The EBNA3 family of Epstein-Barr virus nuclear proteins associates with the USP46/USP12 deubiquitination complexes to regulate lymphoblastoid cell line growth.
The EBNA3 family of Epstein-Barr virus nuclear proteins associates with the USP46/USP12 deubiquitination complexes to regulate lymphoblastoid cell line growth.
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DOI：
10.1371/journal.ppat.1004822
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发表时间：
2015-04
期刊：
影响因子：
6.7
通讯作者：
Johannsen E
中科院分区：
文献类型：
--
作者：
Ohashi M;Holthaus AM;Calderwood MA;Lai CY;Krastins B;Sarracino D;Johannsen E
EB病毒（EBV）核蛋白EBNA 3A、EBNA 3B和EBNA 3C与细胞DNA结合蛋白RBPJ相互作用并调节细胞和病毒基因。EBNA 3A和EBNA 3C对CDKN 2A肿瘤抑制基因产物p16 INK 4A和p14 ARF的抑制对于EBV介导的静息B淋巴细胞转化为永生化淋巴母细胞样细胞系（LCL）是至关重要的。为了确定内源性EBNA 3蛋白复合物的组成，我们产生了表达flag-HA标记的EBNA 3A、EBNA 3B或EBNA 3C的淋巴母细胞样细胞系（LCL），并使用串联亲和纯化来分离每种EBNA 3复合物。我们的结果表明，每个EBNA 3蛋白与RBPJ形成不同的复合物。质谱分析显示EBNA 3A和EBNA 3B复合物还含有由WDR 48、WDR 20和USP 46（或其副产物USP 12）组成的脱乙酰化复合物，EBNA 3C复合物含有WDR 48。免疫沉淀证实EBNA 3A、EBNA 3B和EBNA 3C与USP 46复合物结合。使用染色质免疫沉淀，我们证明，WDR 48和USP 46招募的p14 ARF启动子在EBNA 3C依赖的方式。作图研究与WDR 48是EBNA 3与DUB复合物结合的主要介质一致。通过ChIP分析，WDR 48以EBNA 3C依赖的方式募集到p14 ARF启动子。重要的是，WDR 48与对LCL生长至关重要的EBNA 3A和EBNA 3C结构域相关，表明USP 46/USP 12在EBV诱导的生长转化中的作用。EB病毒（Epstein-Barr virus，EBV）是一种γ疱疹病毒，与多种恶性肿瘤的发病机制有关，包括伯基特淋巴瘤（Burkitt lymphoma）、霍奇金淋巴瘤（Hodgkin lymphoma）、移植后淋巴增生性疾病（PTLD）、鼻咽癌和胃癌。静息B淋巴细胞的EBV感染驱动它们增殖为淋巴母细胞样细胞系（LCL），这是PTLD的体外模型。LCL表达有限的EBV基因库，包括六种核蛋白（EBNA 1，2，3A，3B，3C和LP），三种整合膜蛋白（LMP 1，2A和2B）和超过30种微RNA。EBNA 2和EBNA 3蛋白是通过细胞DNA结合蛋白RBPJ调节病毒和细胞基因表达的转录因子。在这项研究中，我们建立了由重组EBV基因组转化的LCL，其中Flag-HA表位标签与EBNA 3A、EBNA 3B或EBNA 3C的C末端框内融合。使用这些LCL，我们纯化了内源性EBNA 3复合物，并鉴定了USP 46去泛素化酶（DUB）及其相关的伴侣蛋白WDR 48和WDR 20作为EBNA 3结合蛋白。我们发现EBNA 3s主要与WDR 48蛋白相互作用，并且WDR 48与EBNA 3A或EBNA 3C相互作用的丧失损害LCL生长。这项研究代表了来自LCL的EBNA 3复合物的首次表征，并暗示了USP 46 DUB复合物在EBNA 3介导的基因调控中的作用。
The Epstein-Barr virus (EBV) nuclear proteins EBNA3A, EBNA3B, and EBNA3C interact with the cell DNA binding protein RBPJ and regulate cell and viral genes. Repression of the CDKN2A tumor suppressor gene products p16INK4A and p14ARF by EBNA3A and EBNA3C is critical for EBV mediated transformation of resting B lymphocytes into immortalized lymphoblastoid cell lines (LCLs). To define the composition of endogenous EBNA3 protein complexes, we generated lymphoblastoid cell lines (LCLs) expressing flag-HA tagged EBNA3A, EBNA3B, or EBNA3C and used tandem affinity purification to isolate each EBNA3 complex. Our results demonstrated that each EBNA3 protein forms a distinct complex with RBPJ. Mass-spectrometry revealed that the EBNA3A and EBNA3B complexes also contained the deubquitylation complex consisting of WDR48, WDR20, and USP46 (or its paralog USP12) and that EBNA3C complexes contained WDR48. Immunoprecipitation confirmed that EBNA3A, EBNA3B, and EBNA3C association with the USP46 complex. Using chromatin immunoprecipitation, we demonstrate that WDR48 and USP46 are recruited to the p14ARF promoter in an EBNA3C dependent manner. Mapping studies were consistent with WDR48 being the primary mediator of EBNA3 association with the DUB complex. By ChIP assay, WDR48 was recruited to the p14ARF promoter in an EBNA3C dependent manner. Importantly, WDR48 associated with EBNA3A and EBNA3C domains that are critical for LCL growth, suggesting a role for USP46/USP12 in EBV induced growth transformation. Epstein-Barr virus (EBV) is a gammaherpesvirus implicated in the pathogenesis of multiple malignancies, including Burkitt lymphoma, Hodgkin lymphoma, post-transplant lymphoproliferative disease (PTLD), nasopharyngeal carcinoma, and gastric carcinoma. EBV infection of resting B-lymphocytes drives them to proliferate as lymphoblastoid cell lines (LCLs), an in vitro model of PTLD. LCLs express a limited EBV gene repertoire, including six nuclear proteins (EBNA1, 2, 3A, 3B, 3C, and LP), three integral membrane proteins (LMP1, 2A, and 2B), and more than 30 micro RNAs. EBNA2 and the EBNA3 proteins are transcription factors that regulate viral and cell gene expression through the cell DNA binding protein RBPJ. In this study, we established LCLs transformed by recombinant EBV genomes in which a Flag-HA epitope tag is fused in-frame to the C-terminus of EBNA3A, EBNA3B or EBNA3C. Using these LCLs, we purified endogenous EBNA3 complexes and identified the USP46 deubiquitinating enzyme (DUB) and its associated chaperones WDR48 and WDR20 as EBNA3 binding proteins. We find that EBNA3s interact primarily with the WDR48 protein and that loss of WDR48 interaction with EBNA3A or EBNA3C impairs LCL growth. This study represents the first characterization of EBNA3 complexes from LCLs and implicates the USP46 DUB complex in EBNA3 mediated gene regulation.
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