Regulation of Nuclear Signaling Pathways by the Adenovirus E4-ORF3 Protein

描述（由申请人提供）：具有线性，dsDNA基因组的病毒，如腺病毒（Ad）和疱疹病毒，遇到许多可能严重抑制病毒复制的宿主细胞反应。本研究的重点是通过对抗细胞抗病毒反应促进病毒复制的腺病毒E4-ORF3蛋白。近年来，腺病毒已被认为是免疫功能低下患者的重要病原体。目前尚无针对Ad感染的病毒特异性治疗方法。因此，为了促进抗病毒治疗的发展，充分了解宿主对Ad感染的反应和用于抑制这些反应的病毒策略变得越来越重要。Ad E4-ORF3蛋白通过将多种细胞蛋白重新定位到核结构（称为轨道）中来抑制其活性。在感染期间，E4-ORF3抑制DNA损伤反应并抵消干扰素（IFN）反应。E4-ORF3蛋白诱导两种细胞蛋白Nbs1和Mre11的sumo化，这两种蛋白在细胞DNA损伤反应中是至关重要的效应物。E4-ORF3也结合SUMO并利用SUMO结合基序将细胞蛋白重新定位到核轨道上。这些结果表明E4-ORF3处于细胞酰化系统的联系中。SUMO通过翻译后蛋白修饰调节多种细胞过程，包括转录、DNA复制、DNA修复、亚细胞定位和泛素化。E4-ORF3诱导细胞蛋白sumo化可能用于抑制宿主抗病毒活性。我们将进一步研究E4-ORF3诱导Nbs1和Mre11 sumoylation的机制，以及这些修饰的功能后果，并鉴定和研究其他由E4-ORF3诱导sumoylation的细胞蛋白。E4-ORF3蛋白与SUMO的相互作用可能代表了E4-ORF3将sumylated细胞蛋白招募到核轨道的基础，并提出了实验来验证这一想法。在Ad感染期间，E4-ORF3蛋白通过隔离蛋白PML和Daxx抑制IFN反应，这两种蛋白是两种主要的抗病毒效应器。在ifn诱导的抗病毒状态下，PML和Daxx抑制Ad复制的机制将被研究。在IFN应答过程中，E4-ORF3的另外两个靶点，转录抑制因子TIF1¿和TIF1¿的作用将被确定。E4- ORF3蛋白将大量与多种功能相关的细胞蛋白招募到核轨道中。

DESCRIPTION (provided by applicant): Viruses with linear, dsDNA genomes, such as the adenoviruses (Ad) and herpesviruses, encounter a number of host cell responses that may severely inhibit virus replication. This proposal focuses on the adenovirus E4-ORF3 protein which promotes virus replication by counteracting cellular antiviral responses. Adenoviruses have been recognized in recent years as significant pathogens in immunocompromised patients. There is no virus-specific therapy for Ad infection. It has become increasingly important, therefore, to fully understand host responses to Ad infection and viral strategies used to inhibit these responses in order to foster the development of antiviral therapies. The Ad E4-ORF3 protein functions by relocalizing a variety of cellular proteins into nuclear structures, referred to as tracks, to inhibit their activities. E4-ORF3 inhibits a DNA damage response and counteracts an interferon (IFN) response during infection. The E4-ORF3 protein induces the sumoylation of two cellular proteins, Nbs1 and Mre11, which are critically important effectors in a cellular DNA damage response. E4-ORF3 also binds SUMO and uses SUMO binding motifs to relocalize cellular proteins into nuclear tracks. These results place E4-ORF3 at the nexus of the cellular sumoylation system. Post-translational protein modification by SUMO regulates diverse cellular processes including transcription, DNA replication, DNA repair, subcellular localization, and ubiquitination. The induction of cellular protein sumoylation by E4-ORF3 likely is used to inhibit host antiviral activities. Studies are proposed to investigate the mechanism by which E4-ORF3 induces Nbs1 and Mre11 sumoylation, the functional consequences of these modifications, and identify and investigate other cellular proteins whose sumoylation is induced by E4-ORF3. The interaction of the E4-ORF3 protein with SUMO may represent the foundation by which E4-ORF3 recruits sumoylated cellular proteins into nuclear tracks and experiments are proposed to test this idea. The E4-ORF3 protein inhibits an IFN response during Ad infection by sequestering the proteins PML and Daxx, two prominent antiviral effectors. The mechanism(s) by which PML and Daxx inhibit Ad replication during the IFN-induced antiviral state will be investigated. The contribution of two other targets of E4-ORF3, transcriptional repressors TIF1¿ and TIF1¿, during an IFN response will be determined. The E4- ORF3 protein recruits a large number of different cellular proteins, associated with a variety of functions, into nuclear tracks. How this small viral protein accomplishes this is unknown. Physical analyses of wild type and mutant E4-ORF3 proteins will be conducted with the goal to study E4-ORF3 protein structure as it relates to function. Collectively, these studies will provide unique insight into cellular mechanisms that interfere with virus replication and viral responses that counteract these effects.