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 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.
Highlights
Epstein-Barr Virus (EBV) is a herpesvirus that establishes lifelong asymptomatic infection in up to 95% of the human population [1]
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
Summary
Epstein-Barr Virus (EBV) is a herpesvirus that establishes lifelong asymptomatic infection in up to 95% of the human population [1]. Latency III driven B lymphocyte proliferation in vivo is normally controlled by a vigorous cytotoxic T cell response [4]. Extensive genetic and biochemical data support the model that EBV latency III gene expression usurps growth and survival signaling pathways in B lymphocytes normally triggered by antigen recognition and CD4+ T cell co-stimulation [1,5]. The ability of LMP2A to engage B cell receptor signaling molecules may be important for maintaining viral latency or for the growth and survival of EBV infected cells in vivo [12,13]. EBNA2 and its co-activator EBNALP are the first genes expressed during EBV latent infection and result in upregulation of promoters including c-myc, EBV LMP1, LMP2A, and EBNA essential for latency III transformation [15,16]. The role of the EBNA3 proteins in LCL transformation is less clearly defined
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