Fig 1.
Identification of CAND1 protein network by C60-affinity purification.
(A) Chemical structure of C60 and scheme for generating C60-sepharose using HiTrap NHS resin and an amide linker. (B) Silver stain of C60-affinity purification. Mutu I cell total cell extract (Lysate) was incubated with Linker-Sepharose or C60-Sepharose and eluted with SDS elution buffer and heat (Δ) or with 10 μM C60 (C60 elute). M is marker proteins with Kd indicated. (C) List of proteins identified by LC-MS/MS showing unique peptides in C60 eluted from C60 or Linker Control resin. Only proteins detected with at least 5 spectra counts in C60, but none in blank or control are shown. C60 = spectra counts for C60 sample. MW = molecular weight, pep = number of unique peptides, inter = total number of known protein-protein interactions with other proteins from the list. (D) Known protein-protein interactions among the list of most abundant proteins identified from LC-MS/MS C60 affinity purification shows CAND1 as the main hub of the identified network. (E) Western blot for proteins shown in panel B probed with anti-CAND1 (top panel), or anti-Actin (lower panel). (F) List of proteins involved with CAND1 in protein modification network (ubquitylation, sumoylation) identified in C60 affinity purification, but not shown in panel C.
Fig 2.
Identification of CAND1 by shRNA screen for lytic reactivation.
(A) Mutu I cells containing BHLF1-GFP were transduced with shRNA lentivirus library and FACS sorted for GFP. (B) LCL cells were transduced with shRNA lentivirus library and FACS sorted for VCA. (C) Correlation of Illumina sequencing of shRNAs from GFP+ vs GFP- populations for transduced Mutu I cells described in panel A. (D) Scoring of shRNA clones from combining both GFP+ Mutu I cells and VCA+ LCL cells after transduction with shRNA lentivirus library. Genes with 4 different targeting shRNA are indicated in red, 3 shRNA in green. Lytic vs non-lytic score reflects the percentage of shRNA found in each FACS sorted population. (E) Western blot analysis of Mutu I cells transduced with shCtrl, shZEB1, zhCAND1_1, or shCAND1_2, and probed with antibody to CAND1, viral antigens EA-D or ZTA, or β-Actin. (F) The percentage of EBV lytic cells was measured by FACS sorting for VCA+ Mutu I cells after transduction with shCTRL, shZEB1, shSET, shCAND1_1, or shCAND1_2.
Fig 3.
Gene expression profiling of C60 treated Mutu I cells.
(A) RNA expression heatmap for genes affected >2.5 by C60 treatment. Group indicates if the gene was affected by b = both C60 and NaB, o = opposite by C60 compared to NaB and u = uniquely by C60. (B) Overlap between C60 and NaB transcriptional effect identifies sets of common and specific to C60 genes. (C) Genes affected by both C60 and NaB 2.5 fold or more. (D) Genes affected specifically by C60 2.5 fold or more in opposite or unique effect relative to NaB. (E-F) IPA analysis for regulators, pathways and functions enriched among genes significantly affected by C60. Z = z-score for predicted activation state of the category: positive = activated by C60, negative = inhibited by C60. Where significant, regulators are listed with mRNA level changes in parentheses. (G) DAVID analysis for swiss-prot categories significantly enriched among genes with a specific C60 effect (affected only by C60 or in opposite way compared to NaB) with genes fold changes listed in parenthesis. Enr = fold enrichment, FDR = false discovery rate.
Fig 4.
Stabilization of p53 and destabilization of p21 by C60.
(A) Western blot of extracts from Mutu I, LCL352, Akata EBV+, Akata EBV-, and BJAB cells treated with DMSO (D) or C60 and probed with antibody to either CAND1, p53, p53 pS15, Cul1, CDC25, ZTA, EA-D, p21, γH2AX, or β-Actin, as indicated. (B) Comparison of C60 with TPA/NaB relative to DMSO control at 24 and 48 hrs post treatment in Mutu-LCL (M-LCL) and Mutu I cells. (C) Western blot of LCL352 cells transduced with shCtrl or shCAND1_1 were probed with antibodies to CAND1, p53 S15, p53, EA-D, p21, γH2AX, or β-Actin.
