New Paradigms for Ribosome Recoding in (+)Strand Viruses

( )链病毒中核糖体重新编码的新范例

• 批准号: 8891615
• 负责人: Anne Elizabeth Simon
• 金额: $ 22.26万
• 依托单位: UNIV OF MARYLAND, COLLEGE PARK
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-02-01 至 2017-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8891615
• 关键词: Animal Viruses; Animals; Antiviral Agents; Area; Beryllium; Binding; Biological Assay; Carmovirus; Cells; Collaborations; Coronavirus; Elements; Enhancers; Equilibrium; Event; Genome; Germ; Human; In Vitro; Indium; Length; Link; Maps; Modeling; Molecular Conformation; Mutagenesis; Mutation; Plant Viruses; Plants; Play; Population; Positioning Attribute; Probability; Proteins; RNA; Recycling; Retroviridae; Ribosomes; Role; Severe Acute Respiratory Syndrome; Shapes; Site; Site-Directed Mutagenesis; Structure; Terminator Codon; Testing; Therapeutic; Translation Initiation; Translations; Turnip - dietary; Viral; Virus; Wheat; Work; base; design; in vivo; mortality; pathogen; public health relevance; research study

 DESCRIPTION (provided by applicant): Ribosome recoding (frameshifting, stop-codon readthrough) is used by many plant-, fungal-, animal- and human-infecting viruses to produce two proteins from a single 5'-end translation initiation site. Most recoding is dependent on a pseudoknot-containing Recoding Structured Element (RSE) positioned just downstream from the stop codon/frameshift site. This exploratory proposal is designed to test the overarching hypothesis that similar recoding events necessary to generate the RdRp by ribosome recoding in Turnip crinkle carmovirus (TCV) and SARS-coV involve similar overall mechanisms. We posit that both viruses require similar alternative basal conformations requiring an additional hairpin and sequences upstream of their RSE and similar long-distance interactions between their RSEs and the 3' and 5' ends of their genomes. This hypothesis is based on finding that TCV contains a stable alternative (basal) structure that disrupts its RSE, and that similar alternative structures are predicted for all coronavirus RSEs examined. In addition, a long-distance RNA-RNA interaction that connects RSEs in plant viruses with their 3' terminus is also conserved in coronavirus RSEs. Furthermore, the RSE sequence in carmoviruses and coronaviruses that has the pairing partner near the 3' end also has a possible pairing partner (7-8 nt for coronaviruses) near their 5' ends. We propose that the 3' end interaction stabilizes the active RSE structure allowing ribosomes to readthrough /frameshift, and that the RSE-5' end interaction occurs when the RSE is in the basal conformation to aid in ribosome recycling following translation termination. In Specific Aim 1, we will investigate the importance of a conformational switch between basal and active structures in the RSE region of TCV using SHAPE RNA structure probing of full-length virus combined with selective mutations. We will also determine if the known long-distance interaction between the TCV RSE bulge loop and 3' terminal sequences stabilizes the active RSE structure. Using single and compensatory mutagenesis, we will also test (with collaborator Dr. Ralph Baric) if a basal conformation exists for the RSE of SARS-coV and if a phylogenetically conserved hairpin loop in the RSE of SARS-coV is involved in a very similar interaction with a 3' sequence. In Specific Aim 2, we will investigate if the RSEs in TCV and SARS-coV play an additional role in ribosome recycling by engaging in a predicted long-distance interaction with the 5' end. We will also investigate if release of this interaction in TC promotes a conformational switch to the RSE active structure for ribosome readthrough. In Specific Aim 3, we will use in-line RNA structure probing of isolated TCV and SARS-coV fragments to investigate structural requirements for the basal and active RSE conformations. The results of these experiments will likely transform current models on ribosome recoding and provide evidence for the importance of ribosome recycling, which has long been lacking in the translation field. It should also, importantly, open up new targets for antiviral agents against viruses that are important human pathogens.

 描述（由申请人提供）：许多植物、真菌、动物和人类感染病毒使用核糖体记录（移码、终止密码子通读）从单个 5' 端翻译起始位点产生两种蛋白质。记录依赖于位于终止密码子/移码位点下游的包含假结的重新编码结构元件（RSE）。该探索性提议旨在测试总体假设是，芜菁皱纹车病毒 (TCV) 和 SARS-coV 中通过核糖体记录生成 RdRp 所需的类似记录事件涉及相似的整体机制，我们假设这两种病毒需要类似的基础替代构象，需要额外的发夹和 RSE 上游序列。以及它们的 RSE 和基因组 3' 和 5' 端之间类似的长距离相互作用。该假设基于 TCV 包含稳定替代物（基础）的发现。破坏其 RSE 的结构，以及类似的替代方案 预测了所有检查的冠状病毒 RSE 的结构。此外，将植物病毒中的 RSE 与其 3' 末端连接的长距离 RNA-RNA 相互作用在冠状病毒 RSE 中也是保守的。此外，胡萝卜病毒和冠状病毒中的 RSE 序列具有3' 端附近的配对伙伴在其 5' 端附近也有一个可能的配对伙伴（冠状病毒为 7-8 nt），我们认为 3' 端相互作用稳定了活性 RSE 结构。允许核糖体通读/移码，并且当 RSE 处于基础构象时发生 RSE-5' 末端相互作用，以帮助翻译终止后的核糖体回收。在具体目标 1 中，我们将研究基础构象之间构象转换的重要性。我们还将使用全长病毒的 SHAPE RNA 结构探测结合选择性突变来确定 TCV RSE 区域中的活性结构。 3' 末端序列可稳定活性 RSE 结构，我们还将（与合作者 Ralph Baric 博士一起）测试 SARS-coV 的 RSE 是否存在基础构象，以及 RSE 中是否存在系统发育保守的发夹环。 SARS-coV 的 RSE 与 3' 序列的相互作用非常相似，在特定目标 2 中，我们将研究 TCV 和 SARS-coV 中的 RSE 是否在其中发挥额外作用。通过与 5' 端进行预测的长距离相互作用，我们还将研究 TC 中这种相互作用的释放是否会促进核糖体通读中的 RSE 活性结构的构象转换。对分离的 TCV 和 SARS-coV 片段进行线 RNA 结构探测，以研究基本和活性 RSE 构象的结构要求。这些实验的结果可能会改变当前的核糖体记录模型，并为核糖体记录提供证据。核糖体回收的重要性，这在翻译领域长期以来一直缺乏，它还应该为针对重要人类病原体的病毒的抗病毒药物开辟新的靶点。