Non-canonical translation in plant and animal (+)-strand viruses

植物和动物 ( ) 链病毒中的非规范翻译

基本信息

批准号：
9121934
负责人：
Jared Paul May
金额：
$ 5.43万
依托单位：
UNIV OF MARYLAND, COLLEGE PARK
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-05-01 至 2019-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9121934
关键词：
Address Algorithms Animals Antiviral Agents Beryllium Biological Assay Bypass Capsid Proteins Cardiovirus Code Detection Development Elements Encephalomyocarditis virus Eukaryota Event Family Picornaviridae Frequencies Genome HIV Human In Vitro Internal Ribosome Entry Site Lead Length Modeling Molecular Conformation Mutagenesis Mutation Phenotype Plant Viruses Plants Play Production Protein Isoforms Proteins Protoplasts RNA RNA Viruses RNA-Directed RNA Polymerase Reporter Research Ribosomes Role Scheme Site Small RNA Stretching Structure System Terminator Codon Translation Initiation Translations Turnip - dietary Viral Viral Genome Viral Proteins Virus Virus Replication Western Blotting Work deletion analysis human disease improved in vivo innovation mutant nanoluciferase novel pathogen public health relevance reverse genetics symposium tissue culture translation assay

项目摘要

DESCRIPTION (provided by applicant): Many positive-sense, single-stranded, RNA viruses infecting either plants or animals use ribosome recoding and other forms of non-canonical translation to produce important viral proteins such as the RNA dependent RNA polymerase (RdRp). In addition to viruses, evidence is emerging that ribosome recoding may be more widespread in eukaryotic genomes than initially thought and could have important implications for human diseases. Recoding events occur at a specific frequency and are critical for maintaining efficient replication. Previous animal virus recoding research has focused only on a small region of RNA surrounding the recoding site, assuming that all critical RNA structures are located in that small stretch of RNA. However, work using model plant viruses, including Turnip crinkle virus (TCV), has shown that RNA elements far outside of the recoding region play a critical role in recoding efficiency. Here, the importance of alternative RNA structures and long-range RNA:RNA interactions involving recoding sequences will be determined for Encephalomyocarditis virus (EMCV). EMCV is in the cardiovirus genus of Picornaviridae which includes the recently discovered human pathogen, Saffold virus. TCV will also be used to find novel internal ribosome entry sites, or IRES, which likely promote expression of the coat protein and novel isoforms of the RdRp. These studies will increase our understanding of ribosome recoding and IRES function in small RNA viruses. I will address the following questions in the proposed studies: 1) Are long-range RNA:RNA interactions required for efficient recoding in EMCV? Using full-length genome constructs, an in vitro and in vivo translation reporter assay will be developed to determine the effects of disrupting predicted long-range interactions on frameshifting. Reverse genetics will be used to determine if disrupting long-range RNA:RNA interactions is detrimental for virus accumulation. 2) Do alternative RNA structures in the EMCV recoding region exist? Critical alternative recoding structures exist for TCV and similar structures are predicted by folding algorithms to exist for EMCV. SHAPE RNA structure probing will be used to determine the structure of the EMCV recoding region both in vitro and in vivo. The importance of alternative structures in recoding will be determined using both in vitro and in vivo translation assays. 3) Locate novel IRES in TCV. The coat protein and novel RdRp isoforms are expressed from internal initiation in in vitro translation assays. The IRES sequences responsible for either coat protein or RdRp isoform expression will be determined experimentally using SHAPE, mutagenesis, and in vitro translation assays. The importance of RdRp isoforms for TCV accumulation will also be determined. The proposed studies will greatly benefit the ribosome recoding and IRES-related fields by taking an innovative approach that will bridge animal and plant virus studies.

描述（由申请人提供）：许多感染植物或动物的正义、单链 RNA 病毒使用核糖体记录和其他形式的非规范翻译来产生重要的病毒蛋白，例如 RNA 依赖性 RNA 聚合酶 (RdRp)。除了病毒之外，越来越多的证据表明核糖体记录在真核基因组中可能比最初想象的更广泛，并且可能对人类疾病产生重要影响。先前的动物病毒记录研究仅集中于记录位点周围的一小部分RNA，假设所有关键的RNA结构都位于该小段RNA中，但是，使用模型植物病毒（包括芜菁）进行研究。皱纹病毒 (TCV) 已表明，远离记录区域的 RNA 元件在记录效率中起着至关重要的作用，在此，将确定涉及记录序列的替代 RNA 结构和长程 RNA:RNA 相互作用的重要性。脑心肌炎病毒 (EMCV) 属于小核糖核酸病毒科的心脏病毒属，其中包括最近发现的人类病原体 TCV，也将用于寻找新的内部核糖体进入位点 (IRES)，该位点可能促进外壳蛋白的表达。 RdRp 的新亚型。这些研究将加深我们对小 RNA 病毒中核糖体记录和 IRES 功能的理解。我将在拟议的研究中解决以下问题： 1) EMCV 中的有效记录是否需要长程 RNA:RNA 相互作用？使用全长基因组构建体，将开发体外和体内翻译报告基因测定，以确定破坏预测的长程相互作用对移码的影响。反向遗传学将用于确定破坏长程 RNA:RNA 相互作用是否会导致病毒积累。2) EMCV 记录区域中是否存在 TCV 的关键替代记录结构，并且预测了类似的结构。 EMCV 现有的折叠算法将用于确定 EMCV 记录区域的体外和体内结构。记录中替代结构的重要性将通过体外和体内翻译测定来确定。 3) 在 TCV 中定位新的 IRES。外壳蛋白和新的 RdRp 同种型在体外翻译测定中从内部起始表达。 IRES 序列负责外壳蛋白或 RdRp 同种型的表达。将使用 SHAPE、诱变和体外翻译测定进行实验确定 RdRp 同工型对 TCV 积累的重要性也将通过采取创新方法来确定，这将极大地有益于核糖体记录和 IRES 相关领域。动物和植物病毒研究。