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（应用程序提供）：许多植物，真菌 - ，动物和人类感染病毒使用核糖体重新编码（Frameshifting，stop-Codon Readtrough），从单个5'End翻译起始位点产生两种蛋白质。大多数重新编码取决于含有伪KNOT的重新编码结构化元件（RSE），该元件（RSE）位于终止密码子/移码站点下游。该探索性建议旨在检验总体假设，即通过在萝卜皱纹carmovirus（TCV）中进行核糖体重新编码所需的类似的重新编码事件，而SARS-COV涉及类似的整体机制。我们将这两种病毒都分配给了类似的替代基本构象，需要额外的发夹和RSE上游的序列以及其RSES与基因组的3'和5'端之间的类似长距离相互作用。该假设基于发现TCV包含破坏其RSE的稳定替代（基底）结构，并且该替代方案类似 预测了所有冠状病毒RSE的结构。此外，在冠状病毒RSE中也保留了将植物病毒中RSE与3'末端连接起来的长距离RNA-RNA相互作用。此外，在3'端附近配对伴侣的carmoviruses和冠状病毒中的RSE序列还具有5'端附近的配对伴侣（冠状病毒7-8 nt）。我们建议3'端相互作用稳定活性RSE结构，使核糖体能够读取 /移序，并且当RSE处于基本组成中以帮助翻译终止后的核糖体回收时，RSE-5'端相互作用发生。在特定的目标1中，我们将使用全长病毒的形状RNA结构探测与选择性突变的形状RNA结构探测，从而研究TCV RSE区域中基本和活性结构之间的构象切换的重要性。我们还将确定TCV RSE Bulbi环与3'末端序列之间的已知长距离相互作用是否稳定活性RSE结构。使用单一和补偿性诱变，我们还将（与合作者Ralph Baric博士）进行测试，如果存在SARS-COV的RSE的基本会议，以及在SARS-COV RSE中的系统发育构成的发夹循环是否与与3'序列的非常相似的交互作用有关。在特定目标2中，我们将调查TCV和SARS-COV中的RSE是否在核糖体回收中发挥了额外的作用，通过与5'端进行预测的长距离相互作用。我们还将调查该相互作用在TC中的释放是否促进了核糖体读取的RSE活动结构的会议开关。在特定的目标3中，我们将使用孤立的TCV和SARS-COV片段的在线RNA结构探测来研究基本和主动RSE构象的结构要求。这些实验的结果可能会在核糖体重编码上转变当前模型，并为核糖体回收的重要性提供了证据，而核糖体回收的重要性长期以来一直缺乏翻译领域。重要的是，重要的是，它为针对重要人类病原体的病毒打开了抗病毒药物的新靶标。