Characterization and targeting rift valley fever virus N protein-RNA interactions

裂谷热病毒 N 蛋白-RNA 相互作用的表征和靶向

• 批准号: 7675666
• 负责人: J. Stephen Lodmell
• 金额: $ 30.26万
• 依托单位: COLORADO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-01 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7675666
• 关键词: Adopted; Affinity; Africa; Africa South of the Sahara; Antiviral Agents; Attenuated; Binding; Binding Sites; Biochemical; Biological Assay; Bioterrorism; Bunyaviridae; Cell Culture Techniques; Cessation of life; Clinical; Collaborations; Development; Disease; Disease Outbreaks; Drug Compounding; Drug Delivery Systems; Emerging Communicable Diseases; Epitopes; Event; Exhibits; Family; Fever; Fluorescence; Generations; Goals; HIV; Habitats; Human; In Vitro; Infection Control; Insecta; Knowledge; Lead; Libraries; Ligands; Light; Liver; Malaise; Molecular Conformation; Molecular Virology; Morbidity - disease rate; Mutagenesis; Nervous System Trauma; Nucleocapsid; Nucleocapsid Proteins; Nucleotides; Pharmaceutical Preparations; Preclinical Drug Evaluation; Protein Binding; Proteins; Protocols documentation; RNA; RNA Binding; RNA Recognition Motif; RNA Sequences; RNA Viruses; RNA-Binding Proteins; RNA-Protein Interaction; Research; Research Project Grants; Respiratory syncytial virus; Rift Valley Fever; Rift Valley fever virus; Role; Scheme; Screening procedure; Sequence Analysis; Signal Transduction; Structure; Symptoms; Testing; Therapeutic; Transcript; Universities; Utah; Validation; Viral; Virus; Virus Diseases; Virus Replication; aptamer; base; biodefense; combinatorial; cytotoxicity; detector; member; molecular recognition; mortality; novel; pathogen; small molecule; small molecule libraries; therapeutic target; tool; treatment strategy; viral RNA

Rift Valley Fever is a clinically and agriculturally important illness caused by Rift Valley Fever Virus (RVFV), a member of the Bunyaviridae endemic primarily in sub-Saharan Africa. Because of the severe illness it causes in humans and because of recent instances of natural spread outside of Africa, it has been identified as emerging disease agent and/or a potential agent of bioterrorism. The symptoms of RVF in humans can include severe fever, malaise, neurological damage, liver damage, hemorrhagic events, and death. Currently there are no clinically proven treatments for RVFV infection. Development of antiviral strategies requires understanding of the viral replication cycle so that specific steps can be targeted. In the research proposed here, we will characterize an essential molecular interaction in the RVFV replication cycle wherein nucleocapsid protein must recognize and bind to viral RNA sequences. To achieve this goal, we will perform truncation analysis on viral RNA transcripts to determine the minimum requirements for efficient RNA-protein binding. We will also perform a combinatorial in vitro selection scheme (SELEX) to obtain a high affinity RNA aptamer(s) to nucleocapsid protein. Sequence analysis of the aptamers will shed light on nucleocapsid target recognition and will subsequently be used to construct a tool to screen drugs that inhibit the nucleocapsid-RNA interaction. Specifically, the aptamer will be ligated to an RNA module that emits a fluorescent signal when the aptamer is displaced from its nucleocapsid binding site. The drug screening tool will then be used to screen compounds at NSRB. Molecules giving promising results in the aptamer displacement assay will be tested against RVFV replication in cell culture. Owing to its central role in replication, nucleocapsid protein has been recognized as a good potential antiviral target. A novel class of drug targeted against nucleocapsid protein has recently been shown to be effective in inhibiting replication of respiratory syncytial virus, also a negative-sense RNA virus, strongly supporting the potential for developing nucleocapsid as a viable drug target in RVFV (Chapman et al, 2007). This research project fits within the RMRCE Integrated Research Focus on Viral Therapeutics, and will interact directly with PI Brian Gowen (RP 3.2) and the NSRB drug screening core.

裂谷热是由裂谷热病毒（RVFV）引起的临床和农业上重要的疾病， 布尼亚病毒科的一种主要在撒哈拉以南非洲地区流行的病毒。由于病情严重， 由于最近在非洲以外的自然传播的例子，它已被确定为人类的原因， 作为新出现的疾病病原体和/或生物恐怖主义的潜在病原体。人类裂谷热的症状可 包括严重发烧、不适、神经损伤、肝损伤、出血事件和死亡。 目前还没有临床证明的治疗RVFV感染。抗病毒策略的发展 需要了解病毒复制周期，以便有针对性地采取具体步骤。 在这里提出的研究中，我们将描述RVFV中的一个重要分子相互作用 复制周期，其中核衣壳蛋白必须识别并结合病毒RNA序列。实现 为此，我们将对病毒RNA转录物进行截短分析，以确定最小截短量。 RNA与蛋白质的有效结合。我们还将进行组合体外选择 方案（SELEX）以获得对核衣壳蛋白的高亲和力RNA适体。序列分析 适体将揭示核衣壳靶点识别，并将随后用于构建一种工具， 筛选抑制核帽蛋白-RNA相互作用的药物。具体地，适体将连接到一个 当适体从其核衣壳结合中被置换时，发射荧光信号的RNA模块 绝佳的价钱药物筛选工具将用于在NSRB筛选化合物。分子给有前途的 将针对细胞培养物中的RVFV复制测试适体置换测定中的结果。由于 核衣壳蛋白在病毒复制过程中起着重要作用，是一种很好的抗病毒靶点。一 一种靶向核衣壳蛋白的新型药物最近已被证明在 抑制呼吸道合胞病毒（也是一种负义RNA病毒）的复制，强烈支持 在RVFV中开发核衣壳作为可行药物靶标的潜力（Chapman等人，2007）。 该研究项目符合RMRCE病毒治疗综合研究重点，并将 直接与PI Brian Gowen（RP 3.2）和NSRB药物筛选核心相互作用。