An essential EBV latent antigen 3C binds Bcl6 for targeted degradation and cell proliferation

<div><p>The latent EBV nuclear antigen 3C (EBNA3C) is required for transformation of primary human B lymphocytes. Most mature B-cell malignancies originate from malignant transformation of germinal center (GC) B-cells. The GC reaction appears to have a role in malignant transformation, in which a major player of the GC reaction is Bcl6, a key regulator of this process. We now demonstrate that EBNA3C contributes to B-cell transformation by targeted degradation of Bcl6. We show that EBNA3C can physically associate with Bcl6. Notably, EBNA3C expression leads to reduced Bcl6 protein levels in a ubiquitin-proteasome dependent manner. Further, EBNA3C inhibits the transcriptional activity of the Bcl6 promoter through interaction with the cellular protein IRF4. Bcl6 degradation induced by EBNA3C rescued the functions of the Bcl6-targeted downstream regulatory proteins Bcl2 and CCND1, which resulted in increased proliferation and G1-S transition. These data provide new insights into the function of EBNA3C in B-cell transformation during GC reaction, and raises the possibility of developing new targeted therapies against EBV-associated cancers.</p></div

EBNA3C regulates Bcl6 mRNA expression through inhibition of its promoter activity.

<p>A) 5 million BJAB, BJAB7, BJAB10, LCL1 and LCL2 cells were harvested and extracted total RNA using Trizol reagent. Then cDNA was prepared with reverse transcriptase kit, and detected Bcl6 mRNA expression by quantitative Real-time PCR analysis (SYBR green). GAPDH was set as an internal reference. Each sample was determined in triplicate. B) EBNA3C knock-down (sh-E3C) stable LCL1 or control (sh-Ctrl) LCL1 cells were harvested and Bcl6 mRNA expression was detected using Real-time PCR as mentioned. C) 10 million BJAB10 cells were transfected with specific EBNA3C (sh-E3C) or control (sh-Ctrl) short hairpin RNA. At 48 hours post-transfection, total RNA was extracted, reverse-transcribed, followed by quantitative Real-time PCR analysis. Meanwhile, EBNA3C expression was also detected by western blot analysis. D) HEK293T cells were transfected with the reporter constructs containing wild-type Bcl6 promoter (pLA/B9) and increasing amount of Myc-EBNA3C. Cells were collected and lysed in lysis buffer at 48 hours post-transfection. Luciferase activity was measured according to the dual-luciferase reporter assay kit. Mean values and standard deviations of two independent experiments were presented. Cell lysate was resolved by 10% SDS-PAGE in order to check EBNA3C expression. GAPDH western blot was done as an internal loading control. E) HEK293T cells were transfected with wild-type Bcl6 promoter reporter plasmids in combination with different expression constructs as indicated. Cells were collected and lysed, then the lysate were used to detect luciferase activity as previously described.</p

Down-regulation of Bcl6 facilitates G1-S transition through increasing CCND1 expression in EBV-transformed cells.

<p>A-B) The stable Bcl6 knock-down A) BJAB or B) LCL1 cells were collected and CCND1 mRNA expression was determined with Real-time PCR as mentioned earlier. C-D) The indicated stable cell lines were treated with PI staining buffer and analyzed by flow cytometry. Bar diagrams show the average values of two independent experiments.</p

EBNA3C down-regulates Bcl6 expression in EBV-infected PBMCs.

<p>A-B) 10 million human peripheral blood mononuclear cells (PBMC) from donor 1 and donor 2 were infected with BAC-GFP wild-type EBV or ΔE3C-EBV for 4 hours. Cells were harvested at indicated time points, then total RNA was isolated and subjected to cDNA preparation according to the manufacture's instruction followed by quantitative Real-time PCR for detecting Bcl6 transcription levels. C) 10 million Burkitt’s lymphoma (BL) cells BL41 or Akata and EBV-positive BL41/B95.8 or Akata-EBV cells were lysed with RIPA buffer and western blot analysis was performed with indicated antibodies. The relative density (RD) of Bcl6 protein was quantified and shown. D) 10 million EBV-negative Ramos, BJAB; EBNA3C stably expressed BJAB7, BJAB10; EBV-transformed LCL1, LCL2 cells were harvested and total cell lysates were subjected to western blot analysis using indicated antibodies. E-F) 10 million E) HEK293T and F) Saos-2 cells were transfected in a dose-dependent manner with increasing amounts EBNA3C constructs and western blot analysis was performed using specific antibodies as indicated. G) Lentivirus mediated stable EBNA3C knock-down (sh-E3C) or scramble control (sh-Ctrl) LCL1 cells were subjected to western blot analysis with indicated antibodies. Protein bands from western blot were analyzed by the Odyssey imager software and represented as bar diagrams based on internal loading control GAPDH. These results shown are representative of three independent experiments.</p

Pim-1 silencing by sh-RNA sensitizes EBV transformed lymphoblastoid cells by induction of the intrinsic apoptosis signaling pathway.

<p>A) Wild type LCL1, Lentivirus mediated and puromycin selected stable sh-Ctrl vector transfected and different clones of Pim-1 knocked down cells were observed by fluorescence microscope for monitoring GFP fluorescence B) 50 million different clones of stable sh-Pim-1, sh-Ctrl LCL1, and LCL1 cells were harvested and cell lysates were prepared by RIPA buffer. Western blot analysis was performed to show the expression levels of EBNA3C, Pim-1 and GAPDH. C) sh-Ctrl, sh-Pim-1 LCL1 cells were grown in RPMI medium for 12 hrs with or without serum starvation. Cells were stained with Propidium iodide and subjected to Flowcytometric analysis. D) Bar diagram represents the fold change of apoptosis observed by apoptosis assay using FACS. The results represented here are representative of three independent experiments. E–F) 50 million sh-Ctrl and sh-Pim-1 LCL1 cells were harvested and cell lysates were used for Western blot analysis with indicated antibodies. G) 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.</p

Co-localization of EBNA3C and Bcl6 in human cells.

