Structure/Function Analysis of the TEV IRES and its Interaction With eIF4G, eIF4A, and eIF4B

烟草蚀刻病毒(TEV)是侵染植物的一种马铃薯Y病毒，属于小核糖核酸病毒超群的正链RNA病毒。与脑心肌炎病毒(EMCV)和脊髓灰质炎病毒一样，TEV的基因组RNA是一种多腺苷化的mRNA，天然缺乏5‘帽结构，但仍可有效翻译。TEV 5‘-先导足以赋予mRNA不依赖帽的翻译，它在功能上类似于帽，因为它与聚(A)尾巴相互作用，促进有效的翻译。当双顺反子mRNA存在于顺反子间区时，TEV 5‘-先导还促进体内双顺反子5’-远端(即第二个)顺反子的翻译，这表明TEV 5‘-先导作为内部核糖体进入位点(IRES)发挥作用。TEV IRES功能需要翻译起始因子eIF4G。对于TEV或任何其他植物病毒，IRES介导的帽非依赖性翻译的机制尚未被研究。TEV是一种自然无上限的mRNA，它进化出一个5‘-前导序列，提供帽子无关的翻译，即使定位在双顺反子mRNA的顺反子间区也具有促进内部起始的功能，并且需要eIF4G才能实现IRES功能，这表明这个小核糖核酸病毒超家族的成员可能与动物小核糖核酸病毒(如EMCV和FMDV)所描述的IRES功能相似。该项目的目标是确定TEV IRES功能促进植物中不依赖帽的翻译的机制。该项目将首次确定植物病毒IRES是否具有动物小核糖核酸病毒IRES的功能。由于动物微小核糖核酸病毒IRESS的机制不同，研究将确定TEV IRES的进化更类似于EMCV的IRES(可以直接招募eIF4G)或脊髓灰质炎病毒(可能需要其他因素的协助才能招募eIF4G)。这项研究将确定TEV IRES的结构，并调查eIF4G是直接与TEV IRES结合还是通过额外的反式作用因子招募到TEV IRES。TEV的IRES比动物微小核糖核酸病毒的IRESS小得多，这表明IRES不需要广泛的序列来招募翻译机制。阐明这种简化的IRES如何以帽不依赖的方式有效地招募翻译机制，将为了解高等真核生物对IRES功能的最低要求提供重要的见解。

Tobacco etch virus (TEV) is a potyvirus, a member of the picornavirus supergroup of positive-strand RNA viruses, which infects plants. Like encephalomyocarditis virus (EMCV) and poliovirus, the genomic RNA of TEV is a polyadenylated mRNA that naturally lacks a 5' cap structure but is nevertheless efficiently translated. The TEV 5'-leader is sufficient to confer cap-independent translation to an mRNA and it is functionally analogous to a cap in that it interacts with the poly(A) tail to promote efficient translation. The TEV 5'-leader also promotes translation of the 5'-distal (i.e., second) cistron of a dicistronic mRNA in vivo when it is present in the intercistronic region, an observation indicating that the TEV 5'-leader functions as an internal ribosome entry site (IRES). The translation initiation factor eIF4G is required for TEV IRES function. The mechanism underlying IRES-mediated cap-independent translation has not been investigated for TEV or any other plant virus. The observation that the TEV is a naturally uncapped mRNA that has evolved a 5'-leader sequence that confers cap-independent translation, functions even when positioned in the intercistronic region of a dicistronic mRNA to promote internal initiation, and requires eIF4G for IRES function suggests that this member of the picornaviral superfamily may share similarities to IRES function described for animal picornaviruses such as EMCV and FMDV. The goal of this project is to determine the mechanism by which TEV IRES function promotes cap-independent translation in plants. This project will be the first to establish whether a plant viral IRES functions like animal picornaviral IRES. Because of the mechanistic differences among animal picornaviral IRESs, the research will determine whether the evolution of the TEV IRES is more similar to the IRES of EMCV (which can recruit eIF4G directly) or poliovirus (which may require the assistance of other factors to recruit eIF4G). The research will determine the structure of the TEV IRES and investigate whether eIF4G binds directly or is recruited to the TEV IRES by an additional trans-acting factor. The IRES of TEV is substantially smaller than animal picornaviral IRESs, suggesting that an IRES does not require extensive sequence to recruit the translational machinery. Elucidation of how such a simplified IRES can efficiently recruit the translational machinery in a cap-independent manner will provide important insight into the minimum requirement for IRES function in a higher eukaryote.