Mechanism of Adenovirus E4-ORF3-Mediated SUMO Modifications.
Mechanism of Adenovirus E4-ORF3-Mediated SUMO Modifications.
复制标题
腺病毒 E4-ORF3 介导的 SUMO 修饰机制。
DOI：
10.1128/mbio.00022-19
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发表时间：
2019
期刊：
影响因子：
6.4
通讯作者：
Hearing,Patrick
中科院分区：
文献类型：
--
作者：
Sohn,Sook-Young;Hearing,Patrick
多种不同细胞过程的调节，包括翻译后修饰，对于许多病毒在宿主细胞内有效复制的能力至关重要。腺病毒（Ad）E4-ORF 3蛋白组装成聚合物并形成独特的核支架，其导致细胞蛋白的重新定位和隔离，包括小泛素样修饰物（SUMO）。以前，我们发现E4-ORF 3作为转录中介因子-1 γ（TIF-1γ）的SUMO E3连接酶发挥功能，并促进多聚SUMO链的形成。在这里，我们提出了细胞和生化数据，以进一步了解E4-ORF 3 SUMO连接酶的活性。在体外实验中，发现5种不同Ad的E4-ORF 3蛋白均具有SUMO E3连接酶活性。在感染的细胞中，SUMO修饰的靶蛋白发生时，蛋白质被招募到E4-ORF 3聚合结构。通过对SUMO缺陷型TIF-1γ的分析，我们证明了E4-ORF 3介导的靶蛋白在感染细胞中的重定位不需要SUMO缀合，这意味着在隔离之后是SUMO修饰。体外SUMO缀合试验表明，Ad E1 B-55 K癌蛋白是E4-ORF 3介导的SUMO修饰的新的病毒靶点。我们还验证了E4-ORF 3作为SUMO连接酶对多种细胞蛋白的直接功能，包括转录因子II-I（TFII-I），Nbs 1和Mre 11。此外，我们发现E4-ORF 3与SUMO结合的UBC 9缔合，并且E4-ORF 3聚合对于这种三元相互作用至关重要。总之，我们的研究结果表征E4-ORF 3作为一种新的聚合物型SUMO E3连接酶，并提供了机制的见解E4-ORF 3在SUMO conjugation.IMPORTANCEViruses与宿主SUMO化系统相互作用，以操纵不同的细胞反应。Ad E4-ORF 3蛋白形成动态核网络以干扰和利用不同的宿主过程，包括DNA损伤和干扰素应答。我们以前报道过E4-ORF 3是一种SUMO E3连接酶。在这里，我们证明，这种活动是一个保守的功能，进化上不同的人类广告E4-ORF 3蛋白和E4-ORF 3的功能直接促进SUMO共轭多种细胞蛋白。在体内，SUMO结合需要将细胞底物募集到E4-ORF 3核包涵体中。我们探索了E4-ORF 3作为SUMO E3连接酶的作用机制。E4-ORF 3在体外仅以三聚体形式与SUMO结合，而非二聚体形式与SUMO E2结合酶UBC 9结合。这些结果揭示了一种新的机制，保守的病毒蛋白篡夺细胞SUMO共轭机制。
Regulation of a variety of different cellular processes, including posttranslational modifications, is critical for the ability of many viruses to replicate efficiently within host cells. The adenovirus (Ad) E4-ORF3 protein assembles into polymers and forms a unique nuclear scaffold that leads to the relocalization and sequestration of cellular proteins, including small ubiquitin-like modifiers (SUMOs). Previously, we showed that E4-ORF3 functions as a SUMO E3 ligase of transcriptional intermediary factor-1 gamma (TIF-1γ) and promotes poly-SUMO chain formation. Here, we present cellular and biochemical data to further understand E4-ORF3 SUMO ligase activity. E4-ORF3 proteins from five different Ad species were found to possess SUMO E3 ligase activitiesin vitro. In infected cells, SUMO modifications of target proteins occurred only when the proteins were recruited into E4-ORF3 polymeric structures. By analyzing SUMO-deficient TIF-1γ, we demonstrated that SUMO conjugations are not required for E4-ORF3-mediated relocalization of target proteins in infected cells, implying that sequestration is followed by SUMO modification.In vitroSUMO conjugation assays revealed the Ad E1B-55K oncoprotein as a new viral target of E4-ORF3-mediated SUMOylation. We also verified a direct function of E4-ORF3 as a SUMO ligase for multiple cellular proteins, including transcription factor II-I (TFII-I), Nbs1, and Mre11. Moreover, we discovered that E4-ORF3 associates with SUMO-bound UBC9, and E4-ORF3 polymerization is crucial for this ternary interaction. Together, our findings characterize E4-ORF3 as a novel polymer-type SUMO E3 ligase and provide mechanistic insights into the role of E4-ORF3 in SUMO conjugation.IMPORTANCEViruses interplay with the host SUMOylation system to manipulate diverse cellular responses. The Ad E4-ORF3 protein forms a dynamic nuclear network to interfere with and exploit different host processes, including the DNA damage and interferon responses. We previously reported that E4-ORF3 is a SUMO E3 ligase. Here, we demonstrate that this activity is a conserved function of evolutionarily diverse human Ad E4-ORF3 proteins and that E4-ORF3 functions directly to promote SUMO conjugations to multiple cellular proteins. Recruitment of cellular substrates into E4-ORF3 nuclear inclusions is required for SUMO conjugation to occurin vivo. We probed the mechanism by which E4-ORF3 functions as a SUMO E3 ligase. Only multimeric, but not dimeric, E4-ORF3 binds to the SUMO E2 conjugation enzyme UBC9in vitroonly in a trimeric complex with SUMO. These results reveal a novel mechanism by which a conserved viral protein usurps the cellular SUMO conjugation machinery.