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

基本信息

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
项目类别：
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

项目摘要

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）引起的一种临床和农业上的重要疾病，主要流行于撒哈拉以南非洲的布尼亚病毒科的成员。由于病情严重，人类的原因以及最近在非洲以外自然传播的情况，已确定作为新出现的疾病媒介和/或生物恐怖主义的潜在媒介。人类裂谷热的症状可以包括严重发烧、不适、神经损伤、肝损伤、出血事件和死亡。目前尚无临床证明可治疗 RVFV 感染的方法。抗病毒策略的制定需要了解病毒复制周期，以便有针对性地执行特定步骤。在此提出的研究中，我们将描述 RVFV 中重要的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。达到为了这个目标，我们将对病毒 RNA 转录本进行截断分析，以确定最小有效RNA-蛋白质结合的要求。我们还将进行组合体外选择方案（SELEX）以获得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目标识别，随后将用于构建工具筛选抑制核衣壳-RNA相互作用的药物。具体来说，适体将被连接到当适配体脱离其核衣壳结合时，RNA 模块会发出荧光信号地点。 NSRB 将使用药物筛选工具筛选化合物。有希望的分子适体置换测定的结果将针对细胞培养中的 RVFV 复制进行测试。由于核衣壳蛋白在复制中发挥核心作用，已被认为是良好的潜在抗病毒靶点。一个最近证明针对核衣壳蛋白的新型药物可有效抑制呼吸道合胞病毒（也是一种负义 RNA 病毒）的复制，有力地支持了开发核衣壳作为 RVFV 可行药物靶点的潜力（Chapman 等，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 药物筛选核心互动。