Fig 1.
Proteasome inhibition leads to degradation of EBV essential antigen EBNA3C.
(A) ~10 x 106 LCL#1 and (B) BJAB stably expressing EBNA3C (BJAB-E3C#7) cells were treated with protein synthesis inhibitor, cyclohexamide (CHX) for 24 h in the absence (DMSO control) or in the presence of 1 μM proteasome inhibitor MG132 or 50 μM autophagy-lysosomal inhibitor Chloroquine (CQ). (C) ~10 x 106 HEK293 cells transiently transfected with empty vector (pA3M) or pA3M-EBNA3C expressing myc-tagged EBNA3C. 36 h post-transfection cells were either left untreated (DMSO control) or treated with 20 μM MG132 or 50 μM CQ for another 4 h before harvesting. HEK293 cells transfected with myc-tagged EBNA3C were incubated with (D) EBSS to induce autophagy for 9 h or (E) increasing concentrations of MG132 (0–40 μM) for 4 h. (F) HEK293 cells transiently transfected with empty vector or expression plasmids for myc-tagged EBNA3C or EBNA1, were either left untreated or treated with 20 μM MG132 for additional 4 h. (A-F) In each experiment, cells were harvested after drug treatment, washed with 1 x PBS, lysed in RIPA and fractionated using appropriate SDS-PAGE. For transient transfection studies, cells were additionally transfected with GFP expression vector to monitor the transfection efficiency. Western blots were performed with indicated antibodies. GAPDH blot was performed as loading control. The relative intensities (RI) of protein bands shown as bar diagrams were quantified using the software provided by Odyssey CLx Imaging System. Representative gel pictures are shown of at least two independent experiments.
Fig 2.
Proteasomal inhibition results in degradation of viral oncoproteins—EBNA3A and EBNA3C, but not EBNA3B in LCLs.
(A) ~10 x 106 LCLs–both LCL#1 and LCL#89 were either left untreated (DMSO control) or treated with 1 μM MG132. 12 h post-incubation, cells were harvested, washed with 1 x PBS, lysed in RIPA buffer and subjected for western blot analyses with indicated antibodies. GAPDH blot was used as loading control. The relative intensities (RI) of protein bands shown as bar diagrams were quantitated using the software provided by Odyssey CLx Imaging System. Representative gel pictures are shown of at least two independent experiments. (B) For confocal assays, ~5 x 104 LCLs (LCL#89) either left untreated (DMSO control) or treated with 1 μM MG132 for 12 h, were fixed with 4% paraformaldehyde. Immuno-staining was performed using primary antibodies against viral proteins—EBNA3A, EBNA3B and EBNA3C followed by Alexa Fluor conjugated secondary antibodies for visualization in a Leica DMi8 Confocal Laser Scanning Microscope. All panels are representative pictures of two independent experiments. The bar diagram represents the mean value of staining intensities of EBNA3 proteins from at least 10 cells of 3 different fields. *** indicates P < 0.05. Nuclei were counterstained using DAPI (4’,6’-diamidino-2-phenylindole) before mounting the cells.
Fig 3.
Proteasomal inhibition induces both viral and autophagy gene transcriptions.
(A-D) ~10 x 106 two LCL clones–LCL#1 and LCL#89 were either left untreated (DMSO control) or treated with 1 μM MG132. 12 h post-treatment cells were harvested for (A and C) total RNA or (B and D) genomic DNA isolation as described in the “Materials and Methods” section. (B and D) LCLs were treated with 3 mM sodium butyrate (NaBu) in combination with 20 ng/ml 12-O-tetradecanoylphorbol-13-acetate (TPA) for 24 h to induce viral lytic cycle as positive control. (A and C) Total RNA was subjected to cDNA preparation followed by quantitative real-time PCR (qPCR) analyses for the selected viral genes. The relative changes in transcripts (log10) using the 2−ΔΔCt method are represented as bar diagrams in comparison to DMSO control using GAPDH, B2M and RPLPO as housekeeping genes. Two independent experiments were carried out in similar settings and results represent as an average value for each transcript. (B and D) qPCR was performed for the detection of EBV DNA (BamHW fragment) using the genomic DNA isolated from each sample. The average fold increase of two independent experiments represented as bar diagrams was calculated in comparison to DMSO control using the 2−ΔΔCt method taking GAPDH as genomic control. (A-D and G-H) Average values +/- SEM are plotted. *, **, *** = p-value < 0.01, 0.005 and 0.001 respectively. (E) The cDNA samples similarly prepared in (B and D) were subjected to whole transcriptome analyses (RNA-Seq) using Ion S5 XL System as described in the “Materials and Methods” section. RNASeqAnalysis plugin (v5.2.0.5) was utilized to perform the analysis after align with human genome (hg19) and produce gene counts for all the samples. Differential gene expressions were performed based on p-value as < = 0.05 and log2 Fold Change as 2 and above (upregulated, red) and -2 and below (downregulated, blue). (F) Differentially expressed gene sets were uploaded on DAVID v6.8 webserver for functional analysis. Gene Ontology (GO) was selected from the hits table for DAVID clustering. The bar diagrams (upregulated: red; downregulated: blue) represent top 15 most significantly affected pathways. (G-H) qPCR analyses of the selected cellular genes as described in (A and C).
