Limits...
Influenza A virus inhibits type I IFN signaling via NF-kappaB-dependent induction of SOCS-3 expression.

Pauli EK, Schmolke M, Wolff T, Viemann D, Roth J, Bode JG, Ludwig S - PLoS Pathog. (2008)

Bottom Line: Closer examination revealed that SOCS-3 but not SOCS-1 mRNA levels increase in an RNA- and nuclear factor kappa B (NF-kappaB)-dependent but type I IFN-independent manner early in the viral replication cycle.This direct viral induction of SOCS-3 mRNA and protein expression appears to be relevant for suppression of the antiviral response since in SOCS-3 deficient cells a sustained phosphorylation of STAT1 correlated with elevated expression of type I IFN-dependent genes.The inhibitory effect is at least in part due to the induction of SOCS-3 gene expression, which results in an impaired antiviral response.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Virology (IMV), Centre of Molecular Biology of Inflammation (ZMBE), WWU Muenster, Germany.

ABSTRACT
The type I interferon (IFN) system is a first line of defense against viral infections. Viruses have developed various mechanisms to counteract this response. So far, the interferon antagonistic activity of influenza A viruses was mainly observed on the level of IFNbeta gene induction via action of the viral non-structural protein 1 (NS1). Here we present data indicating that influenza A viruses not only suppress IFNbeta gene induction but also inhibit type I IFN signaling through a mechanism involving induction of the suppressor of cytokine signaling-3 (SOCS-3) protein. Our study was based on the observation that in cells that were infected with influenza A virus and subsequently stimulated with IFNalpha/beta, phosphorylation of the signal transducer and activator of transcription protein 1 (STAT1) was strongly reduced. This impaired STAT1 activation was not due to the action of viral proteins but rather appeared to be induced by accumulation of viral 5' triphosphate RNA in the cell. SOCS proteins are potent endogenous inhibitors of Janus kinase (JAK)/STAT signaling. Closer examination revealed that SOCS-3 but not SOCS-1 mRNA levels increase in an RNA- and nuclear factor kappa B (NF-kappaB)-dependent but type I IFN-independent manner early in the viral replication cycle. This direct viral induction of SOCS-3 mRNA and protein expression appears to be relevant for suppression of the antiviral response since in SOCS-3 deficient cells a sustained phosphorylation of STAT1 correlated with elevated expression of type I IFN-dependent genes. As a consequence, progeny virus titers were reduced in SOCS-3 deficient cells or in cells were SOCS-3 expression was knocked-down by siRNA. These data provide the first evidence that influenza A viruses suppress type I IFN signaling on the level of JAK/STAT activation. The inhibitory effect is at least in part due to the induction of SOCS-3 gene expression, which results in an impaired antiviral response.

Show MeSH

Related in: MedlinePlus

Phosphatases do not mediate inhibition of IFNβ-induced STAT1 phosphorylation in infected cells.(A) Vero cells were infected for 10 h with PR8 (MOI = 5) or left uninfected. Prior stimulation with human IFNβ (500 U/ml for 15 min), cells were treated for 10 min with sodium vanadate at concentrations indicated. Cells were harvested and protein lysates were subjected to Western blot analysis using anti-phospho-STAT1 and anti-STAT1 antibody. H2O2: was used as a control for solvent conditions. (B) Quantification of band intensities in (A). To visualize the effect of sodium vanadate on the STAT1 phosphorylation in infected and uninfected cells, band intensities of IFNβ stimulated samples were determined relative to background. Linear regression was calculated using the Excel software (Microsoft) (s = slope of the regression line). (C) Phosphatase activity in A549 cells infected wit PR8 (MOI = 5) was determined using tyrosine phosphatase assay (Promega) according to manufacturers instructions. For measurement of newly generated free phosphate two different phosphorylated pseudosubstrates (peptide 1 and peptide 2) were used.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2572141&req=5

ppat-1000196-g003: Phosphatases do not mediate inhibition of IFNβ-induced STAT1 phosphorylation in infected cells.(A) Vero cells were infected for 10 h with PR8 (MOI = 5) or left uninfected. Prior stimulation with human IFNβ (500 U/ml for 15 min), cells were treated for 10 min with sodium vanadate at concentrations indicated. Cells were harvested and protein lysates were subjected to Western blot analysis using anti-phospho-STAT1 and anti-STAT1 antibody. H2O2: was used as a control for solvent conditions. (B) Quantification of band intensities in (A). To visualize the effect of sodium vanadate on the STAT1 phosphorylation in infected and uninfected cells, band intensities of IFNβ stimulated samples were determined relative to background. Linear regression was calculated using the Excel software (Microsoft) (s = slope of the regression line). (C) Phosphatase activity in A549 cells infected wit PR8 (MOI = 5) was determined using tyrosine phosphatase assay (Promega) according to manufacturers instructions. For measurement of newly generated free phosphate two different phosphorylated pseudosubstrates (peptide 1 and peptide 2) were used.

