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

 描述(申请人提供)：许多感染植物或动物的阳性单链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，如依赖RNA的RNA聚合酶(RdRp)。除了病毒，越来越多的证据表明，核糖体重新编码可能比最初认为的更广泛地存在于真核基因组中，并可能对人类疾病产生重要影响。重新编码事件以特定频率发生，对于保持高效复制至关重要。以前的动物病毒重编研究只集中在重编码位周围的一小段RNA上，假设所有关键的RNA结构都位于这一小段RNA中。然而，使用包括芜菁皱缩病毒(TCV)在内的模式植物病毒的研究表明，远离重新编码区的RNA元件对重新编码效率起着关键作用。在这里，替代的RNA结构和涉及编码序列的远程RNA：RNA相互作用对于脑心肌炎病毒(EMCV)的重要性将被确定。EMCV属于短小病毒科的心脏病毒属，其中包括最近发现的人类病原体Saffold病毒。TCV还将用于寻找新的内部核糖体进入位点，或IRES，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病毒核糖体重新编码和IRES功能的理解。我将在拟议的研究中解决以下问题：1)EMCV中高效记录所需的长距离RNA：RNA相互作用吗？利用全长基因组结构，将开发一种体外和体内翻译报告分析，以确定扰乱预测的远程相互作用对移码的影响。反向遗传学将被用来确定破坏远程RNA：RNA相互作用是否对病毒积累有害。2)在EMCV重新编码区是否存在替代的RNA结构？对于TCV存在关键的替代编码结构，并且通过折叠算法预测对于EMCV存在类似的结构。形状RNA结构探测将用于确定EMCV重新编码区的结构，无论是在体外还是在体内。将使用体外和体内翻译试验来确定可供选择的结构在重新编码中的重要性。3)在TCV中定位新的IRES。在体外翻译实验中，外壳蛋白和新的RdRp亚型从内部启动表达。负责外壳蛋白或RdRp异构体表达的IRES序列将通过SHAPE、突变和体外翻译分析进行实验确定。RdRp亚型对TCV积累的重要性也将被确定。拟议的研究将极大地有利于核糖体重新编码和IRES相关领域，因为它将采用一种创新的方法，将动物和植物病毒研究联系起来。

 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.