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.

Figure 1. EBNA3C differentially regulates IRF4 and IRF8 expression.

A) 10×10⁶ human PBMC (Peripheral blood mononuclear cells) were infected with BAC-GFP EBV for 4 hrs at 37°C. Cells were harvested after 0, 2, 4, 7, 15 days of post-infection and lysed in RIPA buffer. Western blot analysis was performed with indicated antibodies to detect specific endogenous proteins. B) 50 million EBV negative BJAB, DG75, Ramos and EBV transformed LCL1, LCL2 cells were harvested and total cell lysates were subjected to Western blot analysis (WB) using indicated antibodies. C) 20 million Burkitt's lymphoma (BL) cells BL41 and wild type EBV strain B95.8 infected BL41 cells, type I and III latency expressing BL cell lines-MutuI and MutuIII were lysed with RIPA buffer and Western blot analysis was performed with indicated antibodies. GAPDH was taken as internal loading control. D–F) 50 million D) BJAB, BJAB7, BJAB10 cells were harvested and Western blot analysis was performed using specific antibodies as indicated. E) EBV negative DG75 cells were transfected with increasing amount of EBNA3C expressing construct (0, 5, 10, 15 µg) and Western blot analysis was performed to detect IRF4, IRF8, EBNA3C, GAPDH proteins. F) Lentivirus mediated stable EBNA3C knockdown (Sh-E3C) or scramble control (Sh-Ctrl) LCL1 cells were subjected to Western blot analysis with indicated antibodies. Protein bands from Western blot analysis were analyzed by the Odyssey imager software and represented as bar diagrams based on internal loading control GAPDH.

Figure 2. EBNA3C physically associates with IRF4 and IRF8.

A) 10 million HEK- 293 cells were co-transfected with Myc-tagged EBNA3C and Flag-tagged IRF4, IRF8 vectors. Control samples were balanced by using empty vector. Transfected cells were harvested at 36 hrs of post-transfection and approximately 5% of the lysates were used as input and the residual lysate was immunoprecipitated (IP) with 1 µg of anti-Flag (M2) antibody. Lysates and Immunoprecipitated samples were resolved by 8% SDS-PAGE and western blot (WB) analysis was performed with the indicated antibodies. B) 50 million EBV negative BJAB, BJAB cells stably expressing EBNA3C (BJAB7, BJAB10) and two different clones of EBV transformed lymphoblastoid cell lines as- LCL1, LCL2 cells were harvested and lysed in RIPA buffer. Cell lysates were incubated with either GST control or GST-IRF4 or GST-IRF8 beads. EBNA3C protein was detected by western blot analysis using EBNA3C specific monoclonal antibody (A10). C) Purified control GST and GST-IRF4, GST-IRF8 proteins used in this experiment were resolved by 10% SDS-PAGE and stained with Coomassie Blue. 50 million D–E) BJAB, BJAB7, BJAB10, LCL1, LCL2 cells were lysed and immunoprecipitation was performed by A10, IRF4, IRF8 specific antibodies. Immunoprecipitated samples were resolved by 8% SDS-PAGE and endogenous EBNA3C, IRF4, IRF8 proteins were detected by their specific antibodies.

Figure 3. The N-terminal domain of EBNA3C interacts with IRF4 and IRF8.

