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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.

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Enhanced STAT1 phosphorylation in infected SOCS-3 deficient MEF correlates with elevated induction of IFNβ-stimulated genes.Wild type MEF and SOCS-3 knock out MEF were infected with PR8 (MOI = 5) for the indicated times. Subsequently, cell lysates were analyzed for STAT1 phosphorylation (A). For control of productive virus replication, cell lysates were analyzed for viral protein PB1 expression. In (E, F, G) wild type and knock out cells were lysed at indicated time-points of infection. Subsequently RNA was subjected to reverse transcription. cDNA was analyzed in quantitative real time PCR to assess mRNA amounts of three prototype type I IFN-stimulated genes, SP110 (E), interferon regulatory factor-1 (IRF-1) (F) and OAS1 (G). Equivalent mRNA amounts were normalized to GAPDH mRNA levels and calculated as n-fold of the levels of untreated cells that were arbitrarily set as 1. In (C) wild type MEF and knock out MEF were infected with PR8 (MOI = 5) or left uninfected. Supernatants were taken 6 p.i. and used for stimulation of wild type MEF for 15 minutes. As control wild type MEF were stimulated with 500 U/ml mouse IFNβ for 15 minutes. Cells were harvested and analyzed for the amount of STAT1 and phospho-STAT1 in Western blot analysis by specific antibodies. In (B) and (D) the relative band intensities of phospho-STAT1 of the blots in (A) and (C) were quantified as described.
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ppat-1000196-g007: Enhanced STAT1 phosphorylation in infected SOCS-3 deficient MEF correlates with elevated induction of IFNβ-stimulated genes.Wild type MEF and SOCS-3 knock out MEF were infected with PR8 (MOI = 5) for the indicated times. Subsequently, cell lysates were analyzed for STAT1 phosphorylation (A). For control of productive virus replication, cell lysates were analyzed for viral protein PB1 expression. In (E, F, G) wild type and knock out cells were lysed at indicated time-points of infection. Subsequently RNA was subjected to reverse transcription. cDNA was analyzed in quantitative real time PCR to assess mRNA amounts of three prototype type I IFN-stimulated genes, SP110 (E), interferon regulatory factor-1 (IRF-1) (F) and OAS1 (G). Equivalent mRNA amounts were normalized to GAPDH mRNA levels and calculated as n-fold of the levels of untreated cells that were arbitrarily set as 1. In (C) wild type MEF and knock out MEF were infected with PR8 (MOI = 5) or left uninfected. Supernatants were taken 6 p.i. and used for stimulation of wild type MEF for 15 minutes. As control wild type MEF were stimulated with 500 U/ml mouse IFNβ for 15 minutes. Cells were harvested and analyzed for the amount of STAT1 and phospho-STAT1 in Western blot analysis by specific antibodies. In (B) and (D) the relative band intensities of phospho-STAT1 of the blots in (A) and (C) were quantified as described.

Mentions: To further assess a functional role of SOCS-3 in virus-induced suppression of STAT1 phosphorylation we analyzed mouse cells with a targeted deletion of the SOCS-3 gene [56]. Wild type and SOCS-3 deficient mouse embryonic fibroblasts (MEF) were infected for different time points with PR8. The time of infection was prolonged in comparison to the infection of A549 cells because the human PR8 replicates less efficiently in mouse than in human cells. Following infection lysates of these cells were assessed for STAT1 phosphorylation (Figure 7A). Both cell types showed no phosphorylation of STAT1 in the uninfected state. In contrast, infection of SOCS-3 knock out cells resulted in strongly elevated phosphorylation of STAT1 in a sustained fashion. To rule out that this STAT1 phosphorylation is due to altered secretion of IFNβ or other STAT1-activating cytokines in SOCS-3 deficient cells, we performed conditioned medium experiments (Figure 7C). MEF wild type and MEF SOCS-3 deficient cells were infected for 6 h and supernatants were subsequently harvested. Stimulation of MEF wild type cells with these different supernatants for 15 min. revealed no differences in STAT1 phosphorylation, indicating that both infected cell types secrete similar amounts of IFNβ and other STAT1 activating cytokines. This is a strong indication that the observed differences in virus-induced STAT phosphorylation are directly due to the presence or absence of SOCS-3 in wild type and knock out MEF, respectively. To answer the question whether enhanced STAT phosphorylation in SOCS-3 deficient cells would also lead to enhanced expression of ISGs, total RNA was isolated at different time points p.i. from infected wild type and knock out cells and monitored for induction of SP110, IRF-1 and OAS1 (Figure 7E, 7F and 7G). These genes are described as type I IFN-induced genes [18]. Indeed mRNA levels of all three representative ISGs were elevated in SOCS-3 knock out versus wild type cells at almost every time point during the course of infection. This indicates that enhanced STAT1 phosphorylation and activation in SOCS-3 deficient cells results in elevated expression of ISGs.


