Filovirus replication: initiation mechanism and role of RNA secondary structures

丝状病毒复制：RNA二级结构的启动机制和作用

• 批准号: 8771943
• 负责人: Elke C Muhlberger
• 金额: $ 8.19万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-05 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8771943
• 关键词: Adopted; Antiviral Agents; Case Fatality Rates; Cells; Complex; Containment; Data; Democratic Republic of the Congo; Development; Disease; Event; Family; Filovirus; Genetic Transcription; Genome; Genomics; Human; Infection; Knowledge; Life Cycle Stages; Maps; Messenger RNA; Modeling; Molecular; Mutation; Nucleotides; Paramyxovirus; Polymerase; Positioning Attribute; Primer Extension; Promoter Regions; RNA; RNA Viruses; RNA-Directed RNA Polymerase; Replication Initiation; Research; Respiratory syncytial virus; Rhabdoviridae; Role; Site; Structure; System; Techniques; Testing; Therapeutic; Vesicular stomatitis Indiana virus; Viral; Viral Genome; Viral Hemorrhagic Fevers; Virion; Virus; Work; base; design; drug development; member; pathogen; prevent; promoter; prototype; public health relevance; research study; stem; viral RNA

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.

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，对人类具有高致病性。目前，还没有批准的疗法可以治疗或预防埃博拉病毒出血热。埃博拉病毒使用 RNA 依赖性 RNA 聚合酶来复制和转录其基因组。人们对 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标，合理设计针对病毒聚合酶的抗病毒化合物需要彻底了解基因组复制中涉及的分子事件。在这里，我们建议剖析埃博拉病毒启动复制的分子机制。我们的初步数据表明，埃博拉病毒已经进化出一种与其他 NNS RNA 病毒所使用的复制启动机制不同的复制启动机制。根据我们的模型，EBOV 在模板链的 +2 位点开始复制，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。为了测试这个模型，我们将使用从 EBOV 感染的细胞或病毒粒子中分离的 RNA 来确定复制产物的 3' 和 5' 末端。通过绘制细胞内和病毒颗粒相关病毒 RNA 的末端核苷酸图谱，我们将确定复制起始位点。我们将进一步利用埃博拉病毒小基因组系统在相关区域引入突变，并分析其对复制起始和基因组完整性的影响。我们将使用埃博拉病毒原型扎伊尔埃博拉病毒进行初步实验。然后我们将分析 ZEBOV 使用的已识别复制机制在其他 EBOV 物种中是否保守。最后，我们将确定 EBOV 启动子区域采用的 RNA 二级结构对于启动子功能和复制启动的作用。总体而言，拟议的工作将阐明 EBOV 聚合酶是否已获得与病毒基因组相互作用的独特能力，或者它是否遵循与其他 NNS RNA 病毒共享的通用机制。