Ohashi Makoto, Holthaus Amy M, Calderwood Michael A, Lai Chiou-Yan, Krastins Bryan, Sarracino David, Johannsen Eric
Departments of Medicine and Oncology (McArdle Laboratory for Cancer Research), University of Wisconsin, Madison, Wisconsin, United States of America.
Infectious Disease Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, United States of America.
PLoS Pathog. 2015 Apr 9;11(4):e1004822. doi: 10.1371/journal.ppat.1004822. eCollection 2015 Apr.
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 p16(INK4A) and p14(ARF) 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 p14(ARF) 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 p14(ARF) 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.
爱泼斯坦-巴尔病毒（EBV）核蛋白EBNA3A、EBNA3B和EBNA3C与细胞DNA结合蛋白RBPJ相互作用，并调节细胞和病毒基因。EBNA3A和EBNA3C对细胞周期蛋白依赖性激酶抑制剂2A（CDKN2A）肿瘤抑制基因产物p16（INK4A）和p14（ARF）的抑制作用对于EBV介导的静息B淋巴细胞转化为永生化淋巴母细胞系（LCLs）至关重要。为了确定内源性EBNA3蛋白复合物的组成，我们构建了表达带有Flag-HA标签的EBNA3A、EBNA3B或EBNA3C的淋巴母细胞系（LCLs），并使用串联亲和纯化法分离每个EBNA3复合物。我们的结果表明，每个EBNA3蛋白都与RBPJ形成独特的复合物。质谱分析显示，EBNA3A和EBNA3B复合物还包含由WDR48、WDR20和USP46（或其旁系同源物USP12）组成的去泛素化复合物，而EBNA3C复合物包含WDR48。免疫沉淀证实了EBNA3A、EBNA3B和EBNA3C与USP46复合物的结合。使用染色质免疫沉淀法，我们证明WDR48和USP46以EBNA3C依赖的方式被招募到p14（ARF）启动子。定位研究表明WDR48是EBNA3与去泛素化酶复合物结合的主要介导因子。通过染色质免疫沉淀分析，WDR48以EBNA3C依赖的方式被招募到p14（ARF）启动子。重要的是，WDR48与对LCL生长至关重要的EBNA3A和EBNA3C结构域相关，这表明USP46/USP12在EBV诱导的生长转化中发挥作用。
