title: The EBNA3 family of Epstein-Barr virus nuclear proteins associates with the USP46/USP12 deubiquitination complexes to regulate lymphoblastoid cell line growth.
abstract: 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.
authors: Departments of Medicine and Oncology (McArdle Laboratory for Cancer Research), University of Wisconsin, Madison, Wisconsin, United States of America.
authors: Makoto
authors: M
authors: Ohashi
authors: Infectious Disease Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, United States of America.
authors: Amy M
authors: AM
authors: Holthaus
authors: Infectious Disease Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, United States of America.
authors: Michael A
authors: MA
authors: Calderwood
authors: Infectious Disease Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, United States of America.
authors: Chiou-Yan
authors: CY
authors: Lai
authors: Biomarker Research Initiatives in Mass Spectrometry (BRIMS), Thermo Fisher Scientific, Cambridge, Massachusetts, United States of America.
authors: Bryan
authors: B
authors: Krastins
authors: Biomarker Research Initiatives in Mass Spectrometry (BRIMS), Thermo Fisher Scientific, Cambridge, Massachusetts, United States of America.
authors: David
authors: D
authors: Sarracino
authors: Departments of Medicine and Oncology (McArdle Laboratory for Cancer Research), University of Wisconsin, Madison, Wisconsin, United States of America.
authors: Eric
authors: E
authors: Johannsen
mesh_terms: D015153:Blotting, Western; D002460:Cell Line; D049109:Cell Proliferation; D002472:Cell Transformation, Viral / Q000235:genetics*; D047369:Chromatin Immunoprecipitation; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D019309:Epstein-Barr Virus Nuclear Antigens / Q000378:metabolism*; D015967:Gene Expression Regulation, Viral / Q000235:genetics*; D004854:Herpesvirus 4, Human / Q000235:genetics / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D013058:Mass Spectrometry; D043222:Ubiquitin Thiolesterase / Q000235:genetics / Q000378:metabolism*
chemical_list: C508151:EBNA-3A antigen; C422762:EBNA-3B antigen; C498774:EBNA-3C, epstein-barr virus; D019309:Epstein-Barr Virus Nuclear Antigens; C486402:USP12 protein, human; D010450:Endopeptidases; C562185:ubiquitin-specific peptidase 46, human; D043222:Ubiquitin Thiolesterase