Fig 4.
EBNA3C is degraded through autophagy-lysosomal pathway when proteasome is inhibited.
LCLs were either left untreated (DMSO control) or treated with 1 μM MG132, 0.1 μM bafilomycin (Baf) or MG132 plus Baf. 24 h post-treatment cells were subjected for either (A) western blot analysis or (B) immunostaining with the indicated antibodies. Each panel in (B) is representative picture of two independent experiments and nuclei were counterstained by DAPI before mounting the cells. Scale bars, 5 μm. (C) ~10 x 106 HEK293 cells transfected with either empty vector (pEGFP-C1) or GFP-tagged EBNA3C expression plasmid, either left untreated (DMSO control), or treated with 20 μM MG132 alone or MG132 plus 50 μM chloroquine (CQ). (D) HEK293 cells stably transfected with pTripz-mCherry-Sh-Beclin1 construct expressing sh-Beclin 1 under doxycycline (Dox) inducible promoter were further transfected either empty vector (pA3M) or myc-tagged EBNA3C expressing construct. 36 h post-transfection cells were either left untreated or treated with 20 μM MG132. (C-D) 4 h post-treatment cells were harvested, washed with 1 x PBS, lysed in RIPA buffer and subjected for western blot analyses for the indicated antibodies. (E) ~5 x 104 HEK293 cells stably expressing sh-Beclin 1 with or without doxycycline treatment were transfected with GFP-tagged EBNA3C expressing plasmid using Lipofectamine 3000. 24 h post transfection cells were either left untreated (DMSO control) or treated with 20 μM MG132 for another 4h and subjected to live cell confocal analysis after staining the nucleus with Hochest 33342. Doxycycline treatment induces both mCherry and sh-RNA expression. Scale bars, 5 μm. (F) A colony formation assay was conducted as described in the “Materials and Methods” section using a similar experimental setup in doxycycline inducible sh-Beclin1 stably expressing HEK293 cells transiently transfected with GFP-EBNA3C construct.
Fig 5.
EBNA3C participates within the p62-LC3B complex when proteasome is inhibited.
(A) ~10 x 106 HEK293 cells were transfected with either empty vector (pA3F) or flag-tagged EBNA3C expressing construct. 36h post-transfection cells were either left untreated (DMSO control) or treated with 20 μM MG132 for 4 h. After treatment, cells were harvested and subjected for immunoprecipitation with anti-flag antibody (M2). Precipitated products along with 10% whole cell lysates were run on gel and western blot analysis was performed with indicated antibodies. (B) ~20 x 106 LCLs (LCL#89) either left untreated (DMSO control) or treated with 1 μM MG132 for 12 h, were harvested and subjected for immunoprecipitation using EBNA3C specific mouse monoclonal antibody (A10). Rabbit anti-mouse IgG was used as isotype control. (C) ~5 x 104 HEK293 cells were co-transfected with the indicated expression plasmids using Lipofectamine 3000. 36 h post transfection, cells were further treated with DMSO or 20 μM MG132 for 4 h and subjected for live cell confocal analysis after staining the nucleus with Hochest 33342. (D) For confocal analysis in LCLs, ~5 x 104 LCLs (LCL#1) were treated with DMSO control or 1 μM MG132 for 12 h and subjected for immune staining with EBNA3C, p62, LC3B specific primary antibodies followed by incubation with Alexa Fluor conjugated secondary antibodies. Nuclei were counterstained using DAPI (4’,6’-diamidino-2-phenylindole) before mounting the cells. Each panel in (C-D) corresponds to single experiment of three independent experiments. Scale bars, 5 μm.
