Zinc coordination is required for and regulates transcription activation by Epstein-Barr nuclear antigen 1

Abstract

Epstein-Barr Nuclear Antigen 1 (EBNA1) is essential for Epstein-Barr virus to immortalize naïve B-cells. Upon binding a cluster of 20 cognate binding-sites termed the family of repeats, EBNA1 transactivates promoters for EBV genes that are required for immortalization. A small domain, termed UR1, that is 25 amino-acids in length, has been identified previously as essential for EBNA1 to activate transcription. In this study, we have elucidated how UR1 contributes to EBNA1's ability to transactivate. We show that zinc is necessary for EBNA1 to activate transcription, and that UR1 coordinates zinc through a pair of essential cysteines contained within it. UR1 dimerizes upon coordinating zinc, indicating that EBNA1 contains a second dimerization interface in its amino-terminus. There is a strong correlation between UR1-mediated dimerization and EBNA1's ability to transactivate cooperatively. Point mutants of EBNA1 that disrupt zinc coordination also prevent self-association, and do not activate transcription cooperatively. Further, we demonstrate that UR1 acts as a molecular sensor that regulates the ability of EBNA1 to activate transcription in response to changes in redox and oxygen partial pressure (pO(2)). Mild oxidative stress mimicking such environmental changes decreases EBNA1-dependent transcription in a lymphoblastoid cell-line. Coincident with a reduction in EBNA1-dependent transcription, reductions are observed in EBNA2 and LMP1 protein levels. Although these changes do not affect LCL survival, treated cells accumulate in G0/G1. These findings are discussed in the context of EBV latency in body compartments that differ strikingly in their pO(2) and redox potential.

Figure 1. Two conserved cysteines in EBNA1's UR1 domain are essential for transactivation.

(A) Schematic representation of the EBNA1 derivatives used in this study. The positions of the EBNA1's AT-hook domains (ATH1 & ATH2), UR1, DBD are indicated. The NLS spans from a.a 379–386. The EBNA1 derivatives used in this study contain a shortened glycine-alanine repeat that is 15 a.a. in length. EBNA1Δ(71-88) contains a deletion of a.a. 71–88 within the UR1 region. The HMGA1a-DBD protein contains the full length HMGA1a protein fused to the NLS, and the DBD. UR1-DBD contains a.a. 65–89 fused to a.a. 379–641 of EBNA1. The EBNA1(CC→SS) protein is described in detail below. (B) Transactivation of the FR-TKp-Luciferase reporter plasmid by the derivatives of EBNA1 shown in Figure 1A. C33a cells were individually co-transfected with the FR-TKp-Luciferase reporter plasmid, and expression plasmids for the indicated EBNA1 derivatives as described in the Methods section. Luciferase assays were performed at 48 hours post-transfection. Luciferase activity is expressed as fold over the level observed with when DBD is used as the effector protein. The data represents the average of at least three experiments for each EBNA1 derivative. (C) The UR1 region of EBNA1 is conserved in the EBNA1 orthologs of EBV-like gammaherpesviruses. Amino acids 75–85 of EBNA1 from EBV strain B95-8 (accession no. CA24816) (red) are aligned with the corresponding regions from EBNA1 proteins of other gammaherpesviruses (accession nos. YP_067973, BAB03281, AAA66373) (blue), and a portion of a C4 zinc-finger contained in the catalytic subunit of DNA polymerase δ from Xenopus laevis (accession no. NP_001087694), Danio rerio (accession no. CAM46996), Nematostella vectensis (accession no. XP_001641357), Drosophila mojavensis (accession no. XP_002008314), and Homo sapiens (accession no. P28340), identified by BLAST searches. Three or more alike amino-acids in the aligned sequences are shaded the same color. The Genbank accession number for each protein is indicated adjacent to it. (D) Schematic depiction of EBNA1 and the EBNA1(CC→SS) mutant, in which conserved cysteines at position 79 and 82 are mutated to serine. The sequences of wild-type (WT), and mutant (M) peptides used in this study are also shown.

Figure 2. Zinc is coordinated by EBNA1's UR1 domain, and is required for EBNA1 to transactivate.