A) 10 million HEK-293 cells were transfected with either control vector or Full length and different truncated mutants of Myc-tagged EBNA3C with A) Flag-tagged IRF4 or, B) Flag-tagged IRF8 plasmid constructs. After 36 hours of post-transfection, cells were harvested and immunoprecipitation performed with 1?g of anti-Myc antibody. IP samples were resolved in 10% SDS-PAGE. Western blot was performed with anti-Myc and anti-Flag antibody. C) Full length and different domains truncated mutant constructs of EBNA3C (residues 1-992, 1-365, 366-620 and 621-992) were in vitro translated using a T7-TNT translation kit. After pre-clearing with GST-beads, all S35-radiolabeled in vitro translated proteins incubated with either GST control or GST-IRF4, GST-IRF8 beads. Reaction samples were washed with Binding Buffer and resolved by 10% SDS-PAGE, exposed to phosphoimager plate and scanned by Typhoon Scanner. D) A series of different N-terminal truncated mutants of EBNA3C were used for in vitro translated S35-radiolabeled protein production and their binding affinity determined with GST-IRF4 or GST-IRF8 as similar to C). E) Flag-IRF4 and IRF8 constructs were used for in vitro translation and S35-radiolabeled in vitro translated proteins were incubated with either GST control or different GST-EBNA3C truncated mutants specific for residues 90-129, 130-159,130-190, 160-190 beads. Coomassie staining of SDS-PAGE resolved purified GST proteins is shown in the bottom panel of E). F) Wild type GST-EBNA3C (residues 130-159) and different GST-mutant EBNA3C were expressed in E. coli and purified with Glutathione Sepharose beads.Flag-IRF4 and IRF8 plasmid constructs were used for in vitro translation and S35-radiolabeled in vitro translated proteins were incubated with either GST control or Wild type GST-EBNA3C (residues 130-159) and different GST-mutant EBNA3C. Coomassie staining of SDS-PAGE resolved purified control, wild type and mutant GST proteins are shown in the bottom panel.In each case, 5% of IVT input was used for the comparison. Relative binding units (RBU) were indicated as numerical values at the bottom of IVT gel.

Figure 4. The C-terminal domain of IRF4 and IRF8 binds with EBNA3C and Spi-1/B-like motif is important for IRF4 interaction.

A) 10 million HEK-293 cells were transfected with either control vector or different truncated mutants of Flag-tagged IRF4 with Myc-EBNA3C and B) Myc-tagged IRF8 truncated mutants with Flag-tagged EBNA3C. After 36 hours of post transfection, cells were harvested and immunoprecipitation was performed with 1 µg of anti-Flag or anti-Myc antibodies. IP samples were resolved in 10% SDS-PAGE. Western blot was performed with anti-Myc and anti-Flag antibody. C) The schematic represents different structural and interactive domains of IRF4 and IRF8. Binding affinities of different domains of IRF4 and IRF8 with EBNA3C was also summarized. +, binding; −, no binding. D) Hydrophobicity graph of Spi-1, Spi-B and IRF4 sequence alignment (upper panel). Spi-1, Spi-B and IRF4 protein sequences alignment (lower panel). Spi-1/B-like consensus sequence is underlined and based on the hydrophobic homology. E) 10 million HEK-293 cells were transfected with either control vector or Myc-tagged EBNA3C with either wild type or Spi-1/B motif deleted Flag-IRF4 plasmid constructs. IP was performed with 1 µg anti-Flag antibody. Western blot was performed with anti-Myc and anti-Flag antibodies.

Figure 5. IRF4 co-localizes with EBNA3C in human cells.

A) 0.3 million HEK-293 cells plated on coverslips and transiently transfected with Control vector, GFP-EBNA3C and Flag-IRF4 expression vectors by using Lipofectamine 2000 transfection reagent. B) BJAB, BJAB10, LCL1 cells were plated on slides and air-dried. C) 0.3 million HEK-293 cells were grown on coverslips and transiently transfected with different truncated domains of GFP-EBNA3C (residues 1–365, 366–620, 621–992) and Flag-IRF4 expression vectors by using Lipofectamine 2000 transfection reagent. Cells were fixed using 3% PFA. Ectopic and endogenous expressions of IRF4 was detected using anti-Flag (M2)-antibody (1∶200 dilution) and IRF4 specific antibody (1∶50 dilution) respectively, followed by anti-Rabbit Alexa Fluor 594 (red) as secondary antibody. Endogenous EBNA3C was detected using A10 ascites (1∶150 dilution) followed by anti-mouse Alexa Fluor 488 (green). DAPI (49, 69-diamidino-2-phenylindole) was used (1∶500 dilution) to stain nuclei. The images were captured by Olympus Fluoview confocal microscope.

Figure 6. EBNA3C stabilizes IRF4.

