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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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Alterations in SOD1 protein following infection by MP12 virus.A) Viral infection was confirmed by western blot analysis of HSAEC extracts using anti-RVFV antibody. B) HSAECs infected with MP12 virus (MOI of 3) were harvested at 24, 30, 48 and 72 h post infection. Cell extracts were analyzed by western blot using anti-SOD1 antibody. Actin was utilized as a loading control. C) Quantification of fold differences in SOD1 band intensities between uninfected control cells and MP12 infected cells over the time course of infection is indicated. The quantification is representative of three independent experiments. * indicates p≤0.01. D) Total RNA was obtained from HSAECs infected with MP12 virus at 4, 6, 24 and 30 h post infection and analyzed by RT-PCR with primers against SOD1. GAPDH was used as a loading control. M indicates mock-infected cells and I indicates MP12-infected cells. E) Total RNA from MP12 and mock infected samples were analyzed by q-RT-PCR with primers to SOD1. Data was normalized to GAPDH RNA levels. The results include data from three experiments. F) MP12-infected HSAEC extracts were analyzed by western blot using anti-SOD2 antibody. Actin was used as a loading control.
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pone-0020354-g001: Alterations in SOD1 protein following infection by MP12 virus.A) Viral infection was confirmed by western blot analysis of HSAEC extracts using anti-RVFV antibody. B) HSAECs infected with MP12 virus (MOI of 3) were harvested at 24, 30, 48 and 72 h post infection. Cell extracts were analyzed by western blot using anti-SOD1 antibody. Actin was utilized as a loading control. C) Quantification of fold differences in SOD1 band intensities between uninfected control cells and MP12 infected cells over the time course of infection is indicated. The quantification is representative of three independent experiments. * indicates p≤0.01. D) Total RNA was obtained from HSAECs infected with MP12 virus at 4, 6, 24 and 30 h post infection and analyzed by RT-PCR with primers against SOD1. GAPDH was used as a loading control. M indicates mock-infected cells and I indicates MP12-infected cells. E) Total RNA from MP12 and mock infected samples were analyzed by q-RT-PCR with primers to SOD1. Data was normalized to GAPDH RNA levels. The results include data from three experiments. F) MP12-infected HSAEC extracts were analyzed by western blot using anti-SOD2 antibody. Actin was used as a loading control.

Mentions: Oxidative stress resulting from viral infections is a well characterized phenomenon. Our phosphoproteomic analysis of RVFV infected cells revealed that multiple signaling and transcription factors that are subject to regulation by ROS were altered after infection [28]. So, we wished to determine if exposure of human cells to RVFV would result in significant oxidative stress in the infected cell. We exposed human lung epithelial cells to the MP12 strain of RVFV and first evaluated the levels of the two major enzymes involved in the maintenance of cellular oxidative homeostasis, SOD1 and SOD2 at multiple time points after infection. Whole cell extracts were obtained at 0, 24, 30, 48 and 72 h post infection and were analyzed by western blot analysis for changes in SOD1 (Figure 1B). We observed that in comparison with uninfected cells, the infected cells showed a consistent decrease in SOD1 at early time points (24 and 30 h) (Figure 1B and C). We confirmed viral infection in these cells by analyzing for viral proteins by western blots using anti-RVFV antibody (Figure 1A). We then asked whether this reduced protein levels at early time points was due to transcriptional or post transcriptional events. To answer that question, we carried out RT-PCR studies with primers specific to SOD1. GAPDH expression levels were analyzed as a control. We did not find any striking decrease in the SOD1 expression levels (Figure 1D) suggesting that the alteration in protein level is likely to be post transcriptional. In order to sensitively evaluate whether there is any transcriptional down regulation of SOD1, we carried out additional quantitative RT-PCR (q-RT-PCR) analyses with SOD1 and GAPDH primers. Our q-RT-PCR studies confirmed our RT-PCR analysis (Figure 1E) that no significant changes (p-values>0.5) in SOD1 transcripts could be observed in the infected samples. Next, we carried out western blot analyses with antibodies to superoxide dismutase 2 (SOD2) and found that SOD2 did not display early changes in protein levels (Figure 1F). Collectively, our data indicates that following exposure to MP12, human cells display a lowered abundance of the host antioxidant protein SOD1 and the down regulation appears to be due to post transcriptional effects.


