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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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APE1/Ref-1 increases transactivation by EBNA1, but does not alter transactivation by Tat. (A) C33a cells were co-transfected with reporter and effector plasmids, and the indicated amount of an APE1/Ref-1 expression plasmid. The backbone expression plasmid, pcDNA3.1 was used to normalize the amount of DNA used in each transfection. Reporter activity was measured 24 hours post-transfection. Transactivation is expressed as a percent of transactivation observed in the absence of co-transfected pAPE1 (control conditions). The inset legend indicates the columns corresponding to each effector/reporter combination. (B) Immunoblots indicate that expression of APE1/Ref-1 did not alter 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 APE1/Ref-1 transfected cells compared to control cells where pcDNA3.1 was co-transfected with the effector and reporter plasmids.
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Figure 5: APE1/Ref-1 increases transactivation by EBNA1, but does not alter transactivation by Tat. (A) C33a cells were co-transfected with reporter and effector plasmids, and the indicated amount of an APE1/Ref-1 expression plasmid. The backbone expression plasmid, pcDNA3.1 was used to normalize the amount of DNA used in each transfection. Reporter activity was measured 24 hours post-transfection. Transactivation is expressed as a percent of transactivation observed in the absence of co-transfected pAPE1 (control conditions). The inset legend indicates the columns corresponding to each effector/reporter combination. (B) Immunoblots indicate that expression of APE1/Ref-1 did not alter 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 APE1/Ref-1 transfected cells compared to control cells where pcDNA3.1 was co-transfected with the effector and reporter plasmids.

Mentions: The DNA repair enzyme APE1 (also known as Ref-1) has two functions. It cleaves DNA at apurinic/apyrimidinic sites, and regulates the function of multiple transactivators whose activities are redox-dependent [5-9]. APE1/Ref-1 reduces sulfenic acid back to sulfhydryl [31], although it is unknown whether it can also reduce a disulfide bond. C33a cells were co-transfected with reporter and effector plasmids and variable amounts of an APE1/Ref-1 expression plasmid. Reporter activity was assayed 24 hours post-transfection. As shown in Figure 5A, APE1/Ref-1 significantly augments EBNA1's ability to transactivate to as much as ~200% of control. Transactivation was augmented as a function of increasing the levels of a co-transfected APE1/Ref-1 expression plasmid. This observation, made with epitope-tagged EBNA1 is similar to our previous observations with untagged EBNA1 [18]. In contrast to EBNA1, APE1/Ref-1 had no effect on transactivation by Tat (Figure 5A). APE1/Ref-1 did not augment EBNA1's ability to transactivate by increasing EBNA1 expression (Figure 5B).


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

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

APE1/Ref-1 increases transactivation by EBNA1, but does not alter transactivation by Tat. (A) C33a cells were co-transfected with reporter and effector plasmids, and the indicated amount of an APE1/Ref-1 expression plasmid. The backbone expression plasmid, pcDNA3.1 was used to normalize the amount of DNA used in each transfection. Reporter activity was measured 24 hours post-transfection. Transactivation is expressed as a percent of transactivation observed in the absence of co-transfected pAPE1 (control conditions). The inset legend indicates the columns corresponding to each effector/reporter combination. (B) Immunoblots indicate that expression of APE1/Ref-1 did not alter 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 APE1/Ref-1 transfected cells compared to control cells where pcDNA3.1 was co-transfected with the effector and reporter plasmids.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: APE1/Ref-1 increases transactivation by EBNA1, but does not alter transactivation by Tat. (A) C33a cells were co-transfected with reporter and effector plasmids, and the indicated amount of an APE1/Ref-1 expression plasmid. The backbone expression plasmid, pcDNA3.1 was used to normalize the amount of DNA used in each transfection. Reporter activity was measured 24 hours post-transfection. Transactivation is expressed as a percent of transactivation observed in the absence of co-transfected pAPE1 (control conditions). The inset legend indicates the columns corresponding to each effector/reporter combination. (B) Immunoblots indicate that expression of APE1/Ref-1 did not alter 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 APE1/Ref-1 transfected cells compared to control cells where pcDNA3.1 was co-transfected with the effector and reporter plasmids.
Mentions: The DNA repair enzyme APE1 (also known as Ref-1) has two functions. It cleaves DNA at apurinic/apyrimidinic sites, and regulates the function of multiple transactivators whose activities are redox-dependent [5-9]. APE1/Ref-1 reduces sulfenic acid back to sulfhydryl [31], although it is unknown whether it can also reduce a disulfide bond. C33a cells were co-transfected with reporter and effector plasmids and variable amounts of an APE1/Ref-1 expression plasmid. Reporter activity was assayed 24 hours post-transfection. As shown in Figure 5A, APE1/Ref-1 significantly augments EBNA1's ability to transactivate to as much as ~200% of control. Transactivation was augmented as a function of increasing the levels of a co-transfected APE1/Ref-1 expression plasmid. This observation, made with epitope-tagged EBNA1 is similar to our previous observations with untagged EBNA1 [18]. In contrast to EBNA1, APE1/Ref-1 had no effect on transactivation by Tat (Figure 5A). APE1/Ref-1 did not augment EBNA1's ability to transactivate by increasing EBNA1 expression (Figure 5B).

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