(A) Evaluation of Zinc⁶⁵ binding by the wild type (WT) and mutant (M) UR1 peptides. The indicated amounts of WT and M peptides were dot-blotted on a 0.2 mM PVDF membrane, and probed with radioactive zinc (∼50 µCi) as described in the Methods section. Washed membranes were dried and visualized using a Phosphorimager. WT peptide was observed to bind Zn⁶⁵ in a concentration-dependent manner. In contrast, the M peptide failed to bind zinc. To confirm that both peptides bound the PVDF membrane, a blot prepared in parallel was dried and stained using Amido Black. (B) The metal chelator TPEN reduces the ability of EBNA1 to transactivate FR-TKp-Luciferase. C33a cells were co-transfected the FR-TKp-Luciferase reporter plasmid, an EBNA1-expression plasmid, and a CMV-EGFP expression plasmid. Cells were treated with the indicated levels of TPEN at the time of transfection, and harvested 15 hours later. Harvested cells were analyzed by flow cytometry to determine the fraction of live-transfected cells, the level of EGFP expression in that fraction. For cell-cycle analysis, the cells were fixed and then PI-stained. An aliquot of harvested cells were used to determine the expression level EBNA1 by immunoblot upon treatment with the indicated amounts of TPEN, shown as an inset in the graph. The rest of the cells were processed to determine luciferase activity, which is expressed as a percent of the luciferase activity observed in the absence of TPEN treatment, and shown in the grey bars. The open bars indicate the percent of live EGFP-positive cells at each concentration of TPEN. The asterisks indicate that the significantly lower levels of luciferase activity were observed in the presence of 5, 15 and 45 µM TPEN (p<0.05, Wilcoxon rank-sum test). The cell-cycle profile of TPEN-treated and control cells was obtained for one experiment, and is shown below the graph. (C) TPEN does not affect transactivation by DBD-VP16. C33a cells were co-transfected with the FR-TKp-Luciferase reporter plasmid, and expression plasmids for DBD, and DBD-VP16. Some DBD-VP16 transfected cells were treated with the indicated amounts of TPEN at the time of transfection, and analyzed 15 hours later to determine the fraction of live-transfected cells, and the level of luciferase expression. The activity observed with DBD-VP16 in the absence and presence of TPEN is expressed as the fold-over the luciferase level observed with DBD alone. (D) Zinc and Cadmium reverse TPEN-mediation inhibition of transactivation by EBNA1. C33a cells were co-transfected with the FR-TKp-Luciferase reporter plasmid, an EBNA1 expression plasmid, and a CMV-EGFP expression plasmid, and treated with 5 µM TPEN at the time of transfection. Fifteen hours post-TPEN addition, 5 µM of Zn (CH₃COO)₂, Cd(CH₃COO)₂, CaCl₂, MgSO₄, MnCl₂, or Fe(CH₃COO)₂, was added to the cells for an additional 15 hours. Cells were harvested and analyzed by flow cytometry to determine the level of live-transfected cells, followed by determination of luciferase activity. The relative activation is shown in the grey bars, and is expressed as a percent of the luciferase activity observed in the untreated sample.

Figure 3. EBNA1(1-450)-E2DBD does not squelch transactivation by EBNA1.

C33a cells were co-transfected with the FR-TKp-Luciferase reporter plasmid, and control vector pcDNA3 (dark grey bars), an expression plasmid for EBNA1(1-450)-E2DBD (light grey bars), or an expression plasmid for DBD (open bars, in the amounts indicated below each bar. Cells were harvested at 48 hours post-transfection, normalized by flow cytometry for the number of live-transfected cells, and analyzed for luciferase activity, which is expressed as percent luciferase activity relative to the activity obtained by co-transfection with the same amount pcDNA3.

Figure 4. EBNA1 can self-associate through its UR1 domain.

(A) The amino-terminal 450 amino-acids of EBNA1 can self-associate. 293 cells were co-transfected with expression vectors for 3xF-EBNA1(1-450)-E2DBD, and either EBNA1 or EBNA1(CC→SS). 48 hours post-transfection, lysates were prepared from harvested cells. EBNA1(1-450)-E2DBD was immunoprecipitated using the M2 monoclonal anti-FLAG antibody conjugated to sepharose beads. Immunoprecipitates were separated on an 8% SDS-PAGE gel, transferred to PVDF membranes and immunoblotted using M2 mouse monoclonal anti-FLAG antibody (α-FLAG/α-FLAG), or rabbit polyclonal antibodies directed the EBNA1 DBD (α-FLAG/α-DBD). 5% of the lysates were directly immunoblotted for EBNA1 or EBNA1(CC→SS) (5% lysate/α-DBD). (B) Self-association of UR1 can be detected by bimolecular fluorescence complementation: 293 cells were transfected with expression plasmids for (A) EYFP(2-154) and EYFP(155-238), or (B) & (C) expression plasmids for UR1-EYFP(2-154) and UR1(155-38). Transfected cells were treated with (B) vehicle, or (C) 80 µM 1,10-phenanthroline. 48 hours after transfection, cells were visualized by fluorescence for EYFP (YFP) or by light microscopy (DIC). The scale bars indicate a length of 10 microns. The fraction of fluorescent cells observed under each condition in five independent measurements is shown along with the standard deviation. The number of fluorescent cells observed with the UR1-containing EYFP derivatives is significantly greater than the number seen with the EYFP derivatives by themselves (p<0.01). A significant decrease in the number of florescent cells is observed after treatment with 1,10-phenanthroline (p<0.05) when compared to the untreated cells.

