Limits...
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.

Show MeSH

Related in: MedlinePlus

Selenium and thioredoxin reductase 1 (TRR1) reduce transactivation by Tat. (A) Transfected cells were split 6 hours post-transfection into aliquots and exposed to the indicated concentrations of sodium selenite for 18 hours before analysis. The inset legend indicates the columns corresponding to each effector/reporter combination. Asterisks indicate statistical significance by the Wilcoxon rank-sum test (p < 0.05) for selenium treated samples compared to vehicle treated samples (B) C33a cells were co-transfected with Tat expression and reporter plasmids and indicated amounts of a TRR1 expression plasmid. pcDNA3.1 was used to normalize the amount of DNA used per transfection. Transfections was split six hours post-transfection, and half the transfected cells were exposed to 0.03 μM sodium selenite for an additional 18 hours before analysis. Transactivation is expressed as a percent of transactivation observed in the absence of co-transfected pTRR1 or added sodium selenite (control conditions). The inset legend indicates the columns corresponding to co-transfected pTRR1 alone, or co-transfected pTRR1 with sodium selenite addition. Asterisks indicate statistical significance by the Wilcoxon rank-sum test (p < 0.05) for TRR1 transfected cells compared to controls in which pcDNA3.1 was co-transfected with reporter and effector plasmids, and cells were not exposed to sodium selenite. (C) Immunoblot analysis indicates that treatment with sodium selenite does not alter the expression of EBNA1 or Tat. In addition, co-transfected pTRR1 does not affect the expression of Tat in the presence of absence of 0.03 μM sodium selenite added to the media. β-actin was used as a loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2874542&req=5

Figure 6: Selenium and thioredoxin reductase 1 (TRR1) reduce transactivation by Tat. (A) Transfected cells were split 6 hours post-transfection into aliquots and exposed to the indicated concentrations of sodium selenite for 18 hours before analysis. The inset legend indicates the columns corresponding to each effector/reporter combination. Asterisks indicate statistical significance by the Wilcoxon rank-sum test (p < 0.05) for selenium treated samples compared to vehicle treated samples (B) C33a cells were co-transfected with Tat expression and reporter plasmids and indicated amounts of a TRR1 expression plasmid. pcDNA3.1 was used to normalize the amount of DNA used per transfection. Transfections was split six hours post-transfection, and half the transfected cells were exposed to 0.03 μM sodium selenite for an additional 18 hours before analysis. Transactivation is expressed as a percent of transactivation observed in the absence of co-transfected pTRR1 or added sodium selenite (control conditions). The inset legend indicates the columns corresponding to co-transfected pTRR1 alone, or co-transfected pTRR1 with sodium selenite addition. Asterisks indicate statistical significance by the Wilcoxon rank-sum test (p < 0.05) for TRR1 transfected cells compared to controls in which pcDNA3.1 was co-transfected with reporter and effector plasmids, and cells were not exposed to sodium selenite. (C) Immunoblot analysis indicates that treatment with sodium selenite does not alter the expression of EBNA1 or Tat. In addition, co-transfected pTRR1 does not affect the expression of Tat in the presence of absence of 0.03 μM sodium selenite added to the media. β-actin was used as a loading control.

Mentions: The minimal TK promoter used in the TKp-TAR-luciferase reporter described here lacks recognition sites for Sp1 or any other major redox-regulated transcription factor. Therefore, we tested whether activating TRR1 by the addition of selenium (Figure 6A), or over-expression of TRR1 (Figure 6B), would decrease Tat's ability to transactivate. For the data shown in Figure 6A, C33a cells transfected with effector and reporter plasmids were split six hours post-transfection, and aliquots exposed to increasing concentrations of selenium (0.01 - 0.1 μM). As shown in Figure 6A, the addition of 0.01 μM and higher concentration of selenium significantly decreased Tat's capacity to transactivate. At 0.1 μM, Tat transactivated TKp-TAR-luciferase at 55% the level observed in the absence of selenium. Selenium did not affect EBNA1's ability to transactivate FR-TKp-luciferase. Next, the effect of TRR1 over-expression was tested (Figure 6B). Over-expressed TRR1 negatively affected Tat's capacity to transactivate significantly, even in the absence of additional added selenium (Figure 6B), such that co-transfection of 1 μg of a TRR1 expression plasmid reduced Tat's capacity to transactivate to 45% of control. No further effect was observed with higher amounts of the co-transfected TRR1 expression plasmid. Over-expression of TRR1 had no effect on EBNA1's ability to transactivate (data not shown). While we were initially surprised that over-expression of TRR1 decreased Tat's capacity to transactivate even in the absence of added selenium, it is possible that the over-expressed TRR1 uses the pre-existing intracellular selenium pool to form the active enzyme. Alternatively, it has been reported that TRR1 reduces many disulfide bonds in the absence of selenium [53]. We also tested whether the combination of over-expressed TRR1 and selenium addition would further decrease Tat's capacity to transactivate in cells that over-express TRR1. As shown in Figure 6B; addition of 0.03 μM selenium reduced transactivation by Tat to 25% in cells co-transfected with 1 μg of the TRR1 expression plasmid. Addition of selenium had no effect on the expression of EBNA1 or Tat, and over-expression of TRR1 also had no effect on Tat expression (Figure 6C), confirming that the decrease in transactivation did not result from a decrease in Tat levels.


