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: 9000611
• 负责人: Anne Elizabeth Simon
• 金额: $ 18.73万
• 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 2018-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9000611
• 关键词: Animals; Antiviral Agents; Area; Beryllium; Binding; Biological Assay; Cells; Collaborations; Coronavirus; Elements; Enhancers; Equilibrium; Event; Genome; Germ; Health; 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; 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重新编码（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。大多数重新编码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）。该探索性提案旨在测试总体假设，即在芜菁皱纹胡萝卜病毒 (TCV) 和 SARS-coV 中通过核糖体重新编码生成 RdRp 所需的类似重新编码事件涉及类似的整体机制。我们假设这两种病毒都需要类似的替代基础构象，需要在其 RSE 上游附加发夹和序列，以及其 RSE 与其基因组 3' 和 5' 端之间类似的长距离相互作用。该假设基于发现 TCV 包含破坏其 RSE 的稳定替代（基础）结构，并且类似的替代结构 预测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，Carmovirus 和冠状病毒中的 RSE 序列在 3' 末端附近有配对伙伴，在 5' 末端附近也有可能的配对伙伴（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，RSE-5'端相互作用发生，以帮助翻译终止后的核糖体回收。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 RSE 区域中基础结构和活性结构之间构象转换的重要性。我们还将确定 TCV RSE 凸出环和 3' 末端序列之间已知的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 RSE 是否存在基础构象，以及 SARS-coV RSE 中系统发育保守的发夹环是否参与与 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' 末端进行预测的长距离相互作用，在核糖体回收中发挥额外作用。我们还将研究 TC 中这种相互作用的释放是否会促进构象转变为 RSE 活性结构以进行核糖体通读。在具体目标 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。

项目成果

RNA virus evasion of nonsense-mediated decay.

• DOI: 10.1371/journal.ppat.1007459
• 发表时间: 2018-11
• 期刊: PLoS pathogens
• 影响因子: 6.7
• 作者: May JP;Yuan X;Sawicki E;Simon AE
• 通讯作者: Simon AE

Rapid evolution of in vivo-selected sequences and structures replacing 20% of a subviral RNA.

快速％20进化％20的％20在％20体内选择的％20序列％20和％20结构％20替换％2020％％20的％20a％20亚病毒％20RNA。

• DOI: 10.1016/j.virol.2015.04.002
• 发表时间: 2015
• 期刊: Virology
• 影响因子: 3.7
• 作者: Murawski,AllisonM;Nieves,JohnathanL;Chattopadhyay,Maitreyi;Young,MeganY;Szarko,Christine;Tajalli,HollehF;Azad,Tareq;Jean-Jacques,NinaB;Simon,AnneE;Kushner,DavidB
• 通讯作者: Kushner,DavidB

SELEX and SHAPE reveal that sequence motifs and an extended hairpin in the 5' portion of Turnip crinkle virus satellite RNA C mediate fitness in plants.

SELEX 和 SHAPE 揭示了芜菁皱纹病毒卫星 RNA C 5 部分的序列基序和延伸发夹介导植物的适应性。

• DOI: 10.1016/j.virol.2018.05.010
• 发表时间: 2018
• 期刊: Virology
• 影响因子: 3.7
• 作者: Bayne,CharlieF;Widawski,MaxE;Gao,Feng;Masab,MohammedH;Chattopadhyay,Maitreyi;Murawski,AllisonM;Sansevere,RobertM;Lerner,BryanD;Castillo,RinaldysJ;Griesman,Trevor;Fu,Jiantao;Hibben,JenniferK;Garcia-Perez,AlmaD;Simon,Anne
• 通讯作者: Simon,Anne

Concerted action of two 3' cap-independent translation enhancers increases the competitive strength of translated viral genomes.

• DOI: 10.1093/nar/gkx643
• 发表时间: 2017-09-19
• 期刊: Nucleic acids research
• 影响因子: 14.9
• 作者: Du Z;Alekhina OM;Vassilenko KS;Simon AE
• 通讯作者: Simon AE