A) 10 million HEK-293 cells were co-transfected with Flag-IRF4 and either vector control (lanes 1 and 3) or Myc-EBNA3C (lanes 2 and 4) expression constructs. After 36 hrs of post-transfection, transfected cells were treated with either 40 µM MG132 (+ lanes) or DMSO (- lanes) for additional 6 hrs and cell lysates were resolved by 8% SDS-PAGE and Western blot was performed with the indicated antibodies. B) HEK-293 cells were transfected with Flag-tagged IRF4 and vector control, full length, N-terminal domain expressing or N-terminal domain deleted mutant Myc-tagged EBNA3C plasmid vectors. At 36 hrs of post-transfection, cells were treated with 40 µg/ml cyclohexamide (CHX) for 0, 6, 12 hrs. Cells were lysed and protein samples were resolved by 8% SDS-PAGE. Western blot was performed by specific antibodies shown. C) BJAB, BJAB10, LCL1 cells were treated with 40 µg/ml cyclohexamide (CHX) for indicated time periods. Cell lysates were resolved by 8% SDS-PAGE. Western blot analysis was performed with indicated antibodies. GAPDH blot was shown for internal loading control. Protein bands quantitation was performed by Odyssey imager software as arbitrary numerical values indicated at the bottom of gel.

Figure 7. EBNA3C differentially modulates poly-ubiquitination of IRF4 and IRF8.

A, B, C, D) 10 million HEK-293 cells were transiently transfected with different combinations of expression vectors as indicated. Cells were harvested after 36 hrs of post-transfection and total protein was immunoprecipitated (IP) with indicated antibodies and protein samples were resolved by 10% SDS-PAGE. Western blots were performed by stripping and re-probing the same membrane. E) 50 million EBV negative BJAB cells, BJAB cells stably expressing EBNA3C (BJAB7, BJAB10) and EBV transformed LCL1, LCL2 were incubated with proteasome inhibitor MG132 drug (20 µM) for 6 hrs. Cells were harvested and lysed with RIPA buffer. IRF4 was immunoprecipitated (IP) by using specific antibodies. Samples were resolved by 10% SDS-PAGE. Western blotting (WB) was performed by stripping and reprobing the same membrane. F–G) Ubiquitination assay was performed with Lentivirus mediated stable EBNA3C knockdown (Sh-E3C) or scramble control (Sh-Ctrl) LCL1 cells and subjected to Western blot analysis with indicated antibodies.

Figure 8. EBNA3C is important for IRF4-mediated downregulation of IRF8 and activation of IRF4 target proteins.

A–B) 50 million EBV negative DG75 cells were transfected with increasing amount of A) IRF4 and B) IRF8 expression vector (0, 5, 10, 15 µg) without EBNA3C (left panel) or with 10 µg EBNA3C (right panel). After 36 hrs of post-transfection, cells were harvested and lysed with RIPA buffer. Protein samples were subjected to Western blot analysis using EBNA3C, IRF4, IRF8 specific antibodies. GAPDH western blot was performed for loading control. C) 10 million HEK-293 cells were transiently transfected with different combinations of expression vectors as indicated. After 36 hrs of post-transfection, cells were treated with MG132 for additional 6 hrs and total protein was immunoprecipitated (IP) with indicated antibodies. Protein samples were resolved by 10% SDS-PAGE. Western blots were performed by stripping and re-probing the same membrane. D) 50 million Sh-Ctrl, Sh-IRF4 LCL1 cells were incubated with proteasome inhibitor MG132 (20 µM) for 6 hrs. Cells were harvested and lysed with RIPA buffer. IRF8 protein was immunoprecipitated (IP) by using IRF8 specific antibody. Immunoprecipitated samples were resolved by 10% SDS-PAGE. Western blot analysis was performed by using specific antibodies shown. E) 50 million stable Sh-Ctrl, Sh-IRF4, Sh-E3C LCL1 and Ctrl-vector and Flag-IRF4 transfected, Sh-IRF4 LCL1 cells were lysed in RIPA buffer and Western blot was performed to show the expression levels of EBNA3C, IRF4, c-Myc, Cyclin-dependent kinase 6, Cyclin B1 and GAPDH.

Figure 9. EBNA3C is critical for IRF4-mediated cell proliferation by inhibiting apoptosis.

