Epstein-Barr Virus Nuclear Antigen 3C Facilitates Cell Proliferation by Regulating Cyclin D2

Abstract

Cell cycle regulation is one of the hallmarks of virus-mediated oncogenesis. Epstein-Barr virus (EBV)-induced lymphomas express a repertoire of essential viral latent proteins that regulate expression of cell cycle-related proteins to dysregulate this process, thereby facilitating the proliferation of infected cells. We now demonstrate that the essential EBV latent protein 3C (EBNA3C) stabilizes cyclin D2 to regulate cell cycle progression. More specifically, EBNA3C directly binds to cyclin D2 and they colocalize together in nuclear compartments. We show that EBNA3C regulates the promoter of cyclin D2 through cooperation with master transcription factor Bcl6 and enhances its stability by inhibiting its ubiquitin-dependent degradation. EBNA3C also promoted cell proliferation in the presence of cyclin D2, suggesting that cyclin D2 contributes to EBNA3C-mediated cell cycle progression. These results provide new clues as to the role of this essential viral latent protein and its ability to regulate expression of cellular factors, which drives the oncogenic process.IMPORTANCE Epstein-Barr virus (EBV) is the first identified human tumor virus and is associated with a range of human cancers. During EBV-induced lymphomas, the essential viral latent proteins modify the expression of cell cycle-related proteins to disturb the cell cycle process, thereby facilitating the proliferative process. The essential EBV nuclear antigen 3C (EBNA3C) plays an important role in EBV-mediated B-cell transformation. Here we show that EBNA3C stabilizes cyclin D2 to regulate cell cycle progression. More specifically, EBNA3C directly binds to cyclin D2, and they colocalize together in nuclear compartments. EBNA3C enhances cyclin D2 stability by inhibiting its ubiquitin-dependent degradation and significantly promotes cell proliferation in the presence of cyclin D2. Our results provide novel insights into the function of EBNA3C on cell progression by regulating the cyclin D2 protein and raise the possibility of the development of new anticancer therapies against EBV-associated cancers.

Keywords: EBNA3C; Epstein-Barr virus; cell proliferation; cyclin D2.

FIG 1

EBNA3C expression leads to increased levels of cyclin D2 (CCND2). (A) Ten million PBMCs were infected with wide-type EBV or mock infected. Then, the infected cells were collected at the indicated time points, and cyclin D2 mRNAs were detected using real-time PCR. GAPDH was used as the internal control. dpi, day postinfection. (B, C) Ten million HEK293T (B) or BJAB (C) cells were cotransfected with an increased dose of Myc-tagged EBNA3C and Flag-tagged cyclin D2. At 48 h posttransfection, the EBNA3C and cyclin D2 proteins were detected by Western blotting. GAPDH was used as a control protein.

FIG 2

EBNA3C forms a complex with cyclin D2 in cells. (A, B) About 10 million HEK293T (A) or Saos-2 (B) cells were cotransfected with Myc-tagged EBNA3C and Flag-tagged CCND2. Then, cell lysates from the transfected cells were immunoprecipitated with Myc-specific antibody and detected using Western blotting. (C, D) Cell lysates from the indicated B cells (BJAB, BJAB7, BJAB10, LCL1, and LCL2 cells) were immunoprecipitated with EBNA3C-specific antibody and analyzed by Western blotting.

FIG 3

EBNA3C and cyclin D2 colocalize in nuclear compartments. (A) HEK293T, Saos-2, and U2OS cells were cotransfected with Flag-tagged cyclin D2 and DsRed-tagged EBNA3C. The transfected cells were collected after 48 h of transfection, fixed, and incubated with mouse anti-Flag (M2) antibody and Alex Fluor 488-conjugated secondary antibody. DsRed-tagged EBNA3C was directly detected by fluorescence microscope. (B) BJAB, BJAB7, and LCL1 cells were semi-air dried and kept on slides. Endogenous EBNA3C (E3C) or cyclin D2 was incubated with its specific antibody, followed by the corresponding secondary antibodies. Nuclei were stained with DAPI. The images were captured with an Olympus FluoView FV300 microscope and analyzed using its FluoView software.

FIG 4

EBNA3C inhibits cyclin D2 ubiquitination, enhancing its stability. (A) HEK293T cells were cotransfected with Myc-tagged EBNA3C and Flag-tagged cyclin D2. At 36 h posttransfection, the transfected cells were incubated with cycloheximide (CHX) for the indicated times and then lysed, and Western blot analysis was performed. (B) HEK293T cells were cotransfected with Flag-tagged cyclin D2 with or without Myc-tagged EBNA3C. Then, these cells were treated with MG132 or not treated, as shown. Thirty-six hours after transfection, the expression of the targeted protein was analyzed by Western blotting. (C) The indicated plasmids were transfected into HEK293T cells. The transfected cells were incubated with MG132 for 16 h and harvested for Western blot analysis. (D) BJAB, BJAB10, and LCL1 cells were incubated with dimethyl sulfoxide or MG132 for 16 h and collected to detect EBNA3C and cyclin D2 expression. GAPDH was used as a protein control.

FIG 5

EBNA3C can cooperate with Bcl6 to repress the cyclin D2 promoter. (A) HEK293T cells were cotransfected with the cyclin D2 promoter, Myc-tagged EBNA3C, HA-tagged Bcl6, the control vector, as well as the Renilla luciferase-expressing plasmid (pRL-TK) and then lysed after 36 h of transfection, and a dual-luciferase reporter assay was performed. (B) HEK293T cells were transfected with the indicated plasmids, and the luciferase reporter assays were performed as mentioned in the text. The expression of transfected plasmids was monitored by Western blotting.

FIG 6

EBNA3C significantly promotes cell proliferation in the presence of cyclin D2. (A) HEK293 cells were cotransfected with Myc-tagged EBNA3C or HA-tagged cyclin D2, and then the selected cells were treated with G418 after 24 h of transfection and selected for 2 weeks. EBNA3C or cyclin D2 expression in these stable cell lines was detected by Western blotting. Ctrl vec, the control vector. (B) After 2 weeks of selection, the numbers of colonies were monitored by quantifying the intensity of the GFP fluorescence. Representative results from two independent experiments are shown. (C) BJAB or BJAB10 cells were infected with lentivirus and treated with puromycin for 3 weeks. Selected cell colonies with GFP fluorescence are shown here. (D) Selected cyclin D2 knockdown B cells were incubated with PI staining buffer and analyzed by flow cytometry. These results were obtained from two independent experiments. sh-CCND2, cyclin D2-specific short hairpin RNA. FL2-H represents the fluorescence of PI. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001.

FIG 7

Schematic model showing the modulation of the cyclin D2 function by EBNA3C. EBNA3C associates with cyclin D2 and enhances its stability at the protein level through inhibition of its ubiquitin-mediated degradation. The EBNA3C-cyclin D2 interaction ultimately drives cell proliferation, which leads to lymphomagenesis.