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Alteration in superoxide dismutase 1 causes oxidative stress and p38 MAPK activation following RVFV infection.

Narayanan A, Popova T, Turell M, Kidd J, Chertow J, Popov SG, Bailey C, Kashanchi F, Kehn-Hall K - PLoS ONE (2011)

Bottom Line: Understanding early host events to an infectious exposure to RVFV will be of significant use in the development of effective therapeutics that not only control pathogen multiplication, but also contribute to cell survival.Alterations in the host protein SOD1 following RVFV infection appears to be an early event that occurs in multiple cell types.Our data implies that maintaining oxidative homeostasis in the infected cells may play an important role in improving survival of infected cells.

View Article: PubMed Central - PubMed

Affiliation: National Center for Biodefense and Infectious Diseases, George Mason University, Manassas, Virginia, United States of America.

ABSTRACT
Rift Valley fever (RVF) is a zoonotic disease caused by Rift Valley fever virus (RVFV). RVFV is a category A pathogen that belongs to the genus Phlebovirus, family Bunyaviridae. Understanding early host events to an infectious exposure to RVFV will be of significant use in the development of effective therapeutics that not only control pathogen multiplication, but also contribute to cell survival. In this study, we have carried out infections of human cells with a vaccine strain (MP12) and virulent strain (ZH501) of RVFV and determined host responses to viral infection. We demonstrate that the cellular antioxidant enzyme superoxide dismutase 1 (SOD1) displays altered abundances at early time points following exposure to the virus. We show that the enzyme is down regulated in cases of both a virulent (ZH501) and a vaccine strain (MP12) exposure. Our data demonstrates that the down regulation of SOD1 is likely to be due to post transcriptional processes and may be related to up regulation of TNFα following infection. We also provide evidence for extensive oxidative stress in the MP12 infected cells. Concomitantly, there is an increase in the activation of the p38 MAPK stress response, which our earlier published study demonstrated to be an essential cell survival strategy. Our data suggests that the viral anti-apoptotic protein NSm may play a role in the regulation of the cellular p38 MAPK response. Alterations in the host protein SOD1 following RVFV infection appears to be an early event that occurs in multiple cell types. Activation of the cellular stress response p38 MAPK pathway can be observed in all cell types tested. Our data implies that maintaining oxidative homeostasis in the infected cells may play an important role in improving survival of infected cells.

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Depletion of SOD1 and effect on apoptosis.A) 106 HSAECs were transfected with siRNAs against either SOD 1 (si-SOD) or luciferase (si-Luc). After 96 h, total cell extracts were made and western blots carried out using anti-SOD1 antibodies. Actin was used as a loading control. L stands for Si-Luc and S stands for Si-SOD1. B) SOD1 depleted cells and control cells (Si-Luc transfected cells) were infected with MP12 strain of RVFV (MOI of 3). Twenty four hours after infection, cells were harvested for flow cytometry analysis, stained with propidium iodide and analyzed for cell cycle progression. Percentages of cells in various stages of the cell cycle were determined and the fold differences between SOD1-depleted cells and control cells were calculated. Data is represented as average of two independent experiments.
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pone-0020354-g004: Depletion of SOD1 and effect on apoptosis.A) 106 HSAECs were transfected with siRNAs against either SOD 1 (si-SOD) or luciferase (si-Luc). After 96 h, total cell extracts were made and western blots carried out using anti-SOD1 antibodies. Actin was used as a loading control. L stands for Si-Luc and S stands for Si-SOD1. B) SOD1 depleted cells and control cells (Si-Luc transfected cells) were infected with MP12 strain of RVFV (MOI of 3). Twenty four hours after infection, cells were harvested for flow cytometry analysis, stained with propidium iodide and analyzed for cell cycle progression. Percentages of cells in various stages of the cell cycle were determined and the fold differences between SOD1-depleted cells and control cells were calculated. Data is represented as average of two independent experiments.

Mentions: We next evaluated the effect of lowered SOD1 level during a viral infection on apoptosis. SOD1 has been linked to the regulation of apoptosis [38]. Cell cycle progression and apoptosis have implications for the success of a virus in establishing a productive infection and viruses have evolved special means to control cell cycle progression [39], [40]. We depleted SOD1 utilizing SMARTpool siRNAs (si-SOD). As a control, HSAECs were transfected with siRNAs to luciferase (si-Luc). The cells were maintained in serum-free medium for 96 h when endogenous SOD1 was about 90% depleted (Figure 4A). We then infected the siRNA-treated cells with MP12. Strikingly, we were able to observe strong cytopathic effect in the SOD1-depleted cells as early as 24 h post infection (data not shown) suggesting that a combination of lowered SOD1 levels and a viral infection could push cells faster into apoptosis. To affirm this, the cells were trypsinized and stained with propidium iodide containing staining solution and analyzed by flow cytometry. The analysis revealed that in SOD1-depleted cells, the percentage of cells in apoptosis was almost seven times greater than the control cells at 24 h (Figure 4B). This coincided with a decrease in the G1 population in the si-SOD transfected cells (Figure 4B). We also evaluated apoptosis in SOD1 depleted cells that were not infected with virus and found that the fraction of cells in apoptosis was significantly less (1.5 times greater than control cells; data not shown). Collectively, these results indicate that depletion of endogenous SOD1 makes cells more susceptible to apoptosis during MP12 infection.