Mentions: Decrease of STAT phosphorylation might also be due to the action of virus-induced phosphatases. On the one hand these enzymes may cause direct dephosphorylation of STAT proteins. On the other hand phosphatases could act via an indirect mechanism by dephosphorylation and inactivation of JAKs resulting in an attenuated phosphorylation of STATs. Several protein tyrosine phosphatases (PTPs) are known to mediate dephosphorylation of both, JAKs and STATs [41]. In order to investigate whether influenza A virus activates phosphatases that subsequently target JAKs or STATs, we treated infected or uninfected A549 cells with the well-known tyrosine phosphatase inhibitor sodium vanadate [42],[43]. Uninfected cells or cells infected with PR8 for 10 h were incubated with increasing amounts of this compound 10 min prior to stimulation with IFNβ. This time point of infection was chosen since we observed considerable inhibition of IFN-induced STAT1 phosphorylation in the course of infection (Figures 1A and 1E). Increasing concentrations of vanadate lead to a gradual shift of the steady state balance of phosphorylation/dephosphorylation. Accordingly, a gradual increase of STAT1 phosphorylation was observed that was similar in both infected and uninfected cells, albeit starting from different basal levels of phospho-STAT1 (Figures 3A and B). This is illustrated by an almost identical slope of the regression line in the graphical analysis of the band intensities of the IFNβ stimulated samples (Figure 3B). If the blockade of IFNβ-induced STAT1 phosphorylation would be mediated by specific virus-activated phosphatases, a much steeper slope for vanadate-treated infected cells would be expected. However, the result in Figure 3B indicates that the virus-induced suppression of phosphorylation is not compensated by phosphatase inhibition and consequently no virus-activated phosphatase appears to be involved. In support of these data, phosphatase assays revealed that the overall activity of tyrosine phosphatases in infected cells was not elevated compared to uninfected cells. This is indicated by constant levels of free phosphates released from two different phospho-peptides that represent common tyrosine phosphatase substrates (Figure 3C). Thus, involvement of phosphatases in influenza virus-induced alteration of STAT1 phosphorylation can be greatly ruled out.


Influenza A virus inhibits type I IFN signaling via NF-kappaB-dependent induction of SOCS-3 expression.

Pauli EK, Schmolke M, Wolff T, Viemann D, Roth J, Bode JG, Ludwig S - PLoS Pathog. (2008)

Phosphatases do not mediate inhibition of IFNβ-induced STAT1 phosphorylation in infected cells.(A) Vero cells were infected for 10 h with PR8 (MOI = 5) or left uninfected. Prior stimulation with human IFNβ (500 U/ml for 15 min), cells were treated for 10 min with sodium vanadate at concentrations indicated. Cells were harvested and protein lysates were subjected to Western blot analysis using anti-phospho-STAT1 and anti-STAT1 antibody. H2O2: was used as a control for solvent conditions. (B) Quantification of band intensities in (A). To visualize the effect of sodium vanadate on the STAT1 phosphorylation in infected and uninfected cells, band intensities of IFNβ stimulated samples were determined relative to background. Linear regression was calculated using the Excel software (Microsoft) (s = slope of the regression line). (C) Phosphatase activity in A549 cells infected wit PR8 (MOI = 5) was determined using tyrosine phosphatase assay (Promega) according to manufacturers instructions. For measurement of newly generated free phosphate two different phosphorylated pseudosubstrates (peptide 1 and peptide 2) were used.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2572141&req=5