<p>A-B) 0.1 million A) HEK293T or B) Saos-2 cells were plated on coverslips and transfected with Myc-EBNA3C or HA-Bcl6 using jetPRIME transfection reagent. C) BJAB, BJAB7, BJAB10, LCL1 and LCL2 cells were plated on slides and air-dried. All the cells were fixed with 4% PFA, stained with antibodies. Ectopic and endogenous expression of EBNA3C were detected by mouse anit-Myc (9E10) or mouse anti-EBNA3C (A10) antibodies, respectively, followed by the secondary anti-mouse Alexa Fluor 488 (green). Ectopic and endogenous expression of Bcl6 were detected by rabbit anti-Bcl6 antibodies, followed by anti-rabbit Alexa Fluor 594 (red) as the secondary antibody. The nuclei was subsequently stained with DAPI, and the images were captured using an Olympus Fluoview confocal microscope. These results shown are representative of three independent experiments.</p

EBNA3C associates with Bcl6 in human cells.

<p>A-B) 10 million A) HEK293T and B) Saos-2 cells were transfected with Myc-tagged EBNA3C or HA-tagged Bcl6. Empty vectors were used for balance the total DNA per transfection. Transfected cells were harvested 48 hours post-transfection and approximately 5%-10% of the lysates were used as input and the residual lysates were immunoprecipitated (IP) with 1μg anti-Bcl6 antibody. Lysate and immunoprecipitated samples were resolved by 10% SDS-PAGE and western blot analysis was performed using the indicated antibodies. C-D) 30 million BJAB, BJAB7, BJAB10, LCL1 and LCL2 cells were lysed and co-immunoprecipitation was performed with Bcl6 specific antibody. Immunoprecipitated samples were resolved by 10% SDS-PAGE and endogenous EBNA3C and Bcl6 were detected by their specific antibodies. E-F) 10 million HEK293 cells were transfected with either control vector or full length and different truncated mutants of Myc-tagged EBNA3C combined with HA-tagged Bcl6 construct. At 48 hours post-transfection, cells were harvested and immunoprecipitation was performed with 1μg E) anti-Myc or F) anti-HA antibody. IP samples were resolved in 10% SDS-PAGE, and western blots were performed using anti-Myc and anti-HA antibodies. G) The schematic diagram summarizes the binding domains between different regions of EBNA3C and Bcl6. NLS, nuclear localization signal (aa 72–80, 412–418 and 939–945); LZD, leucine zipper domain; AD, acidic domains; PP, Proline-rich; QP, glutamine-proline-rich. +, binding; -, no binding.</p

Bcl6 expression is regulated by EBNA3C via the ubiquitin-proteasome dependent pathway.

<p>A) 10 million HEK293T cells were transfected with HA-Bcl6 in combination with Myc-EBNA3C or control vector, treated with protein synthesis inhibitor cycloheximide (CHX) after 24 hours transfection as 40 μg/ml concentration. Cells were incubated with CHX for indicated times (0, 4, 8, 12 hours), and lysed with RIPA buffer, then cell lysates were quantitated and used for western blot analysis with indicated antibodies. B) 10 million BJAB or BJAB7 cells were incubated with CHX for 0, 2, 4, 6 hours, harvested and lysed with lysis buffer. Protein samples were resolved by 10% SDS-PAGE. Western blot analysis was performed by specific antibodies as shown. C) 10 million HEK293T cells were transfected with HA-Bcl6 and Myc-EBNA3C or control vector. After 24 hours transfection, cells were treated with MG132 for 16 hours and harvested, then western blot analysis was performed with indicated antibodies. D) 10 million HEK293T cells were transfected with the indicated constructs, incubated with MG132 for another 12 hours after 24 hours transfection, collected and detected with western blot using specific antibodies. The relative intensity (RD) of ubiquitinated Bcl6 complex was quantified and shown.</p

EBNA3C promotes cellular proliferation by upregulating Bcl6-targeted Bcl2 expression.

<p>A) Saos-2 cells were transfected with the indicated plasmids in combination with the GFP vector and selected with G418 antibiotic for 2 weeks. The GFP fluorescence of each plate was scanned by PhosphorImager and the colonies were calculated by Image J software. B) 10⁵ selected cells from the former experiment were plated and cultured for 10 days. Viable cells were counted at the indicated time using trypan blue staining. C) HEK293 cells were transfected, selected and counted the numbers. D) 10 million LCL1 cells were treated with 50 μg/ml (110 μM) Bcl6 inhibitor for 15 hours. Then cells were harvested, and western blot analysis was performed with indicated antibodies. These results shown are representative of three independent experiments.</p

Bcl6 knockdown leads to increased transforming activity in LCLs.

<p>A) HEK293T cells were transfected with sh-Ctrl, sh-Bcl6-1 or sh-Bcl6-2 plasmid in combination with HA-tagged Bcl6. After 48 hours transfection, cell were collected, lysed and detected with western blot. B-C) BJAB or LCL1 cells were infected using lentiviruses and selected by puromycin for at least two weeks. The selected cells were monitored the GFP fluorescence (B) and Bcl6 mRNA expression with Real-time PCR (C), respectively. D-E) Bcl6 knock-down BJAB (D) or LCL1 (E) cells was assessed for their ability to promote colony formation using the soft agar assays. Photomicrograph of representative colonies from three independent experiments are shown.</p