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）：核糖体重新编码（移码、终止密码子通读）被许多植物、真菌、动物和人类感染病毒用于从单个5 '端翻译起始位点产生两种蛋白质。大多数重编码依赖于位于终止密码子/移码位点下游的含有假结的重编码结构元件（RSE）。这个探索性的建议是为了测试的总体假设，即类似的编码事件所需的核糖体编码产生RdRp在芜菁皱纹癌病毒（TCV）和SARS冠状病毒涉及类似的整体机制。我们认为，这两种病毒需要类似的替代基础构象，需要一个额外的发夹和序列上游的RSE和类似的长距离相互作用之间的RSE和3'和5'端的基因组。这一假设是基于发现TCV包含一个稳定的替代（基础）结构，破坏其RSE，并且类似的替代结构 预测了所有检测的冠状病毒RSE的结构。此外，连接植物病毒RSE与其3'末端的长距离RNA-RNA相互作用在冠状病毒RSE中也是保守的。此外，在3'端附近具有配对伴侣的卡莫病毒和冠状病毒中的RSE序列在其5'端附近也具有可能的配对伴侣（对于冠状病毒为7-8 nt）。我们提出，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'端相互作用，以帮助翻译终止后的核糖体再循环。在具体目标1中，我们将使用全长病毒的SHAPE RNA结构探测结合选择性突变来研究TCV的RSE区域中基础结构和活性结构之间的构象转换的重要性。我们还将确定TCV RSE凸起环和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。使用单一和补偿诱变，我们还将测试（与合作者Ralph Baric博士）SARS-coV的RSE是否存在基础构象，以及SARS-coV的RSE中是否有一个遗传上保守的发夹环参与与3'序列非常相似的相互作用。在具体目标2中，我们将研究TCV和SARS-coV中的RSE是否通过与5'端进行预测的长距离相互作用而在核糖体回收中发挥额外的作用。我们还将研究TC中这种相互作用的释放是否促进了核糖体通读的RSE活性结构的构象转换。在特定目标3中，我们将使用分离的TCV和SARS-coV片段的在线RNA结构探测来研究基础和活性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重新编码模型，并为核糖体回收的重要性提供证据，而这在翻译领域长期以来一直缺乏。重要的是，它还应该为抗病毒药物开辟新的靶点，以对抗重要的人类病原体病毒。