ppat-1000196-g003: Phosphatases do not mediate inhibition of IFNβ-induced STAT1 phosphorylation in infected cells.(A) Vero cells were infected for 10 h with PR8 (MOI = 5) or left uninfected. Prior stimulation with human IFNβ (500 U/ml for 15 min), cells were treated for 10 min with sodium vanadate at concentrations indicated. Cells were harvested and protein lysates were subjected to Western blot analysis using anti-phospho-STAT1 and anti-STAT1 antibody. H2O2: was used as a control for solvent conditions. (B) Quantification of band intensities in (A). To visualize the effect of sodium vanadate on the STAT1 phosphorylation in infected and uninfected cells, band intensities of IFNβ stimulated samples were determined relative to background. Linear regression was calculated using the Excel software (Microsoft) (s = slope of the regression line). (C) Phosphatase activity in A549 cells infected wit PR8 (MOI = 5) was determined using tyrosine phosphatase assay (Promega) according to manufacturers instructions. For measurement of newly generated free phosphate two different phosphorylated pseudosubstrates (peptide 1 and peptide 2) were used.
Mentions: Decrease of STAT phosphorylation might also be due to the action of virus-induced phosphatases. On the one hand these enzymes may cause direct dephosphorylation of STAT proteins. On the other hand phosphatases could act via an indirect mechanism by dephosphorylation and inactivation of JAKs resulting in an attenuated phosphorylation of STATs. Several protein tyrosine phosphatases (PTPs) are known to mediate dephosphorylation of both, JAKs and STATs [41]. In order to investigate whether influenza A virus activates phosphatases that subsequently target JAKs or STATs, we treated infected or uninfected A549 cells with the well-known tyrosine phosphatase inhibitor sodium vanadate [42],[43]. Uninfected cells or cells infected with PR8 for 10 h were incubated with increasing amounts of this compound 10 min prior to stimulation with IFNβ. This time point of infection was chosen since we observed considerable inhibition of IFN-induced STAT1 phosphorylation in the course of infection (Figures 1A and 1E). Increasing concentrations of vanadate lead to a gradual shift of the steady state balance of phosphorylation/dephosphorylation. Accordingly, a gradual increase of STAT1 phosphorylation was observed that was similar in both infected and uninfected cells, albeit starting from different basal levels of phospho-STAT1 (Figures 3A and B). This is illustrated by an almost identical slope of the regression line in the graphical analysis of the band intensities of the IFNβ stimulated samples (Figure 3B). If the blockade of IFNβ-induced STAT1 phosphorylation would be mediated by specific virus-activated phosphatases, a much steeper slope for vanadate-treated infected cells would be expected. However, the result in Figure 3B indicates that the virus-induced suppression of phosphorylation is not compensated by phosphatase inhibition and consequently no virus-activated phosphatase appears to be involved. In support of these data, phosphatase assays revealed that the overall activity of tyrosine phosphatases in infected cells was not elevated compared to uninfected cells. This is indicated by constant levels of free phosphates released from two different phospho-peptides that represent common tyrosine phosphatase substrates (Figure 3C). Thus, involvement of phosphatases in influenza virus-induced alteration of STAT1 phosphorylation can be greatly ruled out.

Bottom Line: Closer examination revealed that SOCS-3 but not SOCS-1 mRNA levels increase in an RNA- and nuclear factor kappa B (NF-kappaB)-dependent but type I IFN-independent manner early in the viral replication cycle.This direct viral induction of SOCS-3 mRNA and protein expression appears to be relevant for suppression of the antiviral response since in SOCS-3 deficient cells a sustained phosphorylation of STAT1 correlated with elevated expression of type I IFN-dependent genes.The inhibitory effect is at least in part due to the induction of SOCS-3 gene expression, which results in an impaired antiviral response.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Virology (IMV), Centre of Molecular Biology of Inflammation (ZMBE), WWU Muenster, Germany.

ABSTRACT
The type I interferon (IFN) system is a first line of defense against viral infections. Viruses have developed various mechanisms to counteract this response. So far, the interferon antagonistic activity of influenza A viruses was mainly observed on the level of IFNbeta gene induction via action of the viral non-structural protein 1 (NS1). Here we present data indicating that influenza A viruses not only suppress IFNbeta gene induction but also inhibit type I IFN signaling through a mechanism involving induction of the suppressor of cytokine signaling-3 (SOCS-3) protein. Our study was based on the observation that in cells that were infected with influenza A virus and subsequently stimulated with IFNalpha/beta, phosphorylation of the signal transducer and activator of transcription protein 1 (STAT1) was strongly reduced. This impaired STAT1 activation was not due to the action of viral proteins but rather appeared to be induced by accumulation of viral 5' triphosphate RNA in the cell. SOCS proteins are potent endogenous inhibitors of Janus kinase (JAK)/STAT signaling. Closer examination revealed that SOCS-3 but not SOCS-1 mRNA levels increase in an RNA- and nuclear factor kappa B (NF-kappaB)-dependent but type I IFN-independent manner early in the viral replication cycle. This direct viral induction of SOCS-3 mRNA and protein expression appears to be relevant for suppression of the antiviral response since in SOCS-3 deficient cells a sustained phosphorylation of STAT1 correlated with elevated expression of type I IFN-dependent genes. As a consequence, progeny virus titers were reduced in SOCS-3 deficient cells or in cells were SOCS-3 expression was knocked-down by siRNA. These data provide the first evidence that influenza A viruses suppress type I IFN signaling on the level of JAK/STAT activation. The inhibitory effect is at least in part due to the induction of SOCS-3 gene expression, which results in an impaired antiviral response.

Show MeSH
Related in: MedlinePlus