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

黄病毒包括登革热、西尼罗河病毒和寨卡病毒，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。我们的长期目标是获得关于黄病毒复制复合物的详细结构和生化信息，并将这些信息用于抗病毒治疗和疫苗的开发。黄病毒复制复合物由病毒编码的非结构蛋白（NS）、未鉴定的细胞蛋白和病毒RNA基因组组成，负责复制病毒基因组和5' RNA加帽。然而，复制机器的各个组成部分是如何排列成一个功能复合体的，我们还不知道。具体地，5' RNA加帽机构作为多结构域（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（即，NS3和NS5）复合物，但关于RNA合成和RNA加帽是如何协调的知之甚少。我们最近确定了全长登革病毒（DENV）NS5的晶体结构，其中MTase和RdRp可以协调其各自的活动在单体和二聚体配置。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，这是病毒复制复合物中的另外两个重要组分。在目的1中，我们将探索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合成。我们将通过冷冻电子显微镜确定SLA的结构，并通过竞争结合试验确定其与NS5的相互作用。在目的2中，我们将确定在RNA合成和使用感染性DENV RNA的5' RNA加帽的协调期间NS3和NS5结构域之间的特异性相互作用。在目标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和同时与MTase和RdRp结构域结合以代表病毒复制途径沿着的不同步骤的“双重”底物。结合结构，生物化学和病毒学的研究将有助于阐明协调的RNA合成和加帽反应在NS5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.