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

烟草蚀纹病毒（TEV）是马铃薯Y病毒属的一种，属于正链RNA病毒的小核糖核酸病毒超组成员，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是多腺苷酸化的mRNA，其天然缺乏5'帽结构，但仍然被有效翻译。 TEV 5 '-前导序列足以赋予mRNA帽非依赖性翻译，并且其在功能上类似于帽，因为其与poly（A）尾相互作用以促进有效翻译。 TEV 5 '-前导序列还促进5'-远端（即，第二）当双顺反子mRNA存在于顺反子间区域时，体内双顺反子mRNA的顺反子，观察结果表明TEV 5 '-前导序列作为内部核糖体进入位点（IRES）起作用。 翻译起始因子eIF 4G是TEV IRES功能所必需的。 IRES介导的帽非依赖性翻译的潜在机制尚未针对TEV或任何其他植物病毒进行研究。 TEV是一种天然脱帽的mRNA，其已经进化出赋予帽非依赖性翻译的5 '-前导序列，即使位于双顺反子mRNA的顺反子间区域也能发挥作用以促进内部起始，并且IRES功能需要eIF 4G，这一观察结果表明，小核糖核酸病毒超家族的该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。 该项目将是第一个确定植物病毒IRES是否像动物小核糖核酸病毒IRES一样发挥功能的项目。 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的进化是否与EMCV（可以直接招募eIF 4G）或脊髓灰质炎病毒（可能需要其他因素的帮助来招募eIF 4G）的IRES更相似。 该研究将确定TEV IRES的结构，并研究eIF 4G是否直接结合或通过额外的反式作用因子招募到TEV IRES。 TEV的IRES显著小于动物小核糖核酸病毒IRES，表明IRES不需要广泛的序列来募集翻译机器。 阐明这样一个简化的IRES可以有效地招募翻译机器在帽独立的方式将提供重要的洞察IRES功能在高等真核生物中的最低要求。

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.