Fig 6.
EBNA3C is predominantly K63-linked polyubiquitinated when proteasome is inhibited.
LCLs were either left untreated (DMSO control) or treated with 1 μM MG132 for 12 h and subjected for (A) immunostaining with EBNA3C, total ubiquitin (Ub), K48- and K63-linked polyubiqitination specific antibodies; (B) western blot analyses with indicated antibodies. (A) Nuclei were counterstained with DAPI before mounting the cells for confocal analyses. (C-D) HEK293 cells transiently transfected with flag-tagged EBNA3C expression vector, either left untreated (DMSO control) or treated with 20 μM MG132 for 4h were subjected for (C) western blot analyses or (D) immunoprecipitation (IP) under denatured conditions using anti-flag antibody followed by western blot analyses using indicated antibodies. Each panel in (A) corresponds to single experiment of two independent experiments. Scale bars, 5 μm. Western blots in (B-C) were performed by stripping and reprobing the membranes with different antibodies.
Fig 7.
N-terminal domain of EBNA3C is important for autophagy mediated degradation in response to proteasomal inhibition.
(A) HEK293 cells transiently transfected with plasmids expressing flag-tagged EBNA3C truncations–residues 1–365, 366–620 and 621–992, either left untreated (DMSO control) or treated with 20 μM MG132 or 50 μM Chloroquine (CQ), were harvested and subjected for western blot analyses with the indicated antibodies. (B) Using a similar experimental set up as described in (A), HEK293 cells transiently transfected with full-length (residues 1–992) and different domains of flag-tagged EBNA3C expression constructs, were subjected to immunoprecipitation (IP) with anti-flag antibody followed by western blot analyses with the indicated antibodies after stripping and reprobing the same membrane. (C) The schematic illustrates known structural motifs and different domains of EBNA3C that are used to determine residues important for autophagy mediated degradation upon proteasomal inhibition. Predicted PEST motifs were derived by using http://emboss.bioinformatics.nl/cgi-bin/emboss/epestfind (green: potential; red: poor). (D) HEK293 cells transiently transfected with expression plasmids for flag-tagged EBNA3C N-terminal trunactions (residues 1–365, 50–300, 1–300 and 1–250) either were subjected to western blot analyses with the indicated antibodies after a similar treatment as described in (B). Protein band intensities in (A and D) were quantified by Odyssey imager software and indicated either as bar diagrams or LC3-II/I ratio at the bottom of each corresponding lane. Representative gel pictures are shown of two independent experiments. GAPDH and GFP blots were performed as loading and transfection efficiency controls, respectively.
Fig 8.
EBNA3C N-terminal domain is degraded in both nuclear and cytoplsamic fractions upon proteasomal inhibition.
(A) Schematic shows the predicted nuclear export signal (NES) sequences of EBNA3C, derived from http://www.cbs.dtu.dk/services/NetNES/. (B-C) HEK293 cells were transiently transfected with the indicated expression plasmids. 36 h post-transfection cells were further treated with DMSO control or 20 μM MG132 for 4 h and subjected to (B) subcellular fractionation followed by western blot analyses using the indicated antibodies and (C) live cell confocal analyses. (D-E) LCLs were either left untreated (DMSO control) or treated with 0.5 μM MG132, 20 ng/ml leptomycin B (LMB) or MG132 plus LMB. 24 h post-treatment cells were subjected for either (D) western blot analysis or (E) immunostaining with the indicated antibodies. (C, F-G) Nuclei and lysosomes were counterstained with Hoechst 33342 and LysoTracker Red DND-99, respectively. Nuclei in (D) were counterstained by DAPI before mounting the cells. Each panel of confocal images in (C, E-G) is representative pictures of two independent experiments. Scale bars, 5 μm. (H-I) HEK293 cells transiently transfected with plasmids expressing flag-tagged EBNA3C N-terminal (residues 1–365) and C-terminal (residues 621–992) domains in a similar experimental set up as described in (B) were subjected to subcellular fractionation as per Manufacturer’s instruction, followed by western blot analyses with the indicated antibodies. (B and H-I) GAPDH was used as reference protein for cytoplasmic fraction, while lamin A/C (B) and histone (H-I) blots were performed as nuclear reference proteins. Protein bands in (B, D, H-I) were quantified by Odyssey imager software and indicated as bar diagrams at the bottom of corresponding lanes.
