Control of cap independent translation by a viral 3' UTR

描述(申请人提供)：许多RNA病毒通过绕过细胞翻译控制系统来篡改宿主的蛋白质合成(翻译)机制。细胞mRNAs上的5‘端帽和聚(A)尾与翻译因子相互作用，形成一个封闭的mRNA结构，这是招募核糖体和启动翻译所必需的调控过程。许多病毒RNA通过缺少5‘帽或聚(A)尾而避免了这一控制步骤，而是在通过非规范方式控制翻译的非翻译区(UTRs)中含有序列。了解病毒是如何做到这一点的，可能会导致针对独特的病毒翻译机制的抗病毒药物的开发。本研究的重点是大麦黄矮病毒(BYDV)RNA 3‘端非翻译帽依赖的翻译元件(TE)，它促进了5’端的翻译起始。本研究旨在确定TE(1)如何招募翻译机器，以及(2)如何与启动开始的5‘端进行交流。BYDV RNA通过一种新的方式形成闭环结构：3‘TE和5’UTR之间的直接碱基配对。这种碱基配对是必要的，但不足以调节体内的翻译。第一个目的是确定这种闭环碱基配对对序列变化的耐受性，并在体内区分BYDV RNA序列和模仿5‘帽和PolyA尾巴功能的结构。第二个目标是识别与TE结合的蛋白质，绘制它们的结合位点，并确定它们的作用。初步证据表明，TE可能通过通常仅与5‘端帽结合的因子招募核糖体。待测试的模型是，因子和核糖体被招募到3‘UTR，并通过碱基配对传递到5’端。这项研究将采用(并改进)建立的体外和体内翻译分析、RNA复制分析、RNA结构分析、RNA-蛋白质结合和核糖体结合分析、蛋白质组学和结构导向突变。这项对模型病毒的研究可能有助于控制包括脊髓灰质炎病毒和丙型肝炎病毒在内的许多人类病原体，这些病原体也使用受UTRs之间相互作用调节的帽非依赖性翻译。它也适用于通过UTRs之间的远距离RNA碱基配对来调节基因表达和复制的NidoVirus和FlaviVirus(如登革热、西尼罗河病毒)。最后，本研究将为真核生物翻译机制的研究提供基础性的见解。

DESCRIPTION (provided by applicant): Many RNA viruses usurp the host's protein synthesis (translation) machinery by bypassing the cellular translational control systems. The 5' cap and poly(A) tail on cellular mRNAs interact with translation factors to form a closed-loop mRNA structure in a regulated process necessary to recruit the ribosome and initiate translation. Many viral RNAs avoid this control step by lacking a 5' cap or poly(A) tail and instead harboring sequences in the untranslated regions (UTRs) that control translation by non-canonical means. Understanding how viruses do this could lead to development of antiviral agents specific to the unique viral translation mechanisms. This proposal focuses on the novel cap-independent translation element (TE) in the 3' UTR of barley yellow dwarf virus (BYDV) RNA that facilitates translation initiation at the 5' end. This research aims to determine how the TE (1) recruits translational machinery, and (2) communicates with the 5' end where initiation ensues. BYDV RNA forms the closed-loop structure by a novel means: direct base pairing between the 3' TE and the 5' UTR. This base pairing is necessary but insufficient to mediate translation in vivo. The first aim is to determine the tolerance of this closed-loop base pairing for sequence changes, and to distinguish the BYDV RNA sequences and structures that mimic 5' cap and poly(A) tail functions in vivo. The second aim is to identify the proteins that bind the TE, map their binding sites, and determine their roles. Preliminary evidence indicates that the TE may recruit the ribosome via factors that normally bind only to the 5' cap. The model to be tested is that factors and the ribosome are recruited to the 3' UTR and delivered to the 5' end by base pairing. The research will employ (and improve upon) established in vitro and in vivo translation assays, RNA replication assays, RNA structural analysis, RNA-protein binding and ribosome binding assays, proteomics, and structure-guided mutagenesis. This research on a model virus may contribute to means of controlling many human pathogens, including poliovirus and hepatitis C virus, that also employ cap-independent translation regulated by interactions between the UTRs. It also applies to nidoviruses and flaviviruses (e.g. Dengue, West Nile) that regulate gene expression and replication by long-distance RNA base pairing between UTRs. Finally, the research will provide fundamental insight on eukaryotic translation mechanisms.