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

Aras S, Singh G, Johnston K, Foster T, Aiyar A - PLoS Pathog. (2009)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Immunology & Parasitology, LSU Health Sciences Center, New Orleans, LA, USA.

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.

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Related in: MedlinePlus

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.
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ppat-1000469-g003: 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.

Mentions: We initially postulated that UR1 might facilitate a zinc-dependent interaction between EBNA1 and a cellular transcription co-activator. To identify such proteins, a yeast two-hybrid screen was performed using the first 94 amino acids of wild-type EBNA1 as the bait protein against a cDNA library from HeLa cells, a cell-line in which EBNA1 has been observed to activate transcription. No protein identified in this screen associated specifically with the bait protein, but not an UR1-deleted derivative (data not shown). In an effort to test our hypothesis that UR1 associates with cellular transcription factors, we tested whether co-expression of EBNA1's amino-terminal 450 a.a. interferes with EBNA1's ability to activate transcription from an FR-dependent reporter plasmid, by squelching its interaction with putative cellular co-activators or general transcription factors [24],[25]. As shown in Figure 3, co-transfecting increasing amounts of an expression plasmid encoding a fusion between the first 450 a.a. of EBNA1 and the DNA binding domain of the BPV-1 E2 protein, 3xF-EBNA1(1-450)-E2DBD, did not affect the ability of EBNA1 to activate transcription from the FR-TKp-luciferase reporter, over the pcDNA3 control. In contrast, co-transfecting a DBD expression plasmid decreased the expression observed from FR-TKp-luciferase, by expressing a protein that competes efficiently with EBNA1's ability to bind FR [26]. While 3xF-EBNA1(1-450)-E2DBD does not interfere with the function of wild-type EBNA1, it retains the ability to activate transcription from a TKp-luciferase reporter plasmid containing twenty E2-binding sites (Figure S4), and this transactivation is sensitive to treatment with 5 µM TPEN (Figure S5). Thus, the inability of 3xF-EBNA1(1-450)-E2DBD to squelch transactivation of FR-TKp-luciferase by EBNA1 is not because it is incapable of activating transcription via a mechanism similar to that used by EBNA1.


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

Aras S, Singh G, Johnston K, Foster T, Aiyar A - PLoS Pathog. (2009)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2690687&req=5

ppat-1000469-g003: 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.
Mentions: We initially postulated that UR1 might facilitate a zinc-dependent interaction between EBNA1 and a cellular transcription co-activator. To identify such proteins, a yeast two-hybrid screen was performed using the first 94 amino acids of wild-type EBNA1 as the bait protein against a cDNA library from HeLa cells, a cell-line in which EBNA1 has been observed to activate transcription. No protein identified in this screen associated specifically with the bait protein, but not an UR1-deleted derivative (data not shown). In an effort to test our hypothesis that UR1 associates with cellular transcription factors, we tested whether co-expression of EBNA1's amino-terminal 450 a.a. interferes with EBNA1's ability to activate transcription from an FR-dependent reporter plasmid, by squelching its interaction with putative cellular co-activators or general transcription factors [24],[25]. As shown in Figure 3, co-transfecting increasing amounts of an expression plasmid encoding a fusion between the first 450 a.a. of EBNA1 and the DNA binding domain of the BPV-1 E2 protein, 3xF-EBNA1(1-450)-E2DBD, did not affect the ability of EBNA1 to activate transcription from the FR-TKp-luciferase reporter, over the pcDNA3 control. In contrast, co-transfecting a DBD expression plasmid decreased the expression observed from FR-TKp-luciferase, by expressing a protein that competes efficiently with EBNA1's ability to bind FR [26]. While 3xF-EBNA1(1-450)-E2DBD does not interfere with the function of wild-type EBNA1, it retains the ability to activate transcription from a TKp-luciferase reporter plasmid containing twenty E2-binding sites (Figure S4), and this transactivation is sensitive to treatment with 5 µM TPEN (Figure S5). Thus, the inability of 3xF-EBNA1(1-450)-E2DBD to squelch transactivation of FR-TKp-luciferase by EBNA1 is not because it is incapable of activating transcription via a mechanism similar to that used by EBNA1.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Immunology & Parasitology, LSU Health Sciences Center, New Orleans, LA, USA.

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.

Show MeSH
Related in: MedlinePlus