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: 8904599
• 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/8904599
• 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; Health; 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; genome integrity; member; pathogen; prevent; promoter; prototype; 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）：埃博氏病毒（EBOV）属于非分段阴性（NNS）RNA病毒的组，在人类中是高致​​病性的。目前，尚无批准的治疗或预防EBOV出血热的治疗剂。埃博氏病使用RNA依赖性RNA聚合酶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发育的常见靶标，靶向病毒聚合酶的抗病毒药化合物的合理设计需要透彻了解与基因组复制有关的分子事件。在这里，我们建议剖析EBOV用来启动复制的分子机制。我们的初步数据表明，EBOV已发展出一种与其他NNS RNA病毒所使用的复制起始机制不同。根据我们的模型，EBOV在模板链的位置+2处启动复制，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EBOV感染的细胞或病毒体中分离出的RNA来确定复制产物的3'和5'末端。通过映射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。我们将进一步使用EBOV微型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EBOV原型Zaire Ebolavirus执行初始实验。然后，我们将分析Zebov使用的已鉴定的复制机制在其他EBOV物种中是保守的。最后，我们将确定EBOV启动子区域对启动子功能和复制启动采用的RNA二级结构的作用。总体而言，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功能，或者遵循与其他NNS RNA病毒共享的一般机制。