Mechanisms in Viral RNA Replication Complex Assembly: Novel Targets for Antivira

病毒 RNA 复制复合物组装机制：抗病毒药物的新靶点

• 批准号: 8687938
• 负责人: KYLE L JOHNSON
• 金额: $ 45.3万
• 依托单位: UNIVERSITY OF TEXAS EL PASO
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-01 至 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8687938
• 关键词: Address; Affect; Algorithms; Amino Acids; Antiviral Therapy; Area; Base Pairing; Binding; Biochemical; Biological Assay; Cells; Cellular Membrane; Complex; Confocal Microscopy; Consensus; Coxsackie Viruses; Data; Dengue; Disease; Elements; Employee Strikes; Enterovirus; Family; Family Picornaviridae; Flavivirus; Funding; Genetic; Genome; Genomics; Goals; Human; In Vitro; Indium; Journals; Laboratories; Link; Liquid Chromatography; Mammalian Cell; Measures; Mediating; Membrane; Microscopy; Mitochondria; Modeling; Modification; Morphology; Mutagenesis; Mutation; Paralysed; Poliomyelitis; Post-Translational Protein Processing; Process; Production; Proteins; Publications; RNA; RNA Binding; RNA I; RNA Interference; RNA Viruses; RNA replication; RNA-Directed RNA Polymerase; RNA-Protein Interaction; Reaction; Research; Role; SARS coronavirus; Site; Site-Directed Mutagenesis; Small Ubiquitin-Related Modifier Proteins; Structure; Testing; Ubiquitin; Viral; Viral Genome; Virion; Virus; Virus Replication; West Nile virus; design; human disease; insight; member; membrane assembly; mitochondrial membrane; mutant; novel; pathogen; prevent; public health relevance; research study; response; tandem mass spectrometry; tool; ubiquitin-protein ligase; viral RNA

DESCRIPTION (provided by applicant): The proposed research seeks to decipher the mechanism of viral RNA replication, a key step in production of progeny viruses and their spread to uninfected cells. A greater understanding of these mechanisms will facilitate our design of novel antiviral therapies. We study this process in the nodavirus Nodamura virus (NoV) so that we can harness its ability to replicate its positive-strand RNA genome to tremendously high levels in a wide range of host cells NoV provides an excellent model to study more complex, positive-strand RNA viruses pathogenic to humans. Although this model virus has not yet been associated firmly with human disease, recent studies have linked novel NoV-like viruses to human diarrheal or paralytic illnesses. Thus, we propose that NoV could serve as a model to understand the diseases caused by human Coxsackie viruses and newly designed experiments compare NoV replication complex assembly with that of the enterovirus Coxsackie A virus 21 (CVA21). Replication of NoV and CVA21 RNA occurs via the synthesis of complementary negative strand replication intermediates, which are used as templates for synthesis of additional positive strands. Both reactions are catalyzed by a virus-encoded RNA-dependent RNA polymerase (RdRp). Initiation of viral RNA replication in a host cell requires recognition of the RNA template by the RdRp and assembly of membrane- bound RNA replication complexes (RC's), the sites of RNA replication in the cell. The proposed studies focus on two major areas that impact RC assembly: 1) structural requirements of template recognition by the RdRp and subsequent template recruitment to the RC and 2) the role of RdRp post-translational modification in RC assembly. We hypothesize that recruitment of the required components is controlled by structure-dependent RNA-protein interactions between the RNA template and viral (and/or cellular) proteins and by post- translational modification of the RdRp. We will use a combined genetic and biochemical approach to make mutations that effect RdRp-binding to RNA templates or prevent modification of the RdRp and test their effects on RNA replication, RNA binding studies to measure interactions between the RNA template and the RdRp, microscopy studies to study changes to subcellular localization of the RdRp in response to mutagenesis, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify the amino acids in the RdRp that are post-transnationally modified. We will also design a new RNA structure prediction tool to facilitate identification of long-range base pairing interactions within more complex viral RNA genomes. We propose the following Specific Aims: 1) Define the role of RNA structural elements in recognition by the RdRp and recruitment of RNA templates into mitochondrial RC's and 2) Determine the role of post-translational modification of the nodavirus RdRp in the formation of and recruitment of other proteins to membrane-bound RC's. The results of these studies will provide insight into viral RNA replication mechanisms and identify new targets for antiviral therapies.

描述（由申请人提供）：拟议的研究旨在破译病毒RNA复制的机制，后代病毒生产的关键步骤及其扩散到未感染的细胞。对这些机制的更多了解将有助于我们对新型抗病毒疗法的设计。我们研究了Nodavirus Nodamura病毒（NOV）中的这一过程，以便我们可以利用其将其阳性链RNA基因组复制到广泛的宿主细胞中的高水平，从而为人类提供了更为复杂，阳性链RNA病毒的良好模型。尽管该模型病毒尚未与人类疾病牢固相关，但最近的研究将新型的Nov样病毒与人类腹泻或麻痹性疾病联系起来。因此，我们建议Nov可以作为一个模型，以了解人类Coxsackie病毒引起的疾病和新设计的实验将Nov Replication Complects组成与肠内病毒Coxsackie A A A grope complectbly进行比较A病毒21（CVA21）。通过互补负复制中间体的合成，将NOV和CVA21 RNA复制发生，这些中间体用作合成其他阳性链的模板。这两种反应均由病毒编码的RNA依赖性RNA聚合酶（RDRP）催化。宿主细胞中病毒RNA复制的启动需要通过RDRP和组装膜结合RNA复制复合复合物（RC）识别RNA模板，该复制位置是细胞中RNA复制的位点。拟议的研究集中于影响RC组装的两个主要领域：1）RDRP识别模板识别的结构要求，以及随后对RC的模板募集，以及2）RDRP在RC组装中翻译后修饰的作用。我们假设所需组件的募集受RNA模板与病毒（和/或细胞）蛋白之间的结构依赖性RNA - 蛋白质相互作用以及RDRP的翻译后修饰。我们将使用一种遗传和生化方法来制作突变，从而影响RDRP结合对RNA模板或防止对RDRP的修饰，并测试其对RNA复制的影响，RNA结合研究以测量RNA模板和RDRP之间的相互作用，以研究对RDRP的研究，以研究对RDRP的响应量和液相频谱的变化，以及响应rddrp syprys的变化，以及响应的液相频谱，以及响应的频率谱图，以及响应的变化。 （LC-MS/MS）以识别RDRP中经过转化后修饰的氨基酸。我们还将设计一种新的RNA结构预测工具，以促进在更复杂的病毒RNA基因组中识别远程基础配对相互作用。我们提出以下具体目的：1）定义RNA结构元素在RDRP识别中的作用，并将RNA模板募集到线粒体RC中，并确定2）确定Nodavirus RDRP的翻译后修饰在形成和募集其他蛋白质对其他蛋白质中的作用。这些研究的结果将提供有关病毒RNA复制机制的见解，并确定抗病毒疗法的新靶标。