The EBV Latent Antigen 3C Inhibits Apoptosis through Targeted Regulation of Interferon Regulatory Factors 4 and 8

Abstract

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.

LMP1-deficient Epstein-Barr virus mutant requires T cells for lymphomagenesis

“…The EBV-encoded protein EBNA3C, which inhibits expression of the cell cycle inhibitor p16 (49), may also promote proliferation of the LMP1-KO EBVinfected cells. Notably, EBNA3C was also recently shown to stabilize the IRF4 protein in EBV-infected B cells (32), and we found that the LMP1-KO EBV-induced lymphomas expressed a high level of IRF4. IRF4 is not only an essential growth and survival factor for activated human DLBCLs (34), but is also required for survival of EBV-transformed LCLs in vitro (32,33).…”

“…Notably, EBNA3C was also recently shown to stabilize the IRF4 protein in EBV-infected B cells (32), and we found that the LMP1-KO EBV-induced lymphomas expressed a high level of IRF4. IRF4 is not only an essential growth and survival factor for activated human DLBCLs (34), but is also required for survival of EBV-transformed LCLs in vitro (32,33). T cell-derived CD40 signaling may also help to induce IRF4 expression in LMP1-KO EBVinduced lymphomas (35).…”

“…WT EBV-and LMP1-KO EBV-induced tumors were also both positive for expression of MYC ( Figure 4B), which is known to be activated by the EBNA2 viral protein. WT EBV-and LMP1-KO EBV-induced lymphomas also showed strong staining for the transcription factor IRF4 (also known as MUM-1) (Figure 1 and Figure 4A), a marker for early plasma cell differentiation as well as an essential survival factor both for EBV-transformed LCLs in vitro (32,33) and for activated DLBCLs (34), HLs (35), and multiple myeloma tumors in vivo (36). IRF4 is also a surrogate immunohistochemical (IHC) marker for the more aggressive "activated B cell" type of DLBCL.…”

Epstein-Barr Virus Nuclear Antigen 3 (EBNA3) Proteins Regulate EBNA2 Binding to Distinct RBPJ Genomic Sites

“…Moreover, EBNA3A-and EBNA3B-bound sites are also enriched for IRF4 binding ( (51). However, some lines of evidence suggested that the IRF4 interaction might be more important for EBNA3C, including the increased EBNA3C binding signals at IRF4 sites compared to non-IRF4 sites and the ability of EBNA3C to bind directly to IRF4 and promote its stabilization (35,55). When we restricted our analysis to sites uniquely bound by EBNA3A, EBNA3B, or EBNA3C, we observed enrichment for the IRF4 binding motif only in the EBNA3C subset.…”

Epstein-Barr Virus LMP1-Mediated Oncogenicity

“…For instance, the IRF4 DNA motif is highly enriched at promoter sites occupied only by LMP1/noncanonical pathway-activated p52. EBNA3C and LMP1 upregulate IRF4 expression (17,47), which may then tether p52 to these sites. Similarly, an E-box motif is enriched at LCL promoters bound predominantly by LMP1/canonical pathway-activated cRel, suggesting that an E-box-bound host transcription factor such as c-Myc may tether cRel homodimers to these sites.…”