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。因此，宿主细胞已经进化出许多抗病毒机制来关闭或以其他方式调节翻译。在分子军备竞赛中，病毒又进化出了绕过宿主转译控制的方法。病毒复制的这一重要步骤正是本提案的重点。宿主mRNA包含一个5‘“帽”结构和一个3’ poly(a) tail，它们与翻译起始因子相互作用，将核糖体招募到mRNA中。相比之下，大多数RNA病毒的RNA是无帽的，因此它们进化出的RNA结构通过非规范的、不依赖帽盖的机制来招募核糖体。许多无帽植物病毒rna在3‘非翻译区含有一个帽独立翻译元件（CITE），有助于有效地进入基因组的5’端核糖体。在美国国立卫生研究院资助的研究中，PI的实验室表明，结合翻译起始因子的3‘ CITE和5’非翻译区域之间的长距离碱基配对促进了这一过程。CITE RNA结构如何使其与高亲和力的翻译起始因子结合，从而导致核糖体的招募，目前尚无答案。在这里，将采用各种方法来确定两个不相关的3' CITEs的结构要求，以及它们相互作用的翻译因素。这些包括(i)大麦黄矮病毒样翻译元件（BTE），它结合并需要起始因子eIF4G而不是eIF4E；（ii） Panicum花叶病毒样翻译元件（PTE），其结合并需要eIF4E -一种已知仅与5'帽结构结合的蛋白质。这三个目标都可以独立实现，但从每个目标中获得的知识将为其他两个目标提供帮助。Aim I使用突变的CITEs和与其相互作用的突变同源翻译因子的多个因子耗尽翻译系统。这将揭示所需的关键核苷酸和氨基酸

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.