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，这是产生子代病毒及其传播到未感染细胞的关键步骤。更好地了解这些机制将有助于我们设计新型抗病毒疗法。我们在诺达病毒野田村病毒 (NoV) 中研究这一过程，以便我们能够利用其在多种宿主细胞中以极高水平复制其正链 RNA 基因组的能力。NoV 提供了一个极好的模型来研究更复杂的、正链的 -链RNA病毒对人类致病。尽管这种模型病毒尚未与人类疾病密切相关，但最近的研究已将新型 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 可以作为了解人类柯萨奇病毒引起的疾病的模型，并且新设计的实验将 NoV 复制复合体组装与肠道病毒柯萨奇 A 病毒 21 (CVA21) 的复制复合体组装进行比较。 NoV 和 CVA21 RNA 的复制通过互补负链复制中间体的合成进行，这些中间体用作合成其他正链的模板。这两个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RdRp识别RNA模板并组装膜结合RNA复制复合物(RC)，即细胞中RNA复制的位点。拟议的研究重点关注影响 RC 组装的两个主要领域：1）RdRp 模板识别的结构要求以及随后模板招募到 RC 的结构要求；2）RdRp 翻译后修饰在 RC 组装中的作用。我们假设所需成分的募集是通过 RNA 模板和病毒（和/或细胞）蛋白之间的结构依赖性 RNA-蛋白相互作用以及 RdRp 的翻译后修饰来控制的。我们将使用遗传和生化相结合的方法来产生影响 RdRp 与 RNA 模板结合或防止 RdRp 修饰的突变，并测试它们对 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、显微镜研究研究 RdRp 亚细胞定位响应诱变的变化，并通过液相色谱-串联质谱 (LC-MS/MS) 鉴定 RdRp 中跨国后的氨基酸 修改的。我们还将设计一种新的 RNA 结构预测工具，以促进识别更复杂的病毒 RNA 基因组中的长程碱基配对相互作用。我们提出以下具体目标：1) 定义 RNA 结构元件在 RdRp 识别和将 RNA 模板招募到线粒体 RC 中的作用；2) 确定诺达病毒 RdRp 的翻译后修饰在形成和招募中的作用其他蛋白质与膜结合 RC 的结合。这些研究的结果将深入了解病毒 RNA 复制机制并确定抗病毒治疗的新靶点。