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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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Regulation of SOD1 expression by TNFα.A) 106 HSAECs were maintained in serum-minus media for 48 h. Twenty four h later, they were treated with 10, 50 and 100 ng/ml of TNFα. Total cell extracts were obtained at 4 and 24 h post addition of TNFα. The extracts were run on 4–20% Tris glycine gels and transferred on to nitrocellulose membranes. The membranes were probed with anti-SOD1 antibody. Actin was used as a loading control. B) The band intensities for all SOD1 bands were calculated using the quantity one software and Bio-Rad imaging system. All quantifications represented included normalization to actin band intensity. Quantification of SOD1 is shown for the 24-h time point and is the average of two independent experiments. C) Total RNA was extracted from MP12 infected (I) and uninfected control cells (M) at 1, 2, 4, 6, 24 and 30 h post infection. RT-PCR was performed with primers to TNFα. GAPDH was used as a control. D) Quantification of fold differences in TNFα expression between uninfected control cells and MP12-infected cells over the indicated time course. * indicates p = 0.01. Data comprises results from three experiments.
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pone-0020354-g003: Regulation of SOD1 expression by TNFα.A) 106 HSAECs were maintained in serum-minus media for 48 h. Twenty four h later, they were treated with 10, 50 and 100 ng/ml of TNFα. Total cell extracts were obtained at 4 and 24 h post addition of TNFα. The extracts were run on 4–20% Tris glycine gels and transferred on to nitrocellulose membranes. The membranes were probed with anti-SOD1 antibody. Actin was used as a loading control. B) The band intensities for all SOD1 bands were calculated using the quantity one software and Bio-Rad imaging system. All quantifications represented included normalization to actin band intensity. Quantification of SOD1 is shown for the 24-h time point and is the average of two independent experiments. C) Total RNA was extracted from MP12 infected (I) and uninfected control cells (M) at 1, 2, 4, 6, 24 and 30 h post infection. RT-PCR was performed with primers to TNFα. GAPDH was used as a control. D) Quantification of fold differences in TNFα expression between uninfected control cells and MP12-infected cells over the indicated time course. * indicates p = 0.01. Data comprises results from three experiments.

Mentions: It was recently shown that in U937 human myeloid leukemia cells, increased exposure to TNFα caused a down regulation of SOD1 [37]. We wished to determine if increase in TNFα could contribute to lowered SOD1 protein levels in MP12 infected cells. To first evaluate if treatment of HSAECs with TNFα could lead to down regulation of SOD1, we examined endogenous levels of SOD1 after 4 and 24 h of exposure to increasing concentrations of exogenous TNFα. As seen in Figure 3A, at the 24-h time point, even a low concentration of TNFα reduces the endogenous SOD1 protein level to 60% of that in untreated cells. Increasing TNFα concentration further down regulates SOD1 protein levels (Figure 3A and B). The observed reduction in endogenous SOD1 following a 24 h treatment with TNFα is in agreement with the published results for U937 cells [37]. We then directly determined if viral infection of HSAECs caused up regulation of endogenous TNFα. We performed RT-PCRs with TNFα specific primers at 1, 2, 4, 6, 24 and 30 h post infection and observed up regulation of TNFα expression 24 h after infection (Figure 3C and D). Taken together, our data suggests that an increase in TNFα level may cause down regulation of SOD1 in HSAECs and that MP12 infection causes a modest up regulation of TNFα gene transcription. We had also determined by ELISA and Reverse Phase protein MicroArray (RPMA) that there was an increase in TNFα protein expression following MP12 infection (data not shown).


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)

Regulation of SOD1 expression by TNFα.A) 106 HSAECs were maintained in serum-minus media for 48 h. Twenty four h later, they were treated with 10, 50 and 100 ng/ml of TNFα. Total cell extracts were obtained at 4 and 24 h post addition of TNFα. The extracts were run on 4–20% Tris glycine gels and transferred on to nitrocellulose membranes. The membranes were probed with anti-SOD1 antibody. Actin was used as a loading control. B) The band intensities for all SOD1 bands were calculated using the quantity one software and Bio-Rad imaging system. All quantifications represented included normalization to actin band intensity. Quantification of SOD1 is shown for the 24-h time point and is the average of two independent experiments. C) Total RNA was extracted from MP12 infected (I) and uninfected control cells (M) at 1, 2, 4, 6, 24 and 30 h post infection. RT-PCR was performed with primers to TNFα. GAPDH was used as a control. D) Quantification of fold differences in TNFα expression between uninfected control cells and MP12-infected cells over the indicated time course. * indicates p = 0.01. Data comprises results from three experiments.
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Related In: Results  -  Collection

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pone-0020354-g003: Regulation of SOD1 expression by TNFα.A) 106 HSAECs were maintained in serum-minus media for 48 h. Twenty four h later, they were treated with 10, 50 and 100 ng/ml of TNFα. Total cell extracts were obtained at 4 and 24 h post addition of TNFα. The extracts were run on 4–20% Tris glycine gels and transferred on to nitrocellulose membranes. The membranes were probed with anti-SOD1 antibody. Actin was used as a loading control. B) The band intensities for all SOD1 bands were calculated using the quantity one software and Bio-Rad imaging system. All quantifications represented included normalization to actin band intensity. Quantification of SOD1 is shown for the 24-h time point and is the average of two independent experiments. C) Total RNA was extracted from MP12 infected (I) and uninfected control cells (M) at 1, 2, 4, 6, 24 and 30 h post infection. RT-PCR was performed with primers to TNFα. GAPDH was used as a control. D) Quantification of fold differences in TNFα expression between uninfected control cells and MP12-infected cells over the indicated time course. * indicates p = 0.01. Data comprises results from three experiments.
Mentions: It was recently shown that in U937 human myeloid leukemia cells, increased exposure to TNFα caused a down regulation of SOD1 [37]. We wished to determine if increase in TNFα could contribute to lowered SOD1 protein levels in MP12 infected cells. To first evaluate if treatment of HSAECs with TNFα could lead to down regulation of SOD1, we examined endogenous levels of SOD1 after 4 and 24 h of exposure to increasing concentrations of exogenous TNFα. As seen in Figure 3A, at the 24-h time point, even a low concentration of TNFα reduces the endogenous SOD1 protein level to 60% of that in untreated cells. Increasing TNFα concentration further down regulates SOD1 protein levels (Figure 3A and B). The observed reduction in endogenous SOD1 following a 24 h treatment with TNFα is in agreement with the published results for U937 cells [37]. We then directly determined if viral infection of HSAECs caused up regulation of endogenous TNFα. We performed RT-PCRs with TNFα specific primers at 1, 2, 4, 6, 24 and 30 h post infection and observed up regulation of TNFα expression 24 h after infection (Figure 3C and D). Taken together, our data suggests that an increase in TNFα level may cause down regulation of SOD1 in HSAECs and that MP12 infection causes a modest up regulation of TNFα gene transcription. We had also determined by ELISA and Reverse Phase protein MicroArray (RPMA) that there was an increase in TNFα protein expression following MP12 infection (data not shown).

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