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

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

Selenium and thioredoxin reductase 1 (TRR1) reduce transactivation by Tat. (A) Transfected cells were split 6 hours post-transfection into aliquots and exposed to the indicated concentrations of sodium selenite for 18 hours before analysis. The inset legend indicates the columns corresponding to each effector/reporter combination. Asterisks indicate statistical significance by the Wilcoxon rank-sum test (p < 0.05) for selenium treated samples compared to vehicle treated samples (B) C33a cells were co-transfected with Tat expression and reporter plasmids and indicated amounts of a TRR1 expression plasmid. pcDNA3.1 was used to normalize the amount of DNA used per transfection. Transfections was split six hours post-transfection, and half the transfected cells were exposed to 0.03 μM sodium selenite for an additional 18 hours before analysis. Transactivation is expressed as a percent of transactivation observed in the absence of co-transfected pTRR1 or added sodium selenite (control conditions). The inset legend indicates the columns corresponding to co-transfected pTRR1 alone, or co-transfected pTRR1 with sodium selenite addition. Asterisks indicate statistical significance by the Wilcoxon rank-sum test (p < 0.05) for TRR1 transfected cells compared to controls in which pcDNA3.1 was co-transfected with reporter and effector plasmids, and cells were not exposed to sodium selenite. (C) Immunoblot analysis indicates that treatment with sodium selenite does not alter the expression of EBNA1 or Tat. In addition, co-transfected pTRR1 does not affect the expression of Tat in the presence of absence of 0.03 μM sodium selenite added to the media. β-actin was used as a loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Selenium and thioredoxin reductase 1 (TRR1) reduce transactivation by Tat. (A) Transfected cells were split 6 hours post-transfection into aliquots and exposed to the indicated concentrations of sodium selenite for 18 hours before analysis. The inset legend indicates the columns corresponding to each effector/reporter combination. Asterisks indicate statistical significance by the Wilcoxon rank-sum test (p < 0.05) for selenium treated samples compared to vehicle treated samples (B) C33a cells were co-transfected with Tat expression and reporter plasmids and indicated amounts of a TRR1 expression plasmid. pcDNA3.1 was used to normalize the amount of DNA used per transfection. Transfections was split six hours post-transfection, and half the transfected cells were exposed to 0.03 μM sodium selenite for an additional 18 hours before analysis. Transactivation is expressed as a percent of transactivation observed in the absence of co-transfected pTRR1 or added sodium selenite (control conditions). The inset legend indicates the columns corresponding to co-transfected pTRR1 alone, or co-transfected pTRR1 with sodium selenite addition. Asterisks indicate statistical significance by the Wilcoxon rank-sum test (p < 0.05) for TRR1 transfected cells compared to controls in which pcDNA3.1 was co-transfected with reporter and effector plasmids, and cells were not exposed to sodium selenite. (C) Immunoblot analysis indicates that treatment with sodium selenite does not alter the expression of EBNA1 or Tat. In addition, co-transfected pTRR1 does not affect the expression of Tat in the presence of absence of 0.03 μM sodium selenite added to the media. β-actin was used as a loading control.
Mentions: The minimal TK promoter used in the TKp-TAR-luciferase reporter described here lacks recognition sites for Sp1 or any other major redox-regulated transcription factor. Therefore, we tested whether activating TRR1 by the addition of selenium (Figure 6A), or over-expression of TRR1 (Figure 6B), would decrease Tat's ability to transactivate. For the data shown in Figure 6A, C33a cells transfected with effector and reporter plasmids were split six hours post-transfection, and aliquots exposed to increasing concentrations of selenium (0.01 - 0.1 μM). As shown in Figure 6A, the addition of 0.01 μM and higher concentration of selenium significantly decreased Tat's capacity to transactivate. At 0.1 μM, Tat transactivated TKp-TAR-luciferase at 55% the level observed in the absence of selenium. Selenium did not affect EBNA1's ability to transactivate FR-TKp-luciferase. Next, the effect of TRR1 over-expression was tested (Figure 6B). Over-expressed TRR1 negatively affected Tat's capacity to transactivate significantly, even in the absence of additional added selenium (Figure 6B), such that co-transfection of 1 μg of a TRR1 expression plasmid reduced Tat's capacity to transactivate to 45% of control. No further effect was observed with higher amounts of the co-transfected TRR1 expression plasmid. Over-expression of TRR1 had no effect on EBNA1's ability to transactivate (data not shown). While we were initially surprised that over-expression of TRR1 decreased Tat's capacity to transactivate even in the absence of added selenium, it is possible that the over-expressed TRR1 uses the pre-existing intracellular selenium pool to form the active enzyme. Alternatively, it has been reported that TRR1 reduces many disulfide bonds in the absence of selenium [53]. We also tested whether the combination of over-expressed TRR1 and selenium addition would further decrease Tat's capacity to transactivate in cells that over-express TRR1. As shown in Figure 6B; addition of 0.03 μM selenium reduced transactivation by Tat to 25% in cells co-transfected with 1 μg of the TRR1 expression plasmid. Addition of selenium had no effect on the expression of EBNA1 or Tat, and over-expression of TRR1 also had no effect on Tat expression (Figure 6C), confirming that the decrease in transactivation did not result from a decrease in Tat levels.

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