Filovirus replication: initiation mechanism and role of RNA secondary structures

描述（由申请人提供）：埃博拉病毒（EBOV）属于非节段负义（NNS） RNA病毒群，对人类具有高致病性。目前，没有批准的治疗或预防EBOV出血热的药物。埃博拉病毒使用依赖RNA的RNA聚合酶来复制和转录其基因组。关于EBOV聚合酶与RNA模板相互作用并启动复制的分子机制知之甚少。病毒聚合酶是抗病毒药物开发的常见靶点，针对病毒聚合酶的抗病毒化合物的合理设计需要对基因组复制的分子事件有透彻的了解。在这里，我们建议剖析EBOV启动复制的分子机制。我们的初步数据表明，EBOV已经进化出一种不同于其他NNS RNA病毒的复制启动机制。根据我们的模型，EBOV在模板链的+2位置启动复制，并使用启动子区域采用的RNA二级结构来维持其基因组末端。为了测试该模型，我们将使用从感染ebov的细胞或病毒粒子中分离的RNA来确定复制产物的3‘和5’端。通过绘制细胞内和病毒粒子相关病毒RNA的末端核苷酸，我们将确定复制起始位点。我们将进一步利用EBOV小基因组系统在相关区域引入突变，并分析对复制起始和基因组完整性的影响。我们将使用扎伊尔埃博拉病毒原型进行初步实验。然后，我们将分析ZEBOV所使用的复制机制是否在其他EBOV物种中保守。最后，我们将确定EBOV启动子区域采用的RNA二级结构在启动子功能和复制起始中的作用。总的来说，拟议的工作将阐明EBOV聚合酶是否获得了与病毒基因组相互作用的独特能力，或者是否遵循与其他NNS RNA病毒共享的一般机制。

DESCRIPTION (provided by applicant): Ebolaviruses (EBOV) belong to the group of nonsegmented negative-sense (NNS) RNA viruses and are highly pathogenic in humans. Currently, there are no approved therapeutics to treat or prevent EBOV hemorrhagic fever. Ebolaviruses use an RNA-dependent RNA polymerase to replicate and transcribe their genomes. Little is known about the molecular mechanisms employed by the EBOV polymerase to interact with the RNA template and initiate replication. Viral polymerases are common targets for antiviral drug development, and rational design of antiviral compounds targeting viral polymerases requires a thorough understanding of the molecular events involved in genome replication. Here, we propose to dissect the molecular mechanisms used by EBOV to initiate replication. Our preliminary data suggest that EBOV has evolved a replication initiation mechanism that is different from those used by other NNS RNA viruses. According to our model, EBOV initiates replication at position +2 of the template strand and uses an RNA secondary structure adopted by the promoter region to maintain its genome ends. To test this model, we will use RNA isolated from EBOV-infected cells or virions to determine the 3' and 5' ends of the replication products. By mapping the terminal nucleotides of intracellular and virion-associated viral RNA, we will identify the replication start sites. We will further use EBOV minigenome systems to introduce mutations in relevant regions and analyze the effects on replication initiation and genome integrity. We will perform the initial experiments using the EBOV prototype Zaire ebolavirus. We will then analyze if the identified replication mechanism used by ZEBOV is conserved among other EBOV species. Finally, we will determine the role of an RNA secondary structure adopted by the EBOV promoter region for promoter function and replication initiation. Overall, the proposed work will elucidate if the EBOV polymerase has acquired unique capabilities to interact with the viral genome or if it follows a general mechanism shared with other NNS RNA viruses.