Fig 5.
C60 disrupts CAND1-Cullin1 interaction and increases global ubiquitylation.
(A-C) Extracts from Mutu I cells treated with DMSO or C60 for 48 hrs were subject to IP with antibody to Cullin 1, CAND1, or IgG and analyzed by Western blot for Cullin1 (A) or CAND1 (B). Input (10%) is shown in first two lanes. (C) Quantitation of three independent coIP experiments as represented in panels A and B. (D) Mutu I, LCL352, Akata EBV+, Akata EBV-, and BJAB cells were treated with DMSO or C60 for 48 hrs, and then assayed by Western blot with antibody to Ubiquitin 1 (Ubi-1). (E) Dose dependent activation of global ubiquitylation in Mutu I (left) and EBV-negative 283T (right) cells. Total cell extracts were treated with indicated concentration of C60 for 48 hrs. Actin loading control is shown below.
Fig 6.
ZTA transcription activation domain mediates interactions with CAND1 and Cullins.
(A) Schematic of ZTA transcription activation (TA) indicating position of sumoylation site at K12 and Cul5 box 52-LPEP-54. (B) Luciferase assay for BHLF1-Luc cotransfected with expression vector for ZTA (WT), K12A, or EP53/54AA (MUT) in 293T cells treated with DMSO (black bars) or C60 (grey bars) for 48 hrs. (C) Western blot of 293T cells transfected with ZTA (WT), K12A, or EP53/54AA treated with DMSO or C60 for 48 hrs, as described in panel B. (D-E) 293T cells transfected with FL-CTRL, FL-WT-ZTA, or FL-MUT-ZTA were treated with DMSO or C60 for 48 hrs, and then subject to IP with FLAG and probed with antibodies for Cul 1, Cul 2, CAND1, FLAG, or Actin (D). 10% of input was analyzed by Western for Cul 1, Cul 2, CAND1, FLAG, and Actin (E). (F-G) 293T cells were transfected as in D-E, and subject to IP with either Cul 1, Cul 2, or FLAG, and then analyzed by Western blot with antibody to FLAG (F). 10% of input was analyzed by Western for Cul 1, Cul 2, FLAG, and Actin (G).
Fig 7.
C60 stabilizes ZTA through a mechanism distinct from MG132 and MLN4924.
(A) Timeline of ZTA protein stabilization assay with addition of cyclohexamide (20 μg/ml) in the presence of either C60 (1 μM) or DMSO at 12 hrs post-transfection followed by cell collection at 4 and 24 hrs after addition of cyclohexamide. Western blot for FLAG-ZTA and Actin in 293T cells transfected and treated as shown in timeline above. (B) Quantification of Western blot shown in panel A as FLAG-ZTA relative to Actin at each time point. (C) Mutu I cells treated with DMSO, MG132 (20 μM), MLN4924 (0.5 μM), or C60 (5 μM) were assayed by Western blot at 24 hrs or 48 hrs after drug treatment. Western blots were probed with antibodies to EA-D, ZTA, Cul 1, Cul 2, p53, MDM2, Ubiquitin (Ubi-1) and Actin, as indicated.
Fig 8.
Model of C60 perturbation of CAND1-Cullin function in destabilizing ZTA and p53.
CAND1 recycles Cullins to generate new CRLs that degrade ZTA (right red arrow). C60 alters CAND1 interaction with Cullins to prevents ubiquitin-mediated degradation of ZTA, resulting in EBV reactivation from latency. C60 works through a different arm of the pathway than proteasome inhibitor MG132 or NAE inhibitor MLN4924.