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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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Oxidative stress in cells infected with MP12 strain of RVFV.A) HSAECs were infected with MP12 virus (MOI of 3) and 24 h later stained with 3 µg/ml of Mitosox reagent. After incubation for 30 min the Mitosox reagent was removed, cells washed 3× with HBSS, fixed with paraformaldehyde, visualized. Boxed and magnified inset shows Mitosox staining inside nucleoli. B) MP12 infected HSAECs were stained with Mitosox reagent at 4, 6, 12 and 24 h post infection and visualized as described earlier. C) Mean fluorescence intensity was calculated by averaging flouresence in ten fields picked randomly from quadruplicate samples for every time point. The mean fluorescence intensity of the infected samples was then calculated as fold difference over uninfected cells obtained at comparable time points. * p<0.00005. D) HSAECs were pre- and post treated with DMSO or increasing concentrations of NAC. Untreated and treated cells were infected with MP12 virus and SOD1 protein levels were analyzed by western blot with SOD1 antibody. Actin was used as a control.
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pone-0020354-g002: Oxidative stress in cells infected with MP12 strain of RVFV.A) HSAECs were infected with MP12 virus (MOI of 3) and 24 h later stained with 3 µg/ml of Mitosox reagent. After incubation for 30 min the Mitosox reagent was removed, cells washed 3× with HBSS, fixed with paraformaldehyde, visualized. Boxed and magnified inset shows Mitosox staining inside nucleoli. B) MP12 infected HSAECs were stained with Mitosox reagent at 4, 6, 12 and 24 h post infection and visualized as described earlier. C) Mean fluorescence intensity was calculated by averaging flouresence in ten fields picked randomly from quadruplicate samples for every time point. The mean fluorescence intensity of the infected samples was then calculated as fold difference over uninfected cells obtained at comparable time points. * p<0.00005. D) HSAECs were pre- and post treated with DMSO or increasing concentrations of NAC. Untreated and treated cells were infected with MP12 virus and SOD1 protein levels were analyzed by western blot with SOD1 antibody. Actin was used as a control.

Mentions: Our observation that SOD1 shows a decrease at early time points following infection prompted us to ask the direct question whether infected cells experience oxidative stress at comparable time points. To address that question, we performed Mitosox staining of the infected cells at 24 h post infection. Mitosox is a recently validated fluorogenic dye that is used for sensitive detection of superoxide in cells [32], [33], [34]. Mitosox is a cell permeant dye that is rapidly and selectively targeted to mitochondria, an important intracellular source of ROS. It is oxidized by accumulating superoxide and emits a red fluorescence. Increasing fluorescence intensity is hence indicative of accumulating ROS, which in turn points to defects in oxidative homeostasis. We measured fluorescence intensity in fixed cells following Mitosox treatment and found that there is a striking increase in fluorescence in the infected cells (Figure 2A). Interestingly, we found strong Mitosox staining of the nucleoli of many of these cells (magnified inset, Figure 2A). The nucleolus is considered to be an important sensor of cellular stress and is known to respond to oxidative stress signals [35]. Oxidative stress is known to modulate ribosomal RNA synthesis and hence influence the nucleolus [36]. We then asked the question whether oxidative stress contributes to SOD1 down regulation or oxidative stress is the result of SOD1 down regulation. To distinguish between the two possibilities, we carried out a time course analysis of ROS accumulation in infected cells (0, 2, 4, 6, 12 and 24 h). Our data demonstrates that ROS accumulation occurs as early as 12 h post infection with a strong increase over the uninfected cells at 24 h (Figure 2B and C). Next, in order to directly address the possibility that accumulation of ROS contributes to SOD1 down regulation, we treated cells with an antioxidant, N-acetyl Cysteine (NAC). Increasing concentrations of NAC strongly increased SOD1 protein levels in infected cells (Figure 2D) thus demonstrating that SOD1 down regulation was the result of increasing ROS levels after MP12 infection. Collectively, our data provides evidence that cells experience strong oxidative stress at earlier time points following infection by MP12 virus and this contributes to SOD1 protein down regulation.


