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

烟草蚀刻病毒（TEV）是一种多型病毒，是小核糖核酸病毒超群的一员，感染植物。与脑心肌炎病毒（EMCV）和脊髓灰质炎病毒一样，TEV的基因组RNA是一种聚腺苷化的mRNA，天然缺乏5'帽结构，但仍能有效翻译。TEV 5'-前导体足以赋予mRNA与帽无关的翻译功能，它在功能上类似于帽，因为它与聚(a)尾部相互作用以促进有效的翻译。当TEV 5'-引子存在于双反子间区时，TEV 5'-引子还能促进双反子mRNA的5'-远端（即第二顺子）的翻译，这一观察结果表明TEV 5'-引子具有内部核糖体进入位点（IRES）的功能。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介导的帽无关翻译的机制尚未被研究过。观察到TEV是一种自然无帽的mRNA，已经进化出一个5'-先导序列，赋予帽独立翻译，即使定位在双双子mRNA的顺反子间区域也能促进内部起始，并且需要eIF4G才能实现IRES功能，这表明这一小核糖核酸超家族成员可能与动物小核糖核酸病毒（如EMCV和FMDV）的IRES功能相似。该项目的目标是确定TEV IRES功能促进植物中帽非依赖性翻译的机制。该项目将首次确定植物病毒IRES是否具有与动物小核糖核酸病毒IRES相同的功能。由于动物小核糖核酸病毒IRES之间的机制差异，该研究将确定TEV IRES的进化是否更类似于EMCV的IRES（可以直接招募eIF4G）或脊髓灰质炎病毒的IRES（可能需要其他因素的帮助来招募eIF4G）。该研究将确定TEV IRES的结构，并研究eIF4G是直接结合还是通过额外的交易作用因子被招募到TEV IRES上。TEV的IRES比动物小核糖核酸病毒的IRES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如何以帽独立的方式有效地招募翻译机制，将为了解高等真核生物中IRES功能的最低要求提供重要见解。

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.