Figure 5. UR1-mediated self-interaction is required for EBNA1 to transactivate cooperatively.

(A) Size-exclusion chromatography of the wild-type UR1 peptide indicates zinc-dependent dimerization. WT peptide was subjected to HPLC size-exclusion chromatography, as described in the Methods section, in the absence of zinc (red), or in the presence of 1 mM zinc sulfate in the buffer (green). The retention time is indicated above each peak, and the apparent molecular weight calculated from the retention time is shown in parentheses. The elution profile of molecular weight markers is shown in gray. The known molecular weight of each marker is indicated above the peak, with the observed retention time shown in parentheses. (B) Cooperative transactivation by EBNA1 is UR1-dependent. C33a cells were co-transfected individually with TKp-luciferase reporter plasmids containing one, three, five, seven, ten, or 20 EBNA1 binding sites in FR along with an expression plasmid for EBNA1 or EBNA1(CC→SS). Cells were harvested at 48 hours post-transfection, normalized by flow cytometry for the number of live-transfected cells, and analyzed for luciferase activity, which is expressed as luciferase activity fold-over the activity obtained by co-transfection with vector control. The white bars indicate the predicted levels of transactivation for EBNA1 if it increased additively with increasing numbers of binding sites. The transactivation observed with EBNA1 is indicated by the black bars, and transactivation observed with EBNA1(CC→SS) is indicated by the gray bars. For reporter plasmids containing up to three binding sites, EBNA1 and EBNA1(CC→SS) transactivated equivalently. A small difference was observed for a reporter plasmid with five binding sites, and this difference was greatly accentuated for reporter plasmids containing seven, ten, and 20 binding sites. (C) Cooperative transactivation by EBNA1 fits a sigmoidal dose-response model by non-linear regression analysis. The fold transactivation by EBNA1 observed in (5B) was analyzed for cooperativity as described in the Materials & Methods, and found to fit a sigmoidal dose-response model well, as indicated by the goodness of fit (R²). Positive-cooperativity is inferred from the positive Hill coefficient (HC). (D) EBNA1(CC→SS) does not activate transcription cooperatively. The fold transactivation observed for EBNA1(CC→SS) was fit to the same sigmoidal dose-response model used in 5C. The low R² indicates that transactivation by EBNA1(CC→SS) poorly fits this model. In addition, the large standard deviation observed for the HC also indicates a lack of cooperative transactivation.

Figure 6. Transactivation by EBNA1 is sensitive to oxidative stress, and is augmented by over-expression of APE1/Ref-1.

(A) Menadione reduces the ability of EBNA1 to transactivate FR-TKp-Luciferase. C33a cells were co-transfected the FR-TKp-Luciferase reporter plasmid, and an EBNA1-expression plasmid. Cells were treated with the indicated levels of menadione six hours post-transfection, and harvested 18 hours later. For cell-cycle analysis, an aliquot of cells was fixed and then PI-stained. The rest of the cells were processed to determine luciferase activity, which is expressed as a percent of the luciferase activity observed in the absence of menadione treatment, and shown in the grey bars. The cell-cycle profiles of menadione-treated and control cells were obtained for one experiment, and are shown below the graph. The asterisks indicate statistical significance (p<0.05) compared to control. (B) Menadione affects the ability of UR1 to support bimolecular fluorescence complementation. C33a cells were transfected with expression plasmids for A) EYFP(2-154) and EYFP(155-238), or B) & C) expression plasmids for UR1-EYFP(2-154) and UR1(155-238). Transfected cells were treated with vehicle alone, or 0.6, 1.4 and 2.0 µM menadione six hours after transfection. Cells were visualized by fluorescence for EYFP (YFP) or by light microscopy (DIC) after 18 hours of menadione treatment. The scale bars indicate a length of 10 microns. The fraction of fluorescent cells observed for each menadione concentration in five independent measurements is shown along with the standard deviation. The p-values indicate statistical significances calculated using the Wilcoxon rank-sum test upon a pair-wise comparison to untreated cells. p-values lower than 0.05 indicate significance. (C) Over-expression of Ref-1/APE1 augments transactivation by EBNA1. C33a cells were transfected with an EBNA1 expression plasmid, and increasing amounts of an expression plasmid for Ref-1/APE1 along with the FR-TKp-luciferase reporter plasmid. Luciferase levels were evaluated 48 hours post-transfection, and are expressed as a fraction of transactivation observed in the absence of Ref-1/APE1, which was set to be 100%. The asterisks indicate statistical significance (p<0.05) compared to control. (D) Over-expression of Ref-1/APE1 ameliorates the effect of menadione on EBNA1 mediated transactivation. C33a cells were transfected with an EBNA1 expression plasmid, and either 1 µg of a Ref-1/APE1 expression plasmid or empty vector control plasmid in addition to the FR-TKp-luciferase reporter plasmid. Six hours post-transfection, the cells were split and half the cells were treated with 1.4 µM of menadione. Luciferase levels were evaluated 24 hours post-transfection. p values are indicated in the figure.

