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

项目主任/主要研究者（最后，第一，中间）：听力，帕特里克 项目摘要 DNA肿瘤病毒腺病毒（Ad）已经进化出不同的机制来靶向宿主信号通路， 以优化感染期间的细胞环境。对Ad复制周期的研究表明， 对转录调控和mRNA加工、蛋白质翻译、细胞 增殖和细胞死亡。对Ad感染的研究也提供了对先天宿主的独特见解 对病毒感染的应答包括DNA损伤应答（DDR）和干扰素（IFN）应答。这 该建议是基于我们对DNA损伤和IFN应答如何影响Ad复制周期的研究 以及广告如何抵消这些反应。这两种途径都对细胞的生命和死亡产生重要影响 决策从细胞和病毒的角度对Ad感染的研究，将提供基本的 深入了解调节细胞活力和增殖的基本过程。该提案的重点是一个高度 保守的Ad调节蛋白E4-ORF3。E4-ORF3靶向的许多基因产物中的突变是 与人类癌症有关。E4-ORF 3的功能是抑制参与DDR的不同细胞效应物， IFN反应通过将这些途径所必需的蛋白质隔离到核内含物中。在此 E4-ORF3改变了多种细胞蛋白的翻译后修饰，以诱导其表达。 通过小泛素样修饰物SUMO修饰。SUMO修饰影响多种细胞 流程.在Ad感染的情况下，E4-ORF3诱导的类小泛素化靶向特异性细胞蛋白， 通过蛋白酶体降解。E4-ORF3还调节IFN信号传导。IFN抑制Ad立即早期基因 通过诱导含有细胞周期调节因子E2F的转录阻遏物复合物表达。IFN- E2F轴是IFN应答过程中限制Ad基因表达的关键。这可能与已知的 IFN的抗增殖特性，并提供了一种手段来了解这种分子机制， 过程本建议的具体目的1是研究Ad5 E4-ORF 3蛋白在细胞内表达的机制。 诱导细胞基质降解。假设Ad5 E4-ORF3蛋白取代了细胞的 蛋白酶体降解系统可以指导细胞蛋白的聚类小泛素化，以靶向它们进行蛋白酶体降解。 这可能是一个细胞的SUMO靶向泛素连接酶（STUbL）参与这一过程。也是 假设Ad利用AAA + ATP酶p97/VCP的酶活性提取细胞蛋白质 从E4-ORF 3核包涵体中释放它们用于蛋白酶体降解。具体目标2是调查 E4-ORF3介导的SUMO化机制。E4-ORF 3作为SUMO E3连接酶发挥功能，E4 延伸酶催化底物的单-接着是多-类小泛素化。假设E4-ORF3 功能通过招募相扑机械及其基板在彼此接近的组装 高级蛋白质复合物Ad5 E4-ORF 3蛋白在其SUMO E3和E4活性方面是独特的。研究 对E4-ORF3功能的研究将揭示宿主蛋白类小泛素化机制的基本见解， 它不仅影响DDR，还影响其他重要过程，如转录和DNA复制。 目的3：探讨IFN抑制Ad基因表达和复制的机制 信号IFN α和IFN γ通过E1A中保守的E2F结合位点抑制Ad5 E1A基因表达 增强子区域和IFN诱导阻遏物E2F复合物与该位点的结合。这与 Ad在IFN存在下建立持续感染的能力。IFN已被用于治疗不同的 但是这些作用是如何发挥的在很大程度上是未知的。这一假设是，干扰素调节 细胞增殖涉及阻遏物E2F复合物的形成。这些复合物的调节， 将研究IFN信号传导。IFN在急性和持续性Ad感染的调节中的作用将被 使用体外和体内模型进行研究。 OMB编号0925 - 0001/0002（2016年3月修订版，批准至2018年10月31日）

Program Director/Principal Investigator (Last, First, Middle): Hearing, Patrick Project Summary The DNA tumor virus adenovirus (Ad) has evolved different mechanisms to target host signaling pathways in order to optimize the cellular environment during infection. Studies of the Ad replication cycle have revealed fundamental insights into the regulation of transcription and mRNA processing, protein translation, cell proliferation, and cell death. Studies of Ad infection also have provided unique insights into innate host responses to viral infection including the DNA damage response (DDR) and an interferon (IFN) response. This proposal is based on our studies of how the DNA damage and IFN responses impact the Ad replication cycle and how Ad counteracts these responses. Both of these pathways critically impact cell life and death decisions. Studies of Ad infection, both from the cellular and viral point of view, will provide fundamental insights into essential processes that regulate cell viability and proliferation. The proposal focuses on a highly conserved Ad regulatory protein E4-ORF3. Mutations in many of the gene products targeted by E4-ORF3 are associated with human cancer. E4-ORF3 functions to inhibit different cellular effectors involved in the DDR and IFN responses by sequestering proteins essential for these pathways into nuclear inclusions. During this process, E4-ORF3 alters the post-translational modification of multiple cellular proteins to induce their modification by the Small Ubiquitin-like Modifier SUMO. SUMO modifications affects diverse cellular processes. In the context of Ad infection, E4-ORF3-induced sumoylation targets specific cellular proteins for degradation by the proteasome. E4-ORF3 also regulates IFN signaling. IFNs repress Ad immediate early gene expression by inducing transcriptional repressor complexes containing the cell cycle regulator E2F. The IFN– E2F axis is critical for restriction of Ad gene expression during IFN responses. This likely relates to the known anti-proliferative properties of IFNs and provides a means to understand the molecular mechanisms of this process. Specific Aim 1 of this proposal is to investigate the mechanism by which the Ad5 E4-ORF3 protein induces degradation of cellular substrates. The hypothesis is that the Ad5 E4-ORF3 protein usurps the cellular sumoylation system to direct poly-sumoylation of cellular proteins to target them for proteasomal degradation. It is likely that a cellular SUMO-targeted ubiquitin ligase (STUbL) is involved in this process. It is also hypothesized that Ad uses the enzymatic activity of the AAA+ ATPase p97/VCP to extract cellular proteins from E4-ORF3 nuclear inclusions to release them for proteasomal degradation. Specific Aim 2 is to investigate the mechanism of E4-ORF3-mediated sumoylation. E4-ORF3 functions as a SUMO E3 ligase and E4 elongase to catalyze mono- followed by poly-sumoylation of substrates. The hypothesis is that E4-ORF3 functions by recruiting the SUMO machinery and its substrates in proximity of one another by assembling higher order protein complexes. The Ad5 E4-ORF3 protein is unique in its SUMO E3 and E4 activities. Studies on E4-ORF3 function will reveal fundamental insights into the mechanisms of host protein sumoylation that impacts not only the DDR, but also other essential processes such as transcription and DNA replication. Specific Aim 3 is to investigate the mechanism of inhibition of Ad gene expression and replication by IFN signaling. IFNα and IFNγ repress Ad5 E1A gene expression via a conserved E2F binding site in the E1A enhancer region and IFNs induce the binding of repressor E2F complexes to this site. This correlates with the ability of Ad to establish a persistent infection in the presence of IFNs. IFNs have been used to treat different malignancies but how these effects are exerted is largely unknown. The hypothesis is that IFN regulation of cellular proliferation involves the formation of repressor E2F complexes. The regulation of these complexes by IFN signaling will be investigated. The role of IFNs in the regulation of acute and persistent Ad infections will be investigated using in vitro and in vivo models. OMB No. 0925-0001/0002 (Rev. 03/16 Approved Through 10/31/2018) Page Continuation Format Page