Translational control of gene expression during poxvirus infection

描述（由申请人提供）：在翻译水平上的基因表达的转录后调节对于在包括病毒感染在内的许多生物学环境中蛋白质产生的空间和时间控制是重要的。真核生物翻译起始因子（eIF）F将核糖体连接到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，并且被多种病毒靶向。事实上，由于它们绝对依赖于它们的宿主翻译系统，并且它们可以相对容易地被遗传操纵，病毒系统已经证明在发现和解剖基本生物过程中是非常宝贵的，并且已经定义了许多在感染和未感染细胞中操作的翻译控制范例。然而，仍有许多需要了解的机制，参与调节翻译起始，特别是如何选择性和本地化的mRNA翻译控制。牛痘病毒（VacV）是大的双链DNA病毒的痘病毒家族的成员，其仅在被称为复制区室或病毒工厂的膜结合结构内的感染细胞的细胞质中复制。在这个提议中，我们的目标是利用VacV的分区复制，其mRNA被加帽和聚腺苷酸化类似于它们的宿主对应物，研究翻译因子激活和再分配的机制，以及它们对病毒和宿主蛋白质合成的影响。最近，我们已经表明，VacV激活PI 3 K-Akt-mTOR信号转导，以抑制翻译阻遏蛋白，并增强宿主eIF 4F复合物的形成。这伴随着eIF 4F的核心组分重新分布到病毒工厂内的离散区域，其中认为发生病毒蛋白的合成。我们的初步数据已经确定了一种病毒蛋白，I3结合eIF 4F支架蛋白，eIF 4G，这两种因子共定位在病毒工厂的相同区域内。虽然VacV诱导宿主蛋白质合成的全球抑制，我们初步的多核糖体分析表明，某些宿主mRNA的翻译不仅持续存在，但在某些情况下，增加。我们建议I3在调节eIF 4F分布和局部蛋白质合成中的作用。此外，我们将确定帽结合eIF 4F亚基，eIF 4 E在特定的宿主蛋白质的选择性合成中的作用，并测试其对感染的功能重要性。最后，我们将探讨如何 上游PI 3 K-Akt-mTOR信号传导在VacV的区室化复制期间起作用，补充了我们对eIF 4F再分布的研究，以提供痘病毒感染期间这种帽结合复合物在选择性宿主和病毒mRNA翻译中的作用的综合分析。总的来说，这些目标将为痘病毒的复制提供重要的见解，痘病毒在世纪杀死了估计3亿至5亿人，有可能发现新的治疗靶点，并且还将有助于我们更广泛地理解局部和选择性mRNA翻译控制的一般机制。

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.