Epstein-Barr virus (EBV) is linked to a broad spectrum of B-cell malignancies. EBV nuclear antigen 3C (EBNA3C) is an encoded latent antigen required for growth transformation of primary human B-lymphocytes. Interferon regulatory factor 4 (IRF4) and 8 (IRF8) are transcription factors of the IRF family that regulate diverse functions in B cell development. IRF4 is an oncoprotein with anti-apoptotic properties and IRF8 functions as a regulator of apoptosis and tumor suppressor in many hematopoietic malignancies. We now demonstrate that EBNA3C can contribute to B-cell transformation by modulating the molecular interplay between cellular IRF4 and IRF8. We show that EBNA3C physically interacts with IRF4 and IRF8 with its N-terminal domain in vitro and forms a molecular complex in cells. We identified the Spi-1/B motif of IRF4 as critical for EBNA3C interaction. We also demonstrated that EBNA3C can stabilize IRF4, which leads to downregulation of IRF8 by enhancing its proteasome-mediated degradation. Further, si-RNA mediated knock-down of endogenous IRF4 results in a substantial reduction in proliferation of EBV-transformed lymphoblastoid cell lines (LCLs), as well as augmentation of DNA damage-induced apoptosis. IRF4 knockdown also showed reduced expression of its targeted downstream signalling proteins which include CDK6, Cyclin B1 and c-Myc all critical for cell proliferation. These studies provide novel insights into the contribution of EBNA3C to EBV-mediated B-cell transformation through regulation of IRF4 and IRF8 and add another molecular link to the mechanisms by which EBV dysregulates cellular activities, increasing the potential for therapeutic intervention against EBV-associated cancers.
Interferon regulatory factor (IRF) family members have different roles in context of pathogen response, signal transduction, cell proliferation and hematopoietic development. IRF4 and IRF8 are members of the IRF family and are critical mediators of B-cell development. Enhanced expression of IRF4 is often associated with multiple myeloma and adult T-cell lymphomas. Furthermore, IRF8 can function as a tumor suppressor in myeloid cancers. Epstein-Barr virus (EBV), one of the first characterized human tumor viruses is associated with several lymphoid malignancies. One of the essential antigens, EBV encoded nuclear antigen 3C (EBNA3C), plays a critical role in EBV-induced B-cell transformation. In our study, we now demonstrate that EBNA3C forms a molecular complex with IRF4 and IRF8 specifically through its N-terminal domain. We show that IRF4 is stabilized by EBNA3C, which resulted in downregulation of IRF8 through proteasome-mediated degradation and subsequent inhibition of its tumor suppressive activity. Moreover, si-RNA-mediated inhibition of IRF4 showed a substantial reduction in EBV transformed B-cell proliferation, and also enhanced their sensitivity to DNA-damage induced apoptosis. Therefore, our findings demonstrated that targeted disruption of EBNA3C-mediated differential regulation of IRF4 and IRF8 may have potential therapeutic value for treating EBV induced B-cell malignancies.
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