Translational control of gene expression during poxvirus infection

描述（由申请人提供）：翻译水平上基因表达的转录后调控是包括病毒感染在内的许多生物环境中蛋白质生产的时空控制的核心。真核生物翻译起始因子（eIF） F连接核糖体与mRNA的5'甲基化帽，并提供解旋酶活性以促进核糖体扫描，在调节帽依赖性翻译起始中起关键作用，是多种病毒的靶标。事实上，由于病毒系统完全依赖于宿主翻译系统，并且相对容易进行基因操作，它们在发现和解剖基本生物过程方面被证明是无价的，并定义了许多在感染和未感染细胞中都起作用的翻译控制范式。然而，关于调节翻译起始的机制，特别是如何控制选择性和局部mRNA翻译，仍有许多有待研究。痘苗病毒（VacV）是痘病毒家族的一员，它是一种大型双链DNA病毒，只在被感染细胞的细胞质中复制，这种复制结构被称为复制室或病毒工厂。在本研究中，我们的目标是利用VacV的区隔复制，研究翻译因子的激活和再分配机制，以及它们对病毒和宿主蛋白质合成的影响。最近，我们已经证明，VacV激活PI3K-Akt-mTOR信号通路，使翻译抑制蛋白失活，并增强宿主eIF4F复合物的形成。这伴随着eIF4F的核心成分重新分配到病毒工厂内的离散区域，在那里被认为发生了病毒蛋白的合成。我们的初步数据已经确定了一种结合eIF4F支架蛋白eIF4G的病毒蛋白I3，这两种因子在病毒工厂的同一区域内共定位。虽然VacV诱导宿主蛋白合成的全局抑制，但我们的初步多体分析表明，某些宿主mrna的翻译不仅持续存在，而且在某些情况下增加了。我们提出表征I3在调节eIF4F分布和局部蛋白合成中的作用。此外，我们将确定帽结合eIF4F亚基eIF4E在选择性合成特定宿主蛋白中的作用，并测试其对感染的功能重要性。最后，我们将探讨如何

DESCRIPTION (provided by applicant): Post-transcriptional regulation of gene expression at the level of translation is central to the spatial and temporal control of protein production in may biological contexts, including viral infection. The eukaryotic translation initiation factor (eIF) F, which bridges the ribosome to the 5' methylated cap of the mRNA and provides helicase activity to facilitate ribosome scanning, plays a pivotal role in regulating cap-dependent translation initiation and is targeted by diverse viruses. Indeed, with their absolute dependence on their host translation system and the relative ease with which they can be genetically manipulated, viral systems have proven invaluable in the discovery and dissection of basic biological processes and have defined many translational control paradigms that operate in both infected and uninfected cells. However, much remains to be learned about the mechanisms involved in regulating translation initiation, in particular how selective and localized mRNA translation is controlled. Vaccinia Virus (VacV) is a member of the poxvirus family of large, double stranded DNA viruses that replicate exclusively in the cytoplasm of infected cells within membrane- bound structures termed replication compartments or viral factories. In this proposal we aim to exploit the compartmentalized replication of VacV, whose mRNAs are capped and polyadenylated similar to their host counterparts, to study mechanisms of translation factor activation and redistribution, and their effects on viral and host protein synthesis. Recently, we have shown that VacV activates PI3K-Akt-mTOR signaling to inactivate translational repressor proteins and enhance the formation of host eIF4F complexes. This is accompanied by a redistribution of core components of eIF4F to discrete regions within viral factories where synthesis of viral proteins is thought to occur. Our preliminary data has identified a viral protei, I3 that binds the eIF4F scaffold protein, eIF4G, and both of these factors colocalize within the same regions of viral factories. Although VacV induces global suppression of host protein synthesis, our preliminary polysome profiling shows that translation of certain host mRNAs not only persists but, in select cases, is increased. We propose to characterize the role of I3 in regulating both eIF4F distribution and localized protein synthesis. Furthermore, we will determine the role of the cap-binding eIF4F subunit, eIF4E in the selective synthesis of specific host proteins and test the functional importance of this to infection. Finally, we will explore how upstream PI3K-Akt-mTOR signaling functions during the compartmentalized replication of VacV, complementing our studies of eIF4F redistribution to provide a comprehensive analysis of the role of this cap- binding complex in selective host and viral mRNA translation during poxvirus infection. Overall, these aims will provide important insights into the replication of poxviruses, which killed an estimated 300-500 million people in the 20th century, with the potential to uncover novel therapeutic targets, and will also contribute to our broader understanding of general mechanisms of both localized and selective mRNA translational control.