Role of RNA Structural Switches in the Replication of a (+)-Strand RNA Virus

RNA 结构开关在 ( )-链 RNA 病毒复制中的作用

• 批准号: 7904441
• 负责人: Anne Elizabeth Simon
• 金额: $ 31万
• 依托单位: UNIV OF MARYLAND, COLLEGE PARK
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-31 至 2011-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7904441
• 关键词: Adopted; Affect; Affinity; Animals; Base Pairing; Binding; Cell-Free System; Cells; Complex; Disease; Elements; Encephalitis; Genetic; Genetic Transcription; Genome; Helper Viruses; Hepatitis; Human; In Vitro; Individual; Integration Host Factors; Lead; Length; Link; Mediating; Modeling; Mutagenesis; Mutation; Oligonucleotides; Plants; Protoplasts; RNA; RNA Viruses; RNA-Directed RNA Polymerase; Replicon; Role; Satellite RNA; Site-Directed Mutagenesis; Structure; Temperature; Testing; Turnip - dietary; Viral; Viral Genome; Viral Hemorrhagic Fevers; Virion; Virus; Virus Replication; crosslink; gel electrophoresis; genetic selection; guanylate; in vivo; melting; mutant; novel; pathogen; promoter; research study; viral RNA

DESCRIPTION (provided by applicant): Positive-strand RNA viruses are serious pathogens causing encephalitis, hemorrhagic fever and hepatitis in humans and animals and devastating crop losses in plants. Despite extensive studies, replication of these viruses remains poorly understood. A major stumbling block in efforts to fully understand virus replication is the large size of viral genomes, which complicates efforts to link RNA structure with RNA function. We have discovered that a novel conformational switch activates (-)-strand synthesis in satC (356 nt) associated with the model virus Turnip crinkle (TCV; 4054 nt). Since satC contains all sequences and structures necessary for replication by the TCV RdRp, studying its replication in the past has provided significant information subsequently found to be applicable to much larger viral genomes, including those that cause significant diseases in humans and animals. Analyses of satC and TCV replication elements has revealed astonishing complexities and differences in how satC and TCV use nearly identical sequences to replicate their genomes, which has important implications for interpretation of results using subviral RNA replicons. In this proposal, we will use biophysical and genetic approaches to define secondary and tertiary interactions that characterize the satC pre-active structure and structural transitions of wt and mutant satC. Full length and selected satC fragments will be analyzed by temperature gradient gel electrophoresis, UV melting curves, oligonucleotide accessibility and UV cross-linking. Site-specific mutagenesis and in vivo genetic selection (selex) will help define individual elements and the relationship between elements. We will also use mutagenesis approaches combined with RdRp binding analyses to explore TCV sequences that are uniquely important for (-)-strand synthesis although also found in satC. Finally, we will examine RdRp binding to specific satC and TCV hairpins and determine if elements that flank one satC hairpin affect satC replication while simultaneously interfering with TCV replication and repressing virion accumulation function through an interaction between the two viral RNAs. Successful completion of these experiments will provide new paradigms for virus replication and interactions between helper viruses and associated subviral RNAs and provide the most detailed understanding of RNA conformational switches and the steps that lead to initiation of (-)-strand synthesis for any RNA virus.

描述（由申请人提供）：阳性RNA病毒是严重的病原体，引起人类和动物的脑炎，出血热和肝炎以及植物中毁灭性的作物损失。尽管进行了广泛的研究，但对这些病毒的复制仍然知之甚少。在充分了解病毒复制的努力中，一个主要的绊脚石是病毒基因组的大尺寸，这使将RNA结构与RNA功能联系起来的努力变得复杂。我们已经发现，与模型病毒萝卜皱纹相关的SATC（356 nt）中，一种新型的构象开关激活（ - ） - 链合成（TCV； 4054 NT）。由于SATC包含TCV RDRP复制所必需的所有序列和结构，因此研究其复制曾经提供了随后发现的重要信息，可用于更大的病毒基因组，包括那些在人和动物中引起大量疾病的病毒基因组。 SATC和TCV复制元素的分析揭示了SATC和TCV使用几乎相同序列复制其基因组的惊人复杂性和差异，这对使用亚病毒RNA复制子的解释具有重要意义。在此提案中，我们将使用生物物理和遗传方法来定义二级和第三级相互作用，这些相互作用表征了SATC的活性结构以及WT和突变体SATC的结构过渡。全长和选定的SATC片段将通过温度梯度凝胶电泳，紫外线融化曲线，寡核苷酸可及性和紫外线交联分析。位点特异性诱变和体内遗传选择（SELEX）将有助于定义单个元素以及元素之间的关系。我们还将使用与RDRP结合分析相结合的诱变方法来探索对于（ - ） - 链合成至关重要的TCV序列，尽管在SATC中也发现了。最后，我们将检查与特定SATC和TCV发夹的RDRP结合，并确定一个侧翼SATC发夹的元素是否影响SATC复制，同时通过两个病毒RNA之间的相互作用同时干扰TCV复制并抑制病毒率的积累功能。这些实验的成功完成将为病毒复制和辅助病毒与相关下RNA之间的相互作用提供新的范式，并提供对RNA构象开关的最详细理解以及导致（ - ） - 链接合成的步骤。