Alteration in superoxide dismutase 1 causes oxidative stress and p38 MAPK activation following RVFV infection.

Narayanan A, Popova T, Turell M, Kidd J, Chertow J, Popov SG, Bailey C, Kashanchi F, Kehn-Hall K - PLoS ONE (2011)

Depletion of SOD1 and effect on apoptosis.A) 106 HSAECs were transfected with siRNAs against either SOD 1 (si-SOD) or luciferase (si-Luc). After 96 h, total cell extracts were made and western blots carried out using anti-SOD1 antibodies. Actin was used as a loading control. L stands for Si-Luc and S stands for Si-SOD1. B) SOD1 depleted cells and control cells (Si-Luc transfected cells) were infected with MP12 strain of RVFV (MOI of 3). Twenty four hours after infection, cells were harvested for flow cytometry analysis, stained with propidium iodide and analyzed for cell cycle progression. Percentages of cells in various stages of the cell cycle were determined and the fold differences between SOD1-depleted cells and control cells were calculated. Data is represented as average of two independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3105056&req=5

pone-0020354-g004: Depletion of SOD1 and effect on apoptosis.A) 106 HSAECs were transfected with siRNAs against either SOD 1 (si-SOD) or luciferase (si-Luc). After 96 h, total cell extracts were made and western blots carried out using anti-SOD1 antibodies. Actin was used as a loading control. L stands for Si-Luc and S stands for Si-SOD1. B) SOD1 depleted cells and control cells (Si-Luc transfected cells) were infected with MP12 strain of RVFV (MOI of 3). Twenty four hours after infection, cells were harvested for flow cytometry analysis, stained with propidium iodide and analyzed for cell cycle progression. Percentages of cells in various stages of the cell cycle were determined and the fold differences between SOD1-depleted cells and control cells were calculated. Data is represented as average of two independent experiments.
Mentions: We next evaluated the effect of lowered SOD1 level during a viral infection on apoptosis. SOD1 has been linked to the regulation of apoptosis [38]. Cell cycle progression and apoptosis have implications for the success of a virus in establishing a productive infection and viruses have evolved special means to control cell cycle progression [39], [40]. We depleted SOD1 utilizing SMARTpool siRNAs (si-SOD). As a control, HSAECs were transfected with siRNAs to luciferase (si-Luc). The cells were maintained in serum-free medium for 96 h when endogenous SOD1 was about 90% depleted (Figure 4A). We then infected the siRNA-treated cells with MP12. Strikingly, we were able to observe strong cytopathic effect in the SOD1-depleted cells as early as 24 h post infection (data not shown) suggesting that a combination of lowered SOD1 levels and a viral infection could push cells faster into apoptosis. To affirm this, the cells were trypsinized and stained with propidium iodide containing staining solution and analyzed by flow cytometry. The analysis revealed that in SOD1-depleted cells, the percentage of cells in apoptosis was almost seven times greater than the control cells at 24 h (Figure 4B). This coincided with a decrease in the G1 population in the si-SOD transfected cells (Figure 4B). We also evaluated apoptosis in SOD1 depleted cells that were not infected with virus and found that the fraction of cells in apoptosis was significantly less (1.5 times greater than control cells; data not shown). Collectively, these results indicate that depletion of endogenous SOD1 makes cells more susceptible to apoptosis during MP12 infection.

Bottom Line: Understanding early host events to an infectious exposure to RVFV will be of significant use in the development of effective therapeutics that not only control pathogen multiplication, but also contribute to cell survival.Alterations in the host protein SOD1 following RVFV infection appears to be an early event that occurs in multiple cell types.Our data implies that maintaining oxidative homeostasis in the infected cells may play an important role in improving survival of infected cells.

View Article: PubMed Central - PubMed

Affiliation: National Center for Biodefense and Infectious Diseases, George Mason University, Manassas, Virginia, United States of America.

ABSTRACT
Rift Valley fever (RVF) is a zoonotic disease caused by Rift Valley fever virus (RVFV). RVFV is a category A pathogen that belongs to the genus Phlebovirus, family Bunyaviridae. Understanding early host events to an infectious exposure to RVFV will be of significant use in the development of effective therapeutics that not only control pathogen multiplication, but also contribute to cell survival. In this study, we have carried out infections of human cells with a vaccine strain (MP12) and virulent strain (ZH501) of RVFV and determined host responses to viral infection. We demonstrate that the cellular antioxidant enzyme superoxide dismutase 1 (SOD1) displays altered abundances at early time points following exposure to the virus. We show that the enzyme is down regulated in cases of both a virulent (ZH501) and a vaccine strain (MP12) exposure. Our data demonstrates that the down regulation of SOD1 is likely to be due to post transcriptional processes and may be related to up regulation of TNFα following infection. We also provide evidence for extensive oxidative stress in the MP12 infected cells. Concomitantly, there is an increase in the activation of the p38 MAPK stress response, which our earlier published study demonstrated to be an essential cell survival strategy. Our data suggests that the viral anti-apoptotic protein NSm may play a role in the regulation of the cellular p38 MAPK response. Alterations in the host protein SOD1 following RVFV infection appears to be an early event that occurs in multiple cell types. Activation of the cellular stress response p38 MAPK pathway can be observed in all cell types tested. Our data implies that maintaining oxidative homeostasis in the infected cells may play an important role in improving survival of infected cells.

Show MeSH
Related in: MedlinePlus