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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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Phosphorylation of p38 MAPK following MP12 infection.A) 106 HSAECs were infected with MP12 (MOI of 3). Cell extracts were obtained at 0, 24, 48 and 72 h post infection and analyzed by western blot with anti-phospho-p38 antibody. Actin was used as loading control. B) Total p38 (t-p38) levels in MP12 infected cells were compared with that of control cells at the 24-h time point by western blot with antibody to total p38 MAPK. Actin was used as loading control. C) Phosphorylation status of p38 MAPK in ΔNSm mutant virus infected cells and MP12 infected cells was determined by western blot analysis of cells infected with the NSm mutant strain (ΔNSm) and MP12 (MOI of 3). D) Comparable infection of HSAECs by MP12 virus and the ΔNSm mutant virus was determined by western blot of infected extracts obtained 24 h post infection with anti-RVFV antibody. M refers to mock-infected control cells, MP refers to MP12-infected cells and NSm refers to ΔNSm mutant virus infected cells.
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pone-0020354-g005: Phosphorylation of p38 MAPK following MP12 infection.A) 106 HSAECs were infected with MP12 (MOI of 3). Cell extracts were obtained at 0, 24, 48 and 72 h post infection and analyzed by western blot with anti-phospho-p38 antibody. Actin was used as loading control. B) Total p38 (t-p38) levels in MP12 infected cells were compared with that of control cells at the 24-h time point by western blot with antibody to total p38 MAPK. Actin was used as loading control. C) Phosphorylation status of p38 MAPK in ΔNSm mutant virus infected cells and MP12 infected cells was determined by western blot analysis of cells infected with the NSm mutant strain (ΔNSm) and MP12 (MOI of 3). D) Comparable infection of HSAECs by MP12 virus and the ΔNSm mutant virus was determined by western blot of infected extracts obtained 24 h post infection with anti-RVFV antibody. M refers to mock-infected control cells, MP refers to MP12-infected cells and NSm refers to ΔNSm mutant virus infected cells.

Mentions: Earlier studies have revealed that SOD1 is involved in the regulation of cellular cytoskeleton and siRNA-mediated depletion of SOD1 caused alterations in actin cytoskeleton in neuroblastoma cell lines [41]. As a compensatory measure, activation of the p38 MAPK pathway was observed. It was demonstrated that the activation of this stress response pathway was necessary to counter the cytoskeletal damage that occurred due to an oxidative burst observed under conditions of reduced SOD1. We reasoned that the down regulation of SOD1 (Figure 1A and B) could cause the host to activate the p38 MAPK pathway. Down regulation of SOD1 has been linked to increased phosphorylation of p38 MAPK in disease states like ALS. We next checked the phosphorylation status of p38 MAPK to determine if its activation was increased under these conditions. Western blot analysis carried out on MP12-infected cells revealed strong increase in phosphorylation of p38 MAPK at 24 h post infection (Figure 5A). We had previously demonstrated activation of p38 MAPK following MP12 infection even at lower MOIs of infection [28]. There was no significant difference in total p38 MAPK levels at the 24 h time point between mock-infected and MP12 infected cells (Figure 5B). This suggests that the increase in phosphorylation of p38 MAPK is not dependent on increased expression of p38 MAPK. Interestingly, when HSAECs were infected with a NSm mutant virus (MP12ΔNSm), similar high level of phosphorylation of p38 MAPK was not observed (Figure 5C). We confirmed similar levels of infection with the MP12 virus and the NSm mutant (Figure 5D) to rule out the possibility of lower infectivity by the NSm mutant virus. Collectively, our data suggest that down-regulation of SOD1 and a strong oxidative stress condition elicits the p38 MAPK response in HSAECs. Interestingly, our data also suggest that the viral anti-apoptotic protein NSm may play a role in the activation of p38 MAPK.


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)

Phosphorylation of p38 MAPK following MP12 infection.A) 106 HSAECs were infected with MP12 (MOI of 3). Cell extracts were obtained at 0, 24, 48 and 72 h post infection and analyzed by western blot with anti-phospho-p38 antibody. Actin was used as loading control. B) Total p38 (t-p38) levels in MP12 infected cells were compared with that of control cells at the 24-h time point by western blot with antibody to total p38 MAPK. Actin was used as loading control. C) Phosphorylation status of p38 MAPK in ΔNSm mutant virus infected cells and MP12 infected cells was determined by western blot analysis of cells infected with the NSm mutant strain (ΔNSm) and MP12 (MOI of 3). D) Comparable infection of HSAECs by MP12 virus and the ΔNSm mutant virus was determined by western blot of infected extracts obtained 24 h post infection with anti-RVFV antibody. M refers to mock-infected control cells, MP refers to MP12-infected cells and NSm refers to ΔNSm mutant virus infected cells.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3105056&req=5

pone-0020354-g005: Phosphorylation of p38 MAPK following MP12 infection.A) 106 HSAECs were infected with MP12 (MOI of 3). Cell extracts were obtained at 0, 24, 48 and 72 h post infection and analyzed by western blot with anti-phospho-p38 antibody. Actin was used as loading control. B) Total p38 (t-p38) levels in MP12 infected cells were compared with that of control cells at the 24-h time point by western blot with antibody to total p38 MAPK. Actin was used as loading control. C) Phosphorylation status of p38 MAPK in ΔNSm mutant virus infected cells and MP12 infected cells was determined by western blot analysis of cells infected with the NSm mutant strain (ΔNSm) and MP12 (MOI of 3). D) Comparable infection of HSAECs by MP12 virus and the ΔNSm mutant virus was determined by western blot of infected extracts obtained 24 h post infection with anti-RVFV antibody. M refers to mock-infected control cells, MP refers to MP12-infected cells and NSm refers to ΔNSm mutant virus infected cells.
Mentions: Earlier studies have revealed that SOD1 is involved in the regulation of cellular cytoskeleton and siRNA-mediated depletion of SOD1 caused alterations in actin cytoskeleton in neuroblastoma cell lines [41]. As a compensatory measure, activation of the p38 MAPK pathway was observed. It was demonstrated that the activation of this stress response pathway was necessary to counter the cytoskeletal damage that occurred due to an oxidative burst observed under conditions of reduced SOD1. We reasoned that the down regulation of SOD1 (Figure 1A and B) could cause the host to activate the p38 MAPK pathway. Down regulation of SOD1 has been linked to increased phosphorylation of p38 MAPK in disease states like ALS. We next checked the phosphorylation status of p38 MAPK to determine if its activation was increased under these conditions. Western blot analysis carried out on MP12-infected cells revealed strong increase in phosphorylation of p38 MAPK at 24 h post infection (Figure 5A). We had previously demonstrated activation of p38 MAPK following MP12 infection even at lower MOIs of infection [28]. There was no significant difference in total p38 MAPK levels at the 24 h time point between mock-infected and MP12 infected cells (Figure 5B). This suggests that the increase in phosphorylation of p38 MAPK is not dependent on increased expression of p38 MAPK. Interestingly, when HSAECs were infected with a NSm mutant virus (MP12ΔNSm), similar high level of phosphorylation of p38 MAPK was not observed (Figure 5C). We confirmed similar levels of infection with the MP12 virus and the NSm mutant (Figure 5D) to rule out the possibility of lower infectivity by the NSm mutant virus. Collectively, our data suggest that down-regulation of SOD1 and a strong oxidative stress condition elicits the p38 MAPK response in HSAECs. Interestingly, our data also suggest that the viral anti-apoptotic protein NSm may play a role in the activation of p38 MAPK.

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