Zinc coordination is required for and regulates transcription activation by Epstein-Barr nuclear antigen 1.
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.
Affiliation: Department of Microbiology, Immunology & Parasitology, LSU Health Sciences Center, New Orleans, LA, USA.
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.
- Epstein-Barr Virus Nuclear Antigens/chemistry/genetics/metabolism*
- Herpesvirus 4, Human/genetics*
- Transcriptional Activation*/drug effects
- Amino Acid Sequence
- Cell Hypoxia/physiology
- Cell Line, Transformed
- Cell Line, Tumor
- Data Interpretation, Statistical
- Gene Expression/drug effects/physiology
- Gene Expression Profiling/methods
- Molecular Sequence Data
- Oxidative Stress/physiology
- Protein Binding
- Protein Multimerization
- Protein Structure, Tertiary/genetics
- Vitamin K 3/pharmacology
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ppat-1000469-g002: 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.
To determine if UR1 coordinates zinc, peptides corresponding to a.a 58–89 of EBNA1 and EBNA1(CC→SS), indicated as WT and M in Figure 1D, were tested for their ability to bind radioactive zinc by a zinc-blot procedure previously used to identify and characterize several proteins that bind zinc ,,. As shown in Figure 2A, the wild-type peptide, but not the mutant, was observed to bind Zn65. Parallel blots were stained with amido-black to confirm that both peptides bound PVDF with similar efficiencies.