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)

Alterations in SOD1 protein following infection by MP12 virus.A) Viral infection was confirmed by western blot analysis of HSAEC extracts using anti-RVFV antibody. B) HSAECs infected with MP12 virus (MOI of 3) were harvested at 24, 30, 48 and 72 h post infection. Cell extracts were analyzed by western blot using anti-SOD1 antibody. Actin was utilized as a loading control. C) Quantification of fold differences in SOD1 band intensities between uninfected control cells and MP12 infected cells over the time course of infection is indicated. The quantification is representative of three independent experiments. * indicates p≤0.01. D) Total RNA was obtained from HSAECs infected with MP12 virus at 4, 6, 24 and 30 h post infection and analyzed by RT-PCR with primers against SOD1. GAPDH was used as a loading control. M indicates mock-infected cells and I indicates MP12-infected cells. E) Total RNA from MP12 and mock infected samples were analyzed by q-RT-PCR with primers to SOD1. Data was normalized to GAPDH RNA levels. The results include data from three experiments. F) MP12-infected HSAEC extracts were analyzed by western blot using anti-SOD2 antibody. Actin was used as a loading control.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3105056&req=5

pone-0020354-g001: Alterations in SOD1 protein following infection by MP12 virus.A) Viral infection was confirmed by western blot analysis of HSAEC extracts using anti-RVFV antibody. B) HSAECs infected with MP12 virus (MOI of 3) were harvested at 24, 30, 48 and 72 h post infection. Cell extracts were analyzed by western blot using anti-SOD1 antibody. Actin was utilized as a loading control. C) Quantification of fold differences in SOD1 band intensities between uninfected control cells and MP12 infected cells over the time course of infection is indicated. The quantification is representative of three independent experiments. * indicates p≤0.01. D) Total RNA was obtained from HSAECs infected with MP12 virus at 4, 6, 24 and 30 h post infection and analyzed by RT-PCR with primers against SOD1. GAPDH was used as a loading control. M indicates mock-infected cells and I indicates MP12-infected cells. E) Total RNA from MP12 and mock infected samples were analyzed by q-RT-PCR with primers to SOD1. Data was normalized to GAPDH RNA levels. The results include data from three experiments. F) MP12-infected HSAEC extracts were analyzed by western blot using anti-SOD2 antibody. Actin was used as a loading control.
Mentions: Oxidative stress resulting from viral infections is a well characterized phenomenon. Our phosphoproteomic analysis of RVFV infected cells revealed that multiple signaling and transcription factors that are subject to regulation by ROS were altered after infection [28]. So, we wished to determine if exposure of human cells to RVFV would result in significant oxidative stress in the infected cell. We exposed human lung epithelial cells to the MP12 strain of RVFV and first evaluated the levels of the two major enzymes involved in the maintenance of cellular oxidative homeostasis, SOD1 and SOD2 at multiple time points after infection. Whole cell extracts were obtained at 0, 24, 30, 48 and 72 h post infection and were analyzed by western blot analysis for changes in SOD1 (Figure 1B). We observed that in comparison with uninfected cells, the infected cells showed a consistent decrease in SOD1 at early time points (24 and 30 h) (Figure 1B and C). We confirmed viral infection in these cells by analyzing for viral proteins by western blots using anti-RVFV antibody (Figure 1A). We then asked whether this reduced protein levels at early time points was due to transcriptional or post transcriptional events. To answer that question, we carried out RT-PCR studies with primers specific to SOD1. GAPDH expression levels were analyzed as a control. We did not find any striking decrease in the SOD1 expression levels (Figure 1D) suggesting that the alteration in protein level is likely to be post transcriptional. In order to sensitively evaluate whether there is any transcriptional down regulation of SOD1, we carried out additional quantitative RT-PCR (q-RT-PCR) analyses with SOD1 and GAPDH primers. Our q-RT-PCR studies confirmed our RT-PCR analysis (Figure 1E) that no significant changes (p-values>0.5) in SOD1 transcripts could be observed in the infected samples. Next, we carried out western blot analyses with antibodies to superoxide dismutase 2 (SOD2) and found that SOD2 did not display early changes in protein levels (Figure 1F). Collectively, our data indicates that following exposure to MP12, human cells display a lowered abundance of the host antioxidant protein SOD1 and the down regulation appears to be due to post transcriptional effects.

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