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

项目主任/首席调查员(最后、第一、中间)：听证，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的信号通路 以优化感染期间的细胞环境。对广告复制周期的研究揭示了 对转录和信使核糖核酸的调控、蛋白质翻译、细胞 增殖和细胞死亡。对Ad感染的研究也为研究先天宿主提供了独特的见解 对病毒感染的反应包括DNA损伤反应(DDR)和干扰素(干扰素)反应。这 该建议是基于我们对DNA损伤和干扰素反应如何影响广告复制周期的研究 以及广告如何抵消这些反应。这两条途径对细胞的生存和死亡都有至关重要的影响 决定。从细胞和病毒的角度对Ad感染的研究将提供基础 对调节细胞存活和增殖的基本过程的洞察。该提案将重点放在一个高度 保守的Ad调节蛋白E4-ORF3。E4-ORF3靶向的许多基因产物的突变是 与人类癌症有关。E4-ORF3可抑制参与DDR的不同细胞效应物 干扰素通过将这些途径所必需的蛋白质隔离到核包涵体中而产生反应。在此期间 过程中，E4-ORF3改变多个细胞蛋白的翻译后修饰，以诱导其 用类似泛素的小修饰剂相扑进行修饰。相扑修改影响不同的细胞 流程。在Ad感染的背景下，E4-ORF3诱导的苏莫化作用针对特定的细胞蛋白 蛋白酶体的降解。E4-ORF3还调节干扰素信号转导。干扰素抑制Ad即刻早期基因 通过诱导含有细胞周期调节因子E2F的转录抑制物复合体来表达。干扰素- 在干扰素应答过程中，E2F轴是限制Ad基因表达的关键。这很可能与已知的 并为了解其抗增殖的分子机制提供了手段。 进程。本提案的具体目标1是研究Ad5 E4-ORF3蛋白 导致细胞底物的降解。假设Ad5E4-ORF3蛋白篡夺了细胞 和甲基化系统，指导细胞蛋白质的多-甲基化，以蛋白酶体降解为靶点。 很可能细胞相扑靶向泛素连接酶(STUbL)参与了这一过程。它也是 假设Ad利用AAA+ATPase p97/VCP的酶活性来提取细胞蛋白 从E4-ORF3核包涵体中释放出来，用于蛋白酶体的降解。具体目标2是调查 E4-ORF3介导的苏莫化反应的机制。E4-ORF3作为相扑E3连接酶和E4 延长酶催化底物的单相继多相甲基化。假设是E4-ORF3 通过组装使相扑机及其衬底相互靠近而发挥作用 高阶蛋白质复合体。Ad5 E4-ORF3蛋白在其相扑E3和E4活性中是独一无二的。研究 对E4-ORF3功能的研究将揭示宿主蛋白苏莫化的基本机制 不仅影响DDR，而且还影响其他基本过程，如转录和DNA复制。 目的3：探讨干扰素抑制Ad基因表达和复制的机制 发信号。干扰素α和干扰素γ通过E1a中保守的E2F结合位点抑制Ad5E1a基因的表达 增强子区和IFN诱导抑制子E2F复合体与该位点的结合。这与 在存在干扰素的情况下，Ad建立持续感染的能力。干扰素已经被用来治疗不同的 恶性肿瘤，但这些影响是如何发挥作用的，在很大程度上是未知的。假说是，干扰素调节 细胞增殖涉及抑制因子E2F复合体的形成。通过以下方式调节这些复合体 将对干扰素信号进行调查。IFN在调节急性和持续性Ad感染方面的作用将是 使用体外和体内模型进行了研究。 OMB编号0925-0001/0002(03/16修订版批准至2018年10月31日)页面续格式页面

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