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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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Hypoxia increases transactivation by EBNA1 and reduces transactivation by Tat. (A) Transfected C33a cells were split 6 hours post-transfection into aliquots incubated under normoxia (N) or hypoxia (H). Luciferase activity was assayed at 24 hours post-transfection. Transactivation is expressed as a percent of transactivation observed under normoxic (control) conditions. Hypoxia increased transactivation by EBNA1 increased to 125% of normoxic conditions, but decreased transactivation by Tat to 25% of normoxic conditions. (B) Immunoblots indicate that hypoxia did not alter expression of Tat or EBNA1. β-actin was used as a loading control. Asterisks indicate statistical significance by the Wilcoxon rank-sum test (p < 0.05) when comparing results obtained under hypoxia against normoxia.
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Figure 2: Hypoxia increases transactivation by EBNA1 and reduces transactivation by Tat. (A) Transfected C33a cells were split 6 hours post-transfection into aliquots incubated under normoxia (N) or hypoxia (H). Luciferase activity was assayed at 24 hours post-transfection. Transactivation is expressed as a percent of transactivation observed under normoxic (control) conditions. Hypoxia increased transactivation by EBNA1 increased to 125% of normoxic conditions, but decreased transactivation by Tat to 25% of normoxic conditions. (B) Immunoblots indicate that hypoxia did not alter expression of Tat or EBNA1. β-actin was used as a loading control. Asterisks indicate statistical significance by the Wilcoxon rank-sum test (p < 0.05) when comparing results obtained under hypoxia against normoxia.

Mentions: EBNA1 and Tat contain redox-sensitive cysteines that are essential for transactivation [18,28], and oxidative stress is known to alter the ability of these proteins to transactivate [18,24]. Oxidation modifies cysteines in two distinct ways: 1) by oxidizing adjacent sulfhydryl groups to form inter- or intra-molecular disulfide bods, and 2) by oxidizing cysteines to sulfenic acid and further oxidized derivatives [31]. Hypoxic conditions decrease the generation of intracellular reactive oxygen species and therefore favour the presence of sulfhydryl groups over oxidized derivatives [42]. Therefore, we examined if hypoxia (4% O2) altered transactivation by EBNA1 or Tat, shown in Figure 2. Consistent with previous reports (Figure 2A), for EBNA1, hypoxia significantly increased transactivation to 130% over normoxia transactivation, defined as control conditions, within 24 hours of exposure to 4% O2. In contrast, 4% O2 significantly reduced Tat's capacity to transactivate to 25% of normoxic conditions (Figure 2A), consistent with recently published reports indicating that hypoxia reduces Tat's ability to transactivate whereas depletion of cellular redox modulators increases transactivation [24,30]. The changes in transactivation induced by hypoxic conditions did not result from an altered expression of EBNA1 or Tat during hypoxia (Figure 2B). In addition, this experiment indicates that the augmentative effect of hypoxia on EBNA1 does not result from direct changes to the basal transcription machinery functional at the minimal HSV-1 TK promoter. To confirm that hypoxia does not directly affect the basal transcription machinery active at the TK promoter, expression from reporter AGP47 (TKp-luciferase) was examined under hypoxia and normoxia. No significant difference in reporter expression was observed confirming that hypoxia does not affect basal transcription from TKp (Additional File 1B).


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

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

Hypoxia increases transactivation by EBNA1 and reduces transactivation by Tat. (A) Transfected C33a cells were split 6 hours post-transfection into aliquots incubated under normoxia (N) or hypoxia (H). Luciferase activity was assayed at 24 hours post-transfection. Transactivation is expressed as a percent of transactivation observed under normoxic (control) conditions. Hypoxia increased transactivation by EBNA1 increased to 125% of normoxic conditions, but decreased transactivation by Tat to 25% of normoxic conditions. (B) Immunoblots indicate that hypoxia did not alter expression of Tat or EBNA1. β-actin was used as a loading control. Asterisks indicate statistical significance by the Wilcoxon rank-sum test (p < 0.05) when comparing results obtained under hypoxia against normoxia.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Hypoxia increases transactivation by EBNA1 and reduces transactivation by Tat. (A) Transfected C33a cells were split 6 hours post-transfection into aliquots incubated under normoxia (N) or hypoxia (H). Luciferase activity was assayed at 24 hours post-transfection. Transactivation is expressed as a percent of transactivation observed under normoxic (control) conditions. Hypoxia increased transactivation by EBNA1 increased to 125% of normoxic conditions, but decreased transactivation by Tat to 25% of normoxic conditions. (B) Immunoblots indicate that hypoxia did not alter expression of Tat or EBNA1. β-actin was used as a loading control. Asterisks indicate statistical significance by the Wilcoxon rank-sum test (p < 0.05) when comparing results obtained under hypoxia against normoxia.
Mentions: EBNA1 and Tat contain redox-sensitive cysteines that are essential for transactivation [18,28], and oxidative stress is known to alter the ability of these proteins to transactivate [18,24]. Oxidation modifies cysteines in two distinct ways: 1) by oxidizing adjacent sulfhydryl groups to form inter- or intra-molecular disulfide bods, and 2) by oxidizing cysteines to sulfenic acid and further oxidized derivatives [31]. Hypoxic conditions decrease the generation of intracellular reactive oxygen species and therefore favour the presence of sulfhydryl groups over oxidized derivatives [42]. Therefore, we examined if hypoxia (4% O2) altered transactivation by EBNA1 or Tat, shown in Figure 2. Consistent with previous reports (Figure 2A), for EBNA1, hypoxia significantly increased transactivation to 130% over normoxia transactivation, defined as control conditions, within 24 hours of exposure to 4% O2. In contrast, 4% O2 significantly reduced Tat's capacity to transactivate to 25% of normoxic conditions (Figure 2A), consistent with recently published reports indicating that hypoxia reduces Tat's ability to transactivate whereas depletion of cellular redox modulators increases transactivation [24,30]. The changes in transactivation induced by hypoxic conditions did not result from an altered expression of EBNA1 or Tat during hypoxia (Figure 2B). In addition, this experiment indicates that the augmentative effect of hypoxia on EBNA1 does not result from direct changes to the basal transcription machinery functional at the minimal HSV-1 TK promoter. To confirm that hypoxia does not directly affect the basal transcription machinery active at the TK promoter, expression from reporter AGP47 (TKp-luciferase) was examined under hypoxia and normoxia. No significant difference in reporter expression was observed confirming that hypoxia does not affect basal transcription from TKp (Additional File 1B).

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