10 million HEK-293 were transfected with Ctrl-vector and different combinations of expression plasmids for Myc-tagged EBNA3C, Flag-tagged IRF4 and N-terminal domain deleted mutant Myc-tagged EBNA3C (residues 366–992). In addition, cells were transfected with GFP expression vector. Transfected cells were selected for 2 weeks with G418 antibiotic. A) After 2 weeks of selection, GFP fluorescence of each plate was scanned by a PhosphorImager and the area of the colonies calculated by Image J software. C) The numbers of colonies in different transfected sets are represented in bar diagram. The data represented in bar diagram is the average of three independent experiments. B) 0.1×10⁶ cells from each set of selected samples were plated and cultured for 6 days. Viable cells were counted at indicated time points by trypan blue dye exclusion technique. D) G418 selected stable cells were harvested, lysed in RIPA buffer and subjected to immunoblot analyses with indicated antibodies. E, F, H) Sh-Ctrl, Sh-IRF4 LCL1 cells were grown in RPMI medium for 12 hrs with or without etoposide. E) Cells were harvested and stained with Propidium iodide. Stained cells were subjected to Flowcytometric analysis. G) The bar diagram represents the fold change of apoptosis seen by cell cycle analysis using FACS. The results shown are representative of three independent experiments. F) 1×10⁶ cells were plated and allowed them to grow at 37°C in complete medium without puromycin antibiotic. Viable cells were counted by trypan blue dye exclusion technique at indicated time points. The results shown here are representative of three independent experiments. H) Cells were treated with increasing amount (0, 10, 20, 40, 80, 160 µM) of etoposide drug for 12 hrs. Cell lysates were used for Western blot analysis with PARP-1, GAPDH antibodies.

Figure 10. EBNA3C promotes IRF4-mediated cell proliferation by inhibiting IRF8 induced apoptosis.

A–D) Human Kidney embryonic cells (HEK-293) were transfected with Ctrl-vector and different combinations of expression plasmids for Myc-tagged EBNA3C, Flag-tagged IRF8 and Myc-tagged IRF4. Additionally, cells were transfected with GFP expression vector. Transfected cells were selected for 2 weeks with G418 antibiotic. A) 0.1×10⁶ cells from each set of selected samples were plated and cultured for 6 days. Viable cells were counted at indicated time points by trypan blue dye exclusion technique. (B) G418 selected stable cells were harvested, lysed in RIPA buffer and subjected to immunoblot analyses with indicated antibodies. C–D) HEK-293 cells transfected with different combinations of expression plasmids as indicated in previous experiment and selected similarly as described above with G418 antibiotic. After 2 weeks of selection, GFP fluorescence of each plate was scanned by a PhosphorImager and the area of the colonies was calculated by Image J software. The data represented in bar diagram as the average of three independent experiments. E–J) Lentivirus mediated knock down of IRF4 in EBV negative Ramos cells. Knocked down cells were selected with puromycin to make stable cell lines expressing specific si-RNA against IRF4 and control vector. F) The selected stable cells with GFP fluorescence were monitored by fluorescent microscope. G) 50 million Ramos, stable Sh-Ctrl Ramos, Sh-IRF4 Ramos cells were lysed in RIPA buffer and Western blot was performed to check the expression levels of IRF4. E) Stable Sh-Ctrl Ramos, Sh-IRF4 Ramos, cells were transfected with control vector, Flag-IRF8, Myc-EBNA3C and grown in RPMI medium for 36 hrs. Transfected cells were stained with Propidium iodide and subjected to FACS analysis. H) The bar diagram represents the percentage of apoptosis in each transfected set. The results shown are representative of three independent experiments. I–J) 1×10⁶ stable transfected cells were plated and allowed to grow in RPMI media for 6 days. Viable cells were counted by trypan blue dye exclusion method at indicated time points.

Figure 11. A schematic which illustrates the contribution of EBNA3C to oncogenic transformation of B-cells through stabilization of IRF4 and degradation of IRF8 resulting in activation of IRF4 targeted proteins.

EBNA3C interacts with IRF4 and IRF8 proteins and enhances the stability of IRF4 through inhibition of the poly-ubiquitination process. However, in the presence of EBNA3C, IRF4 targets IRF8 and facilitate the subsequent degradation of IRF8. EBNA3C can also contribute to B-cell proliferation by activating IRF4 downstream signaling as well as inhibiting the growth suppressive and apoptosis inducing properties of IRF8. This molecular strategy potentiates oncogenic activity of IRF4 in EBV transformed B-cell proliferation.