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

植物和动物 ( ) 链病毒中的非规范翻译

• 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
• 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

 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病毒使用核糖体重新编码和其他形式的非规范翻译来产生重要的病毒蛋白，如RNA依赖性RNA聚合酶（RdRp）。除了病毒之外，还有证据表明，核糖体重新编码在真核生物基因组中可能比最初认为的更广泛，并可能对人类疾病产生重要影响。复制事件以特定的频率发生，对于保持高效复制至关重要。以前的动物病毒重编码研究只关注重编码位点周围的一小部分RNA，假设所有关键的RNA结构都位于这一小部分RNA中。然而，使用包括芜菁皱缩病毒（TCV）在内的模型植物病毒的工作已经表明，远在重编码区之外的RNA元件在重编码效率中起关键作用。在这里，替代RNA结构和长程RNA的重要性：RNA相互作用涉及重新编码序列将被确定为脑心肌炎病毒（EMCV）。EMCV属于小核糖核酸病毒科（Picornaviridae）的心脏病毒属，包括最近发现的人类病原体红花病毒（Saffold virus）。TCV还将用于发现新的内部核糖体进入位点或IRES，其可能促进外壳蛋白和RdRp的新亚型的表达。这些研究将增加我们对小RNA病毒中核糖体编码和IRES功能的理解。我将在拟议的研究中解决以下问题：1）在EMCV中有效重编码是否需要长距离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距离RNA：RNA相互作用是否对病毒积累有害。 2)在EMCV编码区是否存在替代的RNA结构？TCV存在关键的替代重编码结构，并且通过折叠算法预测EMCV存在类似的结构。SHAPE RNA结构探测将用于确定体外和体内EMCV编码区的结构。将使用体外和体内翻译测定来确定替代结构在重新编码中的重要性。 3)在TCV中定位新IRES。在体外翻译测定中，外壳蛋白和新型RdRp同种型从内部起始表达。负责外壳蛋白或RdRp亚型表达的IRES序列将使用SHAPE、诱变和体外翻译试验通过实验确定。还将确定RdRp亚型对TCV蓄积的重要性。 拟议的研究将大大有利于核糖体编码和IRES相关领域采取创新的方法，将桥梁动物和植物病毒的研究。