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)

Oxidative stress in cells infected with MP12 strain of RVFV.A) HSAECs were infected with MP12 virus (MOI of 3) and 24 h later stained with 3 µg/ml of Mitosox reagent. After incubation for 30 min the Mitosox reagent was removed, cells washed 3× with HBSS, fixed with paraformaldehyde, visualized. Boxed and magnified inset shows Mitosox staining inside nucleoli. B) MP12 infected HSAECs were stained with Mitosox reagent at 4, 6, 12 and 24 h post infection and visualized as described earlier. C) Mean fluorescence intensity was calculated by averaging flouresence in ten fields picked randomly from quadruplicate samples for every time point. The mean fluorescence intensity of the infected samples was then calculated as fold difference over uninfected cells obtained at comparable time points. * p<0.00005. D) HSAECs were pre- and post treated with DMSO or increasing concentrations of NAC. Untreated and treated cells were infected with MP12 virus and SOD1 protein levels were analyzed by western blot with SOD1 antibody. Actin was used as a control.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020354-g002: Oxidative stress in cells infected with MP12 strain of RVFV.A) HSAECs were infected with MP12 virus (MOI of 3) and 24 h later stained with 3 µg/ml of Mitosox reagent. After incubation for 30 min the Mitosox reagent was removed, cells washed 3× with HBSS, fixed with paraformaldehyde, visualized. Boxed and magnified inset shows Mitosox staining inside nucleoli. B) MP12 infected HSAECs were stained with Mitosox reagent at 4, 6, 12 and 24 h post infection and visualized as described earlier. C) Mean fluorescence intensity was calculated by averaging flouresence in ten fields picked randomly from quadruplicate samples for every time point. The mean fluorescence intensity of the infected samples was then calculated as fold difference over uninfected cells obtained at comparable time points. * p<0.00005. D) HSAECs were pre- and post treated with DMSO or increasing concentrations of NAC. Untreated and treated cells were infected with MP12 virus and SOD1 protein levels were analyzed by western blot with SOD1 antibody. Actin was used as a control.
Mentions: Our observation that SOD1 shows a decrease at early time points following infection prompted us to ask the direct question whether infected cells experience oxidative stress at comparable time points. To address that question, we performed Mitosox staining of the infected cells at 24 h post infection. Mitosox is a recently validated fluorogenic dye that is used for sensitive detection of superoxide in cells [32], [33], [34]. Mitosox is a cell permeant dye that is rapidly and selectively targeted to mitochondria, an important intracellular source of ROS. It is oxidized by accumulating superoxide and emits a red fluorescence. Increasing fluorescence intensity is hence indicative of accumulating ROS, which in turn points to defects in oxidative homeostasis. We measured fluorescence intensity in fixed cells following Mitosox treatment and found that there is a striking increase in fluorescence in the infected cells (Figure 2A). Interestingly, we found strong Mitosox staining of the nucleoli of many of these cells (magnified inset, Figure 2A). The nucleolus is considered to be an important sensor of cellular stress and is known to respond to oxidative stress signals [35]. Oxidative stress is known to modulate ribosomal RNA synthesis and hence influence the nucleolus [36]. We then asked the question whether oxidative stress contributes to SOD1 down regulation or oxidative stress is the result of SOD1 down regulation. To distinguish between the two possibilities, we carried out a time course analysis of ROS accumulation in infected cells (0, 2, 4, 6, 12 and 24 h). Our data demonstrates that ROS accumulation occurs as early as 12 h post infection with a strong increase over the uninfected cells at 24 h (Figure 2B and C). Next, in order to directly address the possibility that accumulation of ROS contributes to SOD1 down regulation, we treated cells with an antioxidant, N-acetyl Cysteine (NAC). Increasing concentrations of NAC strongly increased SOD1 protein levels in infected cells (Figure 2D) thus demonstrating that SOD1 down regulation was the result of increasing ROS levels after MP12 infection. Collectively, our data provides evidence that cells experience strong oxidative stress at earlier time points following infection by MP12 virus and this contributes to SOD1 protein down regulation.

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