Control of cap-independent translation by a viral 3' UTR

描述（由申请人提供）：所有病毒都依赖于宿主的翻译（蛋白质合成）机制。出于这个原因，宿主细胞已经进化出许多抗病毒机制，这些机制关闭或以其他方式调节翻译。在分子军备竞赛中，病毒反过来也进化出绕过宿主翻译控制的方法。正是病毒复制的这一重要步骤是本提案的重点。宿主mRNA含有5'“帽”结构和3' poly（A）尾，其与将核糖体募集到mRNA的翻译起始因子相互作用。相比之下，大多数RNA病毒的RNA是无帽的，因此它们进化出了RNA结构，通过非经典的、不依赖帽的机制来募集核糖体。许多未加帽的植物病毒RNA在3'非翻译区含有帽独立翻译元件（CITE），其促进在基因组的5'末端的有效核糖体进入。在NIH资助的研究中，PI的实验室表明，这是由3' CITE（结合翻译起始因子）和5'非翻译区之间的长距离碱基配对促进的。CITE RNA结构如何使其以高亲和力结合翻译起始因子，导致核糖体的募集，这一问题尚未得到解答。在这里，将应用各种方法来确定两个不相关的3'CITE的结构要求，以及它们相互作用的翻译因子。这些包括（i）大麦黄矮病毒样翻译元件（BTE），其结合并需要起始因子eIF 4G而不是eIF 4 E;和（ii）黍花叶病毒样翻译元件（PTE），其结合并需要eIF 4 E-先前已知仅结合5'帽结构的蛋白质。这三个目标都可以独立完成，但从每个目标中获得的知识将用于其他两个目标。目的我使用多个，因子耗尽的突变CITEs和突变同源翻译因子与它们相互作用的翻译系统。这将揭示关键的核苷酸和氨基酸所需的 互动和翻译功能。第二个目标是使用多种方法来测量突变的CITEs与突变的翻译因子的相互作用。第三个目标是通过离子依赖性RNA折叠和X射线晶体学方法以高分辨率确定CITE结构。该项目将为病毒接管细胞的方式提供新的理解，这反过来可能为抗病毒药物提供潜在的靶点。虽然这项工作的重点是模式植物病毒，但许多正在生长的人类病毒，如登革热和丙型肝炎病毒，也使用类似的机制。此外，这项工作将为翻译机制如何工作提供新的见解，而翻译系统在植物和动物之间是高度保守的。例如，PTE在哺乳动物细胞中起作用，我们将使用人类细胞和提取物来研究它如何使用eIF 4 E来篡夺核糖体。过度活跃的eIF 4 E导致肿瘤，限制其功能可抑制多种类型的癌症。紧密结合的PTE RNA可以为设计eIF 4 E抑制性癌症治疗剂提供结构知识。

DESCRIPTION (provided by applicant): All viruses depend on the host's translation (protein synthesis) machinery. For this reason, host cells have evolved numerous antiviral mechanisms that shut down or otherwise regulate translation. In the molecular arms race, viruses, in turn, have evolved ways to bypass host translational control. It is this essential step in virus replication that is the focus of this proposal. Host mRNAs contain a 5' "cap" structure and a 3' poly(A) tail that interact with translation initiation factors which recruit the ribosome to the mRNA. In contrast, the RNAs of most RNA viruses are uncapped, so they have evolved RNA structures that recruit the ribosome by noncanonical, cap-independent mechanisms. Many uncapped plant viral RNAs contain a cap-independent translation element (CITE) in the 3' untranslated region that facilitates efficient ribosome entry at the 5' end of the genome. In NIH-funded research the PI's lab showed that this is facilitated by long-distance base pairing between the 3' CITE, which binds a translation initiation factor, and the 5' untranslated region. Unanswered is how the CITE RNA structure causes it to bind a translation initiation factor with high affinity, leading to recruitment of the ribosome. Here, a variety of approaches will be applied to determine the structural requirements of two unrelated 3' CITEs, and the translation factors with which they interact. These include (i) the Barley yellow dwarf virus-like translation element (BTE) which binds and requires initiation factor eIF4G and not eIF4E; and (ii) the Panicum mosaic virus-like translation element (PTE), which binds and requires eIF4E - a protein known previously to bind only to the 5' cap structure. The three aims all can be performed independently, but the knowledge gained from each will feed into the other two aims. Aim I uses multiple, factor-depletable translation systems of mutant CITEs and mutant cognate translation factors with which they interact. This will reveal the key nucleotides and amino acids required for interaction and translation function. The second aim uses a variety of methods to measure the interactions of the mutant CITEs with mutant translation factors. The third aim will determine CITE structure at high resolution by ion-dependent RNA folding and X-ray crystallography methods. This project will provide a new understanding of the way in which viruses take over the cell, which may, in turn, suggest potential targets for antiviral drugs. Although this work focuses on model plant viruses, many growing human viruses such as dengue and hepatitis C viruses use similar mechanisms. Also, this work will shed new light on how the translational machinery works, and the translation system is extremely highly conserved between plants and animals. For example, the PTE functions in mammalian cells and we will use human cells and extracts to study how it uses eIF4E to usurp the ribosomes. Over-active eIF4E causes tumors and restriction of its function inhibits many types of cancers. The tightly binding PTE RNA may provide structural knowledge for design of eIF4E-inhibiting cancer therapeutics.