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

描述（由申请方提供）：具有线性双链DNA基因组的病毒，如腺病毒（Ad）和疱疹病毒，会遇到许多可能严重抑制病毒复制的宿主细胞反应。该建议集中于腺病毒E4-ORF 3蛋白，其通过抵消细胞抗病毒应答来促进病毒复制。近年来，腺病毒被认为是免疫功能低下患者的重要病原体。没有针对Ad感染的病毒特异性治疗。因此，充分了解宿主对Ad感染的反应以及用于抑制这些反应的病毒策略以促进抗病毒疗法的发展变得越来越重要。Ad E4-ORF 3蛋白通过将多种细胞蛋白重新定位到核结构（称为轨道）中以抑制其活性来发挥功能。E4-ORF 3在感染期间抑制DNA损伤反应并抵消干扰素（IFN）反应。E4-ORF 3蛋白诱导两种细胞蛋白Nbs 1和Mre 11的类小泛素化，这两种蛋白是细胞DNA损伤反应中至关重要的效应物。E4-ORF 3也结合SUMO，并使用SUMO结合基序将细胞蛋白重新定位到核轨道中。这些结果将E4-ORF 3置于细胞类小泛素化系统的连接处。SUMO对蛋白质的翻译后修饰调节着多种细胞过程，包括转录、DNA复制、DNA修复、亚细胞定位和泛素化。E4-ORF 3诱导细胞蛋白类小泛素化可能用于抑制宿主抗病毒活性。建议进行研究来探讨E4-ORF 3诱导Nbs 1和Mre 11类小化的机制、这些修饰的功能后果，并鉴定和研究E4-ORF 3诱导类小化的其他细胞蛋白质。E4-ORF 3蛋白与SUMO的相互作用可能代表了E4-ORF 3将SUMO化的细胞蛋白招募到核轨道中的基础，并提出实验来验证这一想法。E4-ORF 3蛋白通过隔离蛋白PML和Daxx（两种突出的抗病毒效应物）来抑制Ad感染期间的IFN应答。将研究PML和Daxx在IFN诱导的抗病毒状态期间抑制Ad复制的机制。将确定E4-ORF 3的另外两个靶点，转录抑制因子TIF 1 <$和TIF 1 <$在IFN应答期间的作用。E4-ORF 3蛋白募集大量与各种功能相关的不同细胞蛋白进入核轨道。 这种小病毒蛋白如何实现这一点尚不清楚。将对野生型和突变型E4-ORF 3蛋白进行物理分析，目的是研究E4-ORF 3蛋白结构与功能的关系。总的来说，这些研究将为干扰病毒复制的细胞机制和抵消这些影响的病毒反应提供独特的见解。

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.