Translational control of gene expression during poxvirus infection

在翻译水平上的基因表达的转录后调节是空间和生物学的核心。 在许多生物学背景下，包括病毒感染，蛋白质生产的时间控制。真核 翻译起始因子（eIF）4F，其将核糖体桥接至mRNA的5'甲基化帽， 提供解旋酶活性以促进核糖体扫描，在调节帽依赖性 翻译起始并被多种病毒靶向。事实上，由于它们对宿主的绝对依赖 翻译系统和相对容易的基因操作，病毒系统有 在发现和解剖基本生物过程中被证明是非常宝贵的，并定义了许多 在感染和未感染细胞中均起作用的翻译控制范例。然而，仍有许多工作要做， 了解参与调节翻译起始的机制，特别是如何选择性和 mRNA的翻译受到控制。牛痘病毒（VacV）是痘病毒大家族的成员， 双链DNA病毒，只在膜内感染细胞的细胞质中复制， 被称为复制区室或病毒工厂的结合结构。在本提案中，我们的目标是利用 VacV的区室化复制，其mRNA与其宿主相似地被加帽和聚腺苷酸化 研究翻译因子激活和再分布的机制，以及它们对病毒感染的影响。 和宿主蛋白质合成。最近，我们已经证明VacV激活PI 3 K-Akt-mTOR信号传导， 抑制翻译阻遏蛋白并增强宿主eIF 4F复合物的形成。这是 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， 病毒蛋白质的合成被认为发生。我们的初步数据已经确定了一种病毒蛋白，I3， eIF 4F支架蛋白eIF 4G和这两种因子共定位在病毒的相同区域内， 工厂虽然VacV诱导宿主蛋白质合成的全面抑制，我们的初步多核糖体， 分析表明，某些宿主mRNA的翻译不仅持续存在，而且在选择的情况下增加。我们 建议表征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在选择性合成 特异性宿主蛋白质，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， 补充我们对eIF 4F再分配的研究，以全面分析这一上限的作用- 结合复合物的选择性宿主和病毒的mRNA翻译在痘病毒感染。总的来说，这些目标将 为痘病毒的复制提供了重要的见解，痘病毒在2009年造成约3亿至5亿人死亡。 20世纪世纪，具有发现新的治疗靶点的潜力，也将有助于我们更广泛的 了解局部和选择性mRNA翻译控制的一般机制。

Post-transcriptional regulation of gene expression at the level of translation is central to the spatial and temporal control of protein production in many biological contexts, including viral infection. The eukaryotic translation initiation factor (eIF) 4F, 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 protein, 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.