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Diametrically opposed effects of hypoxia and oxidative stress on two viral transactivators.

Washington AT, Singh G, Aiyar A - Virol. J. (2010)

Bottom Line: Here we have compared the effects of hypoxia, oxidative stress, and cellular redox modulators on EBNA1 and Tat.Conversely, thioredoxin reductase 1 (TRR1) reduces Tat's function without any effect on EBNA1.We conclude that oxygen partial pressure and oxidative stress affects the functions of EBNA1 and Tat in a dramatically opposed fashion.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology, Immunology and Parasitology, LSU Health Sciences Center, New Orleans, LA 70112, USA.

ABSTRACT

Background: Many pathogens exist in multiple physiological niches within the host. Differences between aerobic and anaerobic conditions are known to alter the expression of bacterial virulence factors, typically through the conditional activity of transactivators that modulate their expression. More recently, changes in physiological niches have been shown to affect the expression of viral genes. For many viruses, differences in oxygen tension between hypoxia and normoxia alter gene expression or function. Oxygen tension also affects many mammalian transactivators including AP-1, NFkB, and p53 by affecting the reduced state of critical cysteines in these proteins. We have recently determined that an essential cys-x-x-cys motif in the EBNA1 transactivator of Epstein-Barr virus is redox-regulated, such that transactivation is favoured under reducing conditions. The crucial Tat transactivator of human immunodeficiency virus (HIV) has an essential cysteine-rich region, and is also regulated by redox. Contrary to EBNA1, it is reported that Tat's activity is increased by oxidative stress. Here we have compared the effects of hypoxia, oxidative stress, and cellular redox modulators on EBNA1 and Tat.

Results: Our results indicate that unlike EBNA1, Tat is less active during hypoxia. Agents that generate hydroxyl and superoxide radicals reduce EBNA1's activity but increase transactivation by Tat. The cellular redox modulator, APE1/Ref-1, increases EBNA1's activity, without any effect on Tat. Conversely, thioredoxin reductase 1 (TRR1) reduces Tat's function without any effect on EBNA1.

Conclusions: We conclude that oxygen partial pressure and oxidative stress affects the functions of EBNA1 and Tat in a dramatically opposed fashion. Tat is more active during oxidative stress, whereas EBNA1's activity is compromised under these conditions. The two proteins respond to differing cellular redox modulators, suggesting that the oxidized cysteine adduct is a disulfide bond(s) in Tat, but sulfenic acid in EBNA1. The effect of oxygen partial pressure on transactivator function suggests that changes in redox may underlie differences in virus-infected cells dependent upon the physiological niches they traffic to.

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Oxidative stress induced by menadione and paraquot decrease transactivation by EBNA1, but increase transactivation by Tat. (A) Transfected C33a cells were split 6 hours post-transfection into aliquots and exposed to the indicated concentrations of menadione, or paraquot (B) for an additional 18 hours, prior to reporter analysis. The inset legend indicates the columns corresponding to each effector/reporter combination. Control cells were vehicle treated. Transactivation is expressed as a percent of transactivation observed in the control cells. Immunoblots indicate that neither menadione (C), nor paraquot (D) altered the expression of EBNA1 or Tat. β-actin was used as a loading control. Asterisks indicate statistical significance by the Wilcoxon rank-sum test (p < 0.05) for treated samples compared to vehicle-treated controls.
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Figure 3: Oxidative stress induced by menadione and paraquot decrease transactivation by EBNA1, but increase transactivation by Tat. (A) Transfected C33a cells were split 6 hours post-transfection into aliquots and exposed to the indicated concentrations of menadione, or paraquot (B) for an additional 18 hours, prior to reporter analysis. The inset legend indicates the columns corresponding to each effector/reporter combination. Control cells were vehicle treated. Transactivation is expressed as a percent of transactivation observed in the control cells. Immunoblots indicate that neither menadione (C), nor paraquot (D) altered the expression of EBNA1 or Tat. β-actin was used as a loading control. Asterisks indicate statistical significance by the Wilcoxon rank-sum test (p < 0.05) for treated samples compared to vehicle-treated controls.

Mentions: For the experiments shown in Figure 3, C33a cells transfected with effector and reporter plasmids were split six hours post-transfection into aliquots that were exposed to the indicated ranges of menadione (Figure 3A) and paraquot (Figure 3B) for 18 hours. At this time, reporter expression was assayed and is indicated as percent of reporter expression observed in the absence of menadione or paraquot (control conditions). As observed previously [18], menadione (Figure 3A) decreased transactivation by EBNA1 in a dose-dependent manner with significant decreases at concentrations at or greater than 1.4 μM. EBNA1's capacity to transactivate the FR-TKp-luciferase reporter was reduced to 50% by 2 μM menadione. In striking contrast, menadione caused a dose-dependent increase in transactivation of TKp-TAR-luciferase by Tat, with significant increases at 1.4 μM menadione and higher. At a concentration of 2 μM menadione, Tat-dependent reporter expression increased to 175% of control. Similar to menadione, paraquot treatment (Figure 3B) reduced transactivation by EBNA1 while increasing transactivation by Tat. For example, 400 μM paraquat increased Tat's activity to 150% of control, but reduced EBNA1's activity to 50% of control (Figure 3B). Changes in transactivation caused by menadione and paraquot did not result from altered expression of EBNA1 or Tat. (Figure 3C, 3D). Oxidative stress also did not affect basal transcription from the TKp (Additional Figure 1C).


