小麦黄花叶病毒（WYMV）不依赖帽子翻译的差异化调控机制

The translation of viral proteins of RNA viruses relys on host translation machinery, which favors mRNA with the 5' cap and 3' poly(A). The 5' terminus of some RNA viruses without the 5' cap is linked by viral genome-linked protein(VPg), which is essential for viral replication in vivo. Meanwhile, VPg-encoded RNA viruses often have another cis-element internal ribosome entry site(IRES), which plays crucial role in the translation of viral proteins through recruiting translation initiation complex. It is suggested that VPg and IRES may regulate the translation of viral proteins synergistically. Extensive effort to identify the role of VPg and IRES on translation were performed in vertebrate RNA viruses,while the information of VPg and IRES in the translation of plant RNA viruses was narrow. .In this study, one bipartite virus---Wheat yellow mosaic virus(WYMV) will be used and its cap-independent translation will be identified using methods of virology, molecular biology, biochemistry and RNA structurology as following contents..Part I: 5'UTR of RNA1 or RNA2 of different WYMV isolates will be inserted into upstream of Fluc and its potential IRES activity will be evaluated using in vitro translation of Fluc. In addition, the role of 3'UTRs on translation will be analyzed in the absence or presence of corresponding 5'UTRs. .Part II: The RNA structure of WYMV RNA1 5'UTR with high nucleotide identity will be probed using in-line probing. The core IRES characteristic of RNA1 5'UTR will be identified based on the structure and in vitro translation of mutants. In addition, host factors recruited by IRES will also be identified..Part III: The synergistic regulation of 5'UTR and 3'UTR on translation will be analyzed. The potential RNA-RNA interaction sites between 5'UTR and 3'UTR will be mapped using in-line probing and EMSA..Part IV: The RNA structure of RNA2 5'UTR of different WYMV isolates with high nucleotide variation will be probed using in-line probing. The core IRES characteristic of RNA2 5'UTR differentiallly regulating translation will be identified based on the structure and in vitro translation of mutants..Part V : The interaction between WYMV VPg and eIF4E and its role on translation will be analyzed. In addition, the relationship between VPg and IRES in translation will be analyzed..Part VI: Reverse genetic system of WYMV will be contructed and used to evaluate the role of VPg and IRES on viral replication in vivo. .All results will be used to explain the different mechanism of cap-independent translation between RNA1 IRES and RNA2 IRES, different RNA2 IRESes as well as VPg and IRESes. It will enrich the data of cap-independent translation of plant RNA viruses with VPg. In addition, it will also provide the potential target for the management of virus disease caused by WYMV.

针对植物RNA病毒的翻译调控元件---内部核糖体进入位点（IRES）研究不深入的现状，围绕VPg结合型RNA病毒不依赖帽子翻译的科学问题，采用病毒学、分子生物学和RNA结构学的方法，以双分体病毒小麦黄花叶病毒（WYMV）为对象，主要研究：①不同WYMV分离物RNA1与RNA2的非翻译区（UTR）对翻译的调控②解析序列保守型RNA1IRES的结构，明确调控翻译的核心结构特征，分析与之互作的宿主翻译起始因子③分析IRES相关的远距离RNA-RNA互作及对翻译的调控④解析序列变异型RNA2IRES的结构，明确序列、结构差异与翻译调控效率的关系⑤分析VPg与IRES在翻译调控的关系⑥创建反向遗传学体系，验证翻译调控的核心元件。研究结果将揭示WYMVIRES之间以及VPg和IRES之间的差异化翻译调控机制，丰富植物RNA病毒不依赖帽子翻译调控的数据，也可为小麦黄花叶病毒病的防治提供靶标。

内部核糖体起始位点（IRES）是调控不依赖帽子翻译的2类元件之一，在病毒和宿主细胞的mRNA中均有报道。本研究以小麦黄花叶病毒WYMV为研究对象，全面分析其RNA1和RNA2的IRES元件结构特征及其调控翻译的分子机制。WYMV不同分离物RNA1 5’UTR的IRES活性基本一致，而WYMV不同分离物RNA2 5’UTR的IRES活性则存在明显差异，最大有7倍的差异。RNA结构分析表明，RNA1和RNA2的IRES核心活性区均包括2个茎环及其中间连接区，均通过结合小麦eIF4E调控不依赖帽子翻译；IRES活性均受到相应的3’UTR的正向协同调控，其分子基础为5’UTR和3’UTR的远距离RNA-RNA互作。.RNA1 IRES的茎环1（H1）的茎部稳定性和环部碱基特异性与IRES活性正相关，茎环2的环部碱基特异性与IRES活性正相关，茎环2的环部碱基是小麦eIF4E的结合位点。H1中的C55、C66，H2中的C105、C108与中间连接区的G73、G79和G85存在数量不对等的不连续碱基配对关系，形成三级稳态结构，是世界上首次报道IRES元件的三级平衡稳态结构，暗示WYMV RNA1翻译调控的进化方向是适度，而不是翻译水平的最大化。.不同分离物RNA2的IRES活性呈现明显差异，不同分离物RNA2的IRES共有的活性调控区为H1的顶部茎环，差异化的活性调控区位于H1的基部茎和H2的顶环碱基数量和特异性。H1的基部茎呈现打开状态正调控IRES活性差异，H2顶环的七碱基特异性正调控IRES活性差异。活性差异的结构基础是H1的基部茎的开合状态和H2顶环的碱基数量以及特异性差异，分子机制为结合小麦eIF4E的位点数量差异及多个结合位点的协同效应。H1顶环是不同分离物IRES共有的eIF4E结合位点；H2顶环是差异化的eIF4E结合位点，七特异性碱基可以结合eIF4E，而三碱基无法结合eIF4E。并且H1顶环和H2顶环的eIF4E结合位点存在协同效应，结构基础是H1的基部茎打开状态，以保证不同eIF4E结合位点可协同结合eIF4E。.WYMV的IRES研究暗示，RNA1和 RNA 2的IRES元件的进化策略不同，不同分离物RNA1的IRES元件活性基本一致，而不同分离物RNA2的IRES元件呈现明显的结构和活性差异，可能和其负责表达的病毒蛋白性质有关。