毒NS5聚合酶在一个多肽中同时具有依赖RNA的RNA聚合酶(RdRp)和5‘-RNA甲基转移酶(MTase)活性，这表明RNA合成和封端过程可能是耦合的。关于这两种活动在复制过程中是否或如何协调，人们知之甚少，这主要是因为缺乏关于全长聚合酶的详细结构信息。在目标1中，登革病毒NS5内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)。由此产生的NS5模型将在重组蛋白中进行测试，并在感染病毒中使用定点突变(AIM 1b)进行测试。在目标2中，将研究全长NS5聚合酶与RNA的相互作用。一种可以同时与RdRp和MTase结构域结合的‘双重’底物将被设计并测试其与全长NS5的结合。在目标3中，将确定登革热NS5聚合酶全长及其RNA复合体在催化途径不同步骤的X射线晶体结构。结构、生化和病毒学的联合研究将有助于阐明RNA合成的机制及其与NS5聚合酶中的封端反应的潜在协调。

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.