Diametrically opposed effects of hypoxia and oxidative stress on two viral transactivators.

Washington AT, Singh G, Aiyar A - Virol. J. (2010)

Oxidative stress induced by menadione and paraquot decrease transactivation by EBNA1, but increase transactivation by Tat. (A) Transfected C33a cells were split 6 hours post-transfection into aliquots and exposed to the indicated concentrations of menadione, or paraquot (B) for an additional 18 hours, prior to reporter analysis. The inset legend indicates the columns corresponding to each effector/reporter combination. Control cells were vehicle treated. Transactivation is expressed as a percent of transactivation observed in the control cells. Immunoblots indicate that neither menadione (C), nor paraquot (D) altered the expression of EBNA1 or Tat. β-actin was used as a loading control. Asterisks indicate statistical significance by the Wilcoxon rank-sum test (p < 0.05) for treated samples compared to vehicle-treated controls.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2874542&req=5

Figure 3: Oxidative stress induced by menadione and paraquot decrease transactivation by EBNA1, but increase transactivation by Tat. (A) Transfected C33a cells were split 6 hours post-transfection into aliquots and exposed to the indicated concentrations of menadione, or paraquot (B) for an additional 18 hours, prior to reporter analysis. The inset legend indicates the columns corresponding to each effector/reporter combination. Control cells were vehicle treated. Transactivation is expressed as a percent of transactivation observed in the control cells. Immunoblots indicate that neither menadione (C), nor paraquot (D) altered the expression of EBNA1 or Tat. β-actin was used as a loading control. Asterisks indicate statistical significance by the Wilcoxon rank-sum test (p < 0.05) for treated samples compared to vehicle-treated controls.
Mentions: For the experiments shown in Figure 3, C33a cells transfected with effector and reporter plasmids were split six hours post-transfection into aliquots that were exposed to the indicated ranges of menadione (Figure 3A) and paraquot (Figure 3B) for 18 hours. At this time, reporter expression was assayed and is indicated as percent of reporter expression observed in the absence of menadione or paraquot (control conditions). As observed previously [18], menadione (Figure 3A) decreased transactivation by EBNA1 in a dose-dependent manner with significant decreases at concentrations at or greater than 1.4 μM. EBNA1's capacity to transactivate the FR-TKp-luciferase reporter was reduced to 50% by 2 μM menadione. In striking contrast, menadione caused a dose-dependent increase in transactivation of TKp-TAR-luciferase by Tat, with significant increases at 1.4 μM menadione and higher. At a concentration of 2 μM menadione, Tat-dependent reporter expression increased to 175% of control. Similar to menadione, paraquot treatment (Figure 3B) reduced transactivation by EBNA1 while increasing transactivation by Tat. For example, 400 μM paraquat increased Tat's activity to 150% of control, but reduced EBNA1's activity to 50% of control (Figure 3B). Changes in transactivation caused by menadione and paraquot did not result from altered expression of EBNA1 or Tat. (Figure 3C, 3D). Oxidative stress also did not affect basal transcription from the TKp (Additional Figure 1C).

Bottom Line: Here we have compared the effects of hypoxia, oxidative stress, and cellular redox modulators on EBNA1 and Tat.Conversely, thioredoxin reductase 1 (TRR1) reduces Tat's function without any effect on EBNA1.We conclude that oxygen partial pressure and oxidative stress affects the functions of EBNA1 and Tat in a dramatically opposed fashion.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology, Immunology and Parasitology, LSU Health Sciences Center, New Orleans, LA 70112, USA.

ABSTRACT

Background: Many pathogens exist in multiple physiological niches within the host. Differences between aerobic and anaerobic conditions are known to alter the expression of bacterial virulence factors, typically through the conditional activity of transactivators that modulate their expression. More recently, changes in physiological niches have been shown to affect the expression of viral genes. For many viruses, differences in oxygen tension between hypoxia and normoxia alter gene expression or function. Oxygen tension also affects many mammalian transactivators including AP-1, NFkB, and p53 by affecting the reduced state of critical cysteines in these proteins. We have recently determined that an essential cys-x-x-cys motif in the EBNA1 transactivator of Epstein-Barr virus is redox-regulated, such that transactivation is favoured under reducing conditions. The crucial Tat transactivator of human immunodeficiency virus (HIV) has an essential cysteine-rich region, and is also regulated by redox. Contrary to EBNA1, it is reported that Tat's activity is increased by oxidative stress. Here we have compared the effects of hypoxia, oxidative stress, and cellular redox modulators on EBNA1 and Tat.

Results: Our results indicate that unlike EBNA1, Tat is less active during hypoxia. Agents that generate hydroxyl and superoxide radicals reduce EBNA1's activity but increase transactivation by Tat. The cellular redox modulator, APE1/Ref-1, increases EBNA1's activity, without any effect on Tat. Conversely, thioredoxin reductase 1 (TRR1) reduces Tat's function without any effect on EBNA1.

Conclusions: We conclude that oxygen partial pressure and oxidative stress affects the functions of EBNA1 and Tat in a dramatically opposed fashion. Tat is more active during oxidative stress, whereas EBNA1's activity is compromised under these conditions. The two proteins respond to differing cellular redox modulators, suggesting that the oxidized cysteine adduct is a disulfide bond(s) in Tat, but sulfenic acid in EBNA1. The effect of oxygen partial pressure on transactivator function suggests that changes in redox may underlie differences in virus-infected cells dependent upon the physiological niches they traffic to.

Show MeSH
Related in: MedlinePlus