Fig 9.
Proteasome inhibitors can be used as potential therapeutic strategy against EBV associated B-cell lymphomas.
(A-C) 1 × 105 LCLs (LCL#1 and LCL#89) were either left untreated (DMSO control) or treated with increasing concentrations of MG132 (1–5 μM) or bortezomib (0.5–1 μM) for 24 h and subjected for soft agar colony formation assay as described in the “Materials and Methods” section. After 14 days colonies were stained with 0.1% crystal violet and scanned using Odyssey CLx Imaging System and the number of colonies were measured by Image J software and plotted as bar diagrams in (C). Prior to staining, each well was also photographed (bright-field) using a Fluorescent Cell Imager as shown in (B). (D) ~0.5 × 105 LCLs (LCL#1 and LCL#89) plated into each well of six-well plates were either left untreated (DMSO control) or treated with increasing concentrations (0–10 μM) of MG132 or bortezomib for the indicated time points at 37°C in a humidified CO2 chamber. Viable cells from each well were measured by Trypan blue exclusion method using an automated cell counter. (E) ~0.5 × 105 LCLs (LCL#1 and LCL#89) plated into each well of six-well plates were either left untreated (DMSO control) or treated with 5 μM MG132 or 0.5 μM bortezomib for 24 h were subjected to apoptosis assay using annexin V/propidium iodide (PI) staining. FITC labeled annexin V binding (Ex = 488 nm; Em = 350 nm) and PI staining were detected using BD FACSAria III. Error bars represent standard deviations of duplicate assays of two independent experiments. *, **, *** = p-value < 0.01, 0.005 and 0.001 respectively.
Fig 10.
EBNA3C expressing B-cells are more sensitive to apoptotic cell death induced by proteasome inhibitors.
(A) ~0.5 × 105 BJAB cells stably harboring vector or EBNA3C expressing plasmid plated into each well of six-well plates were either left untreated (DMSO control) or treated with increasing concentrations (0–20 μM) of MG132 or bortezomib for 3 days. Viable cells from each well were measured by Trypan blue exclusion method using an automated cell counter. (B-D) ~10 × 105 BJAB-vector or BJAB-EBNA3C cells were treated with DMSO, or 5 μM of MG132 or bortezomib for 24 h were subjected to (B) apoptosis assay using annexin V/propidium iodide (PI) staining, (C) western blot analyses using indicated antibodies and (D) soft agar colony formation assay as described in the “Materials and Methods” section. GAPDH blot was used as loading control in western blot analyses. Protein bands were quantified by Odyssey imager software and indicated as bar diagrams at the bottom of corresponding lanes. Error bars represent standard deviations of duplicate assays of two independent experiments. *, **, *** = p-value < 0.01, 0.005 and 0.001 respectively.
Fig 11.
Schematic representation of proteasomal inhibition mediated EBNA3C’s degradation.
Due to uncontrolled cell proliferation, cancer cells often encounter excess mis-/un-folded protein aggregates, which are subsequently labeled with either K48-linked or K63-linked polyubiquitination for proteolytic degradation. While K48-linked ubiquitin chains are targeted for the proteasomal pathway, K63-linked directs autophagy mediated protein degradation. Upon proteasomal inhibition, EBV oncoprotein EBNA3C is predominantly tagged with K63-linked polyubiquitin chains, translocated to cytoplasm by an unknown mechanism and degraded through autophagy-lysosomal pathway. The N-terminal domain plays a central role in EBNA3C’s degradation through autophagy mechanism by participating within the p62-LC3B complex. Suppression of autophagy pathway (Beclin1 and p62 knockdown or chloroquine (CQ)/bafilomycin treatment) reverses EBNA3C degradation in response to proteasomal inhibition. Additionally, proteasomal inhibitors (such as MG132) induce both autophagy and viral gene transcription that eventually activate viral lytic replication. The results provide foundation to exploit proteasome inhibitors as potential therapeutic approach for EBV associated B-cell lymphomas, where EBNA3C is expressed, typically diagnosed in immunocompromised individuals.