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

描述(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，宿主细胞进化出了许多可以关闭或以其他方式调节翻译的抗病毒机制。在分子军备竞赛中，病毒反过来进化出绕过宿主翻译控制的方法。正是病毒复制中的这一关键步骤是这项提案的重点。宿主mRNAs含有一个5‘端的“帽”结构和一个3’端的聚(A)尾，它们与翻译起始因子相互作用，从而将核糖体招募到mRNA上。相比之下，大多数RNA病毒的RNA是无帽的，因此它们进化出了通过非规范的、帽无关的机制招募核糖体的RNA结构。许多无帽植物病毒RNA在3‘端非翻译区含有一个帽子非依赖的翻译元件(CITE)，它有助于核糖体在基因组的5’端高效进入。在NIH资助的研究中，PI的实验室表明，这是由3‘Cite和5’非翻译区之间的远程碱基配对促进的，3‘Cite结合了翻译起始因子。尚未回答的是，Cite RNA结构如何使它与高亲和力的翻译起始因子结合，导致核糖体的招募。在这里，将采用多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素。它们包括(I)大麦黄矮病毒样翻译元件(BTE)，它结合并需要启动因子eIF4G而不是eIF4E；以及(Ii)Panicum花叶病毒样翻译元件(PTE)，它结合并需要eIF4E-一种先前已知仅与5‘帽结构结合的蛋白质。这三个目标都可以独立执行，但从每个目标获得的知识将反馈到另外两个目标。目的I使用突变CITES及其相互作用的突变同源翻译因子的多个可耗尽因子的翻译系统。这将揭示关键核苷酸和氨基酸所需的 互动和翻译功能。第二个目的是使用各种方法来测量突变的CITE与突变的翻译因子之间的相互作用。第三个目标将通过离子依赖的RNA折叠和X射线结晶学方法在高分辨率下确定Cite的结构。该项目将提供对病毒接管细胞方式的新理解，这反过来可能为抗病毒药物提供潜在的靶点。尽管这项工作的重点是模式植物病毒，但许多正在成长的人类病毒，如登革热和丙型肝炎病毒，使用类似的机制。此外，这项工作将为翻译机器如何工作提供新的线索，而翻译系统在植物和动物之间是极其保守的。例如，PTE在哺乳动物细胞中发挥作用，我们将使用人类细胞和提取物来研究它如何使用eIF4E来篡夺核糖体。过度激活的eIF4E会导致肿瘤，限制其功能可以抑制许多类型的癌症。紧密结合的PTE RNA可能为eIF4E抑制肿瘤治疗药物的设计提供结构信息。

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.