MECHANISM OF RNA SYNTHESIS AND 5'-CAPPING BY DENGUE VIRUS NS5 POLYMERASE

包括登革热、西尼罗河病毒和寨卡病毒在内的黄病毒，作为新出现的疾病和潜在的生物恐怖制剂，构成了重大威胁。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。我们的长期目标是获得有关黄病毒复制复合体的详细结构和生化信息，并将这些信息用于开发抗病毒疗法和疫苗。黄病毒复制复合体由病毒编码的非结构蛋白（NS）、未识别的细胞蛋白和病毒RNA基因组组成，负责复制病毒基因组和5' RNA盖帽。然而，复制机制的各个组成部分如何作为一个功能复合体排列尚不清楚。具体来说，5' RNA capping机制在功能和物理上与复制机制相关联，作为多结构域（即NS5内的甲基转移酶（MTase）和RNA依赖性RNA聚合酶（RdRp））和更大的多蛋白复合物（即NS3和NS5），但关于RNA合成和RNA capping是如何协调的知之甚少。我们最近确定了全长登革热病毒（DENV） NS5的晶体结构，其中MTase和RdRp可以在单体和二聚体构型中协调各自的活性。在当前的应用中，我们将确定NS5如何与病毒RNA和NS3蛋白相互作用，这是病毒复制复合体中的另外两个重要组成部分。在Aim 1中，我们将探讨NS5如何与病毒RNA的5'端，称为茎环A （SLA）相互作用，以促进病毒RNA的合成。我们将通过低温电子显微镜确定SLA的结构，并通过竞争结合实验确定其与NS5的相互作用。在Aim 2中，我们将确定NS3和NS5结构域在协调RNA合成和使用感染性DENV RNA盖帽的5' RNA过程中的特定相互作用。在Aim 3中，我们将确定NS5及其RNA复合物的x射线晶体结构，包括SLA、dsRNA和同时结合MTase和RdRp结构域的“双重”底物，以代表病毒复制途径的不同步骤。结合结构、生化和病毒学研究将有助于阐明NS5中协调RNA合成和盖帽反应的机制。

Flaviviruses including dengue, West Nile, and Zika 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. Our long-term goal is to obtain detailed structural and biochemical information regarding the flavivirus replication complex and to use this information for the development of antiviral therapeutics and vaccines. The flavivirus replication complex, consisting of virally-encoded non-structural proteins (NS), unidentified cellular proteins, and the viral RNA genome, is responsible for copying the viral genome and 5' RNA capping. However, how the various components of the replication machinery are arranged as a functional complex is not known. Specifically, the 5' RNA capping machinery is functionally and physically linked to the replication machinery as multi-domain (i.e., methyltransferase (MTase) and RNA-dependent RNA polymerase (RdRp) within NS5) and larger multi- protein (i.e., NS3 and NS5) complexes, but little is known regarding how RNA synthesis and RNA capping are coordinated. We have recently determined the crystal structure of the full-length dengue virus (DENV) NS5, in which MTase and RdRp could coordinate their respective activities in both monomer and dimer configurations. In the current application, we will determine how NS5 interacts with viral RNA and the NS3 protein, two other essential components in the viral replication complex. In Aim 1, we will probe how NS5 interacts with the 5' end of viral RNA, called stem loop A (SLA), to promote viral RNA synthesis. We will determine the structure of the SLA by cryo-electron microscopy, and its interactions with NS5 by a competition binding assay. In Aim 2, we will determine specific interactions among NS3 and NS5 domains during coordination of RNA synthesis and 5' RNA capping using infectious DENV RNA. In Aim 3, we will determine the X-ray crystal structures of NS5 and its RNA complexes, including SLA, dsRNA, and a `dual' substrate that binds simultaneously to both MTase and RdRp domains to represent different steps along the viral replication pathway. The combined structural, biochemical, and virological studies will help elucidate the mechanism for coordinated RNA synthesis and capping reactions in NS5.