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（由适用提供）：许多阳性，单链的RNA病毒感染了植物或动物，使用核糖体重新编码和其他形式的非经典翻译来产生重要的病毒蛋白，例如依赖RNA的RNA聚合酶（RDRP）。除病毒外，有证据表明，在真核基因组中，核糖体的重现可能比最初想象的更广泛，并且可能对人类疾病具有重要意义。重新编码事件以特定的频率发生，对于维持有效的复制至关重要。假设所有关键的RNA结构都位于那一小片RNA中，则先前的动物病毒进行了研究仅集中在重新编码位点周围的一小部分RNA区域。但是，使用模型植物病毒（包括萝卜皱纹病毒（TCV））的工作表明，远离回收区域之外的RNA元素在重新编码效率中起着至关重要的作用。在这里，将确定涉及重新编码序列的替代RNA结构和远程RNA的重要性。 EMCV位于Picornaviridae的心脏病毒属中，其中包括最近发现的人类病原体Saffold病毒。 TCV还将用于查找新型的内部核糖体进入位点或IRE，这些位点可能促进RDRP的外套蛋白和新型同工型的表达。这些研究将增加我们对核糖体重现的理解，而IRE在小的RNA病毒中起作用。我将在拟议的研究中解决以下问题：1）是远程RNA：在EMCV中有效重新编码所需的RNA相互作用吗？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法，以确定破坏预测的远程相互作用对Frameshifting的影响。反向遗传学将用于确定是否破坏了远程RNA：RNA相互作用对病毒积累有害。 2）在EMCV重新编码区域中是否存在替代RNA结构？ TCV存在关键的替代重新编码结构，并且通过为EMCV存在折叠算法来预测相似的结构。形状RNA结构探测将用于确定体外和体内EMCV重新编码区域的结构。替代结构在重新编码中的重要性将使用体外和体内翻译分析确定。 3）在TCV中找到新的IRES。外套蛋白和新型RDRP同工型在体外翻译测定中从内部起始表达。负责外套蛋白或RDRP同工型表达的IRES序列将使用形状，诱变和体外翻译测定法确定。还将确定RDRP同工型对TCV积累的重要性。拟议的研究将通过采用一种创新的方法来弥合动物和植物病毒研究，从而极大地使核糖体重现和与IRES相关的领域受益。