Figure 7. Hypoxic conditions prolong EBNA1's capacity to transactivate.

(A) Transactivation by EBNA1 is sustained under hypoxic conditions. C33a cells were co-transfected with an EBNA1-oriP expression plasmid and the oriP-BamHI-Cp-luciferase reporter plasmid. Transfected cells were split six hours post-transfection, and grown under normoxic (filled circles) or hypoxic conditions (filled squares). Luciferase expression was measured at the indicated time-points, and is expressed relative to the luciferase expression observed under normoxic conditions 48 hours post-transfection. (B) Oxygen levels do not affect oriP-plasmid replication and maintenance. A Southern blot was performed to examine the levels of replicated (DpnI-resistant) oriP-BamHI-Cp-luciferase plasmid recovered after six days under normoxic or hypoxic conditions. The arrowhead indicates replicated, full-length oriP-BamHI-Cp-luciferase. (C) Environmental oxygen levels do not alter the activity of DBD-VP16. C33a cells were transfected with a DBD-VP16 expression plasmid and the FR-TKp-luciferase reporter plasmid. Luciferase activity was measured three days post-transfection in cells exposed to normoxic or hypoxic conditions.

Figure 8. Mild oxidative stress reduces transcription from the EBV BamHI-Cp in a lymphoblastoid cell-line.

(A) Paraquat treatment reduces the level of BamHI-Cp transcripts in treated NOLA-1 cells. Total RNA was extracted from control or cells exposed to 150 µM of paraquat for 48 or 72, reverse-transcribed with random hexamers. cDNA was used for real time PCR performed as described in the Methods section to evaluate changes in Cp transcripts relative to the endogenous control GAPDH transcripts. The results are presented relative to the BamHI-Cp transcript level observed in control cells, which was assigned a value of 1. Significant decreases in the level of BamHI-Cp transcripts were observed in cells with paraquat for 48 and 72 hours, such that transcript level was reduced to approximately 60% after 48 hours of treatment, and approximately 40% after 72 hours of treatment. Cell-cycle analyses conducted with several treated samples indicates that paraquat treatment does not increase the sub-G1 population. However, treated cells were consistently observed to accumulate in G0/G1. Treatment caused a small increase in the number of necrotic cells doubly-positive for annexin V and PI staining. Small increases were also seen for cells positive for only annexin V or only PI. (B) Paraquat treatment decreases expression of EBNA2 and LMP1, as evaluated by immunoblot. 2×10⁵ control or cells treated with 150 µM paraquat (P150) for 72 hours were examined as described in Methods for expression of EBNA1, EBNA2 and LMP1. Band intensity was compared to that observed in control cells, and is expressed as percent of control along with standard deviation from two experiments. Expression was normalized to the level of β-actin protein detected in each sample. (C) Paraquat treatment does not alter the expression or localization of EBNA1. Control or treated cells were transferred to slides by cytospin, prior to fixation and permeabilization. EBNA1 expression was evaluated using rabbit polyclonal Ab 2638 as described in the methods, and detected using a TexasRed conjugated secondary anti-rabbit Ab. Cells were counterstained with Hoechst 33342. The absence of paraquat exposure, or length of treatment with 150 µm paraquat is indicated above each panel. Detection of EBNA1-specific signal, Hoechst signal and the merge is also indicated. The scale bar represents a distance of 10 µM.