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)

Enhanced STAT1 phosphorylation in infected SOCS-3 deficient MEF correlates with elevated induction of IFNβ-stimulated genes.Wild type MEF and SOCS-3 knock out MEF were infected with PR8 (MOI = 5) for the indicated times. Subsequently, cell lysates were analyzed for STAT1 phosphorylation (A). For control of productive virus replication, cell lysates were analyzed for viral protein PB1 expression. In (E, F, G) wild type and knock out cells were lysed at indicated time-points of infection. Subsequently RNA was subjected to reverse transcription. cDNA was analyzed in quantitative real time PCR to assess mRNA amounts of three prototype type I IFN-stimulated genes, SP110 (E), interferon regulatory factor-1 (IRF-1) (F) and OAS1 (G). Equivalent mRNA amounts were normalized to GAPDH mRNA levels and calculated as n-fold of the levels of untreated cells that were arbitrarily set as 1. In (C) wild type MEF and knock out MEF were infected with PR8 (MOI = 5) or left uninfected. Supernatants were taken 6 p.i. and used for stimulation of wild type MEF for 15 minutes. As control wild type MEF were stimulated with 500 U/ml mouse IFNβ for 15 minutes. Cells were harvested and analyzed for the amount of STAT1 and phospho-STAT1 in Western blot analysis by specific antibodies. In (B) and (D) the relative band intensities of phospho-STAT1 of the blots in (A) and (C) were quantified as described.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2572141&req=5

ppat-1000196-g007: Enhanced STAT1 phosphorylation in infected SOCS-3 deficient MEF correlates with elevated induction of IFNβ-stimulated genes.Wild type MEF and SOCS-3 knock out MEF were infected with PR8 (MOI = 5) for the indicated times. Subsequently, cell lysates were analyzed for STAT1 phosphorylation (A). For control of productive virus replication, cell lysates were analyzed for viral protein PB1 expression. In (E, F, G) wild type and knock out cells were lysed at indicated time-points of infection. Subsequently RNA was subjected to reverse transcription. cDNA was analyzed in quantitative real time PCR to assess mRNA amounts of three prototype type I IFN-stimulated genes, SP110 (E), interferon regulatory factor-1 (IRF-1) (F) and OAS1 (G). Equivalent mRNA amounts were normalized to GAPDH mRNA levels and calculated as n-fold of the levels of untreated cells that were arbitrarily set as 1. In (C) wild type MEF and knock out MEF were infected with PR8 (MOI = 5) or left uninfected. Supernatants were taken 6 p.i. and used for stimulation of wild type MEF for 15 minutes. As control wild type MEF were stimulated with 500 U/ml mouse IFNβ for 15 minutes. Cells were harvested and analyzed for the amount of STAT1 and phospho-STAT1 in Western blot analysis by specific antibodies. In (B) and (D) the relative band intensities of phospho-STAT1 of the blots in (A) and (C) were quantified as described.
Mentions: To further assess a functional role of SOCS-3 in virus-induced suppression of STAT1 phosphorylation we analyzed mouse cells with a targeted deletion of the SOCS-3 gene [56]. Wild type and SOCS-3 deficient mouse embryonic fibroblasts (MEF) were infected for different time points with PR8. The time of infection was prolonged in comparison to the infection of A549 cells because the human PR8 replicates less efficiently in mouse than in human cells. Following infection lysates of these cells were assessed for STAT1 phosphorylation (Figure 7A). Both cell types showed no phosphorylation of STAT1 in the uninfected state. In contrast, infection of SOCS-3 knock out cells resulted in strongly elevated phosphorylation of STAT1 in a sustained fashion. To rule out that this STAT1 phosphorylation is due to altered secretion of IFNβ or other STAT1-activating cytokines in SOCS-3 deficient cells, we performed conditioned medium experiments (Figure 7C). MEF wild type and MEF SOCS-3 deficient cells were infected for 6 h and supernatants were subsequently harvested. Stimulation of MEF wild type cells with these different supernatants for 15 min. revealed no differences in STAT1 phosphorylation, indicating that both infected cell types secrete similar amounts of IFNβ and other STAT1 activating cytokines. This is a strong indication that the observed differences in virus-induced STAT phosphorylation are directly due to the presence or absence of SOCS-3 in wild type and knock out MEF, respectively. To answer the question whether enhanced STAT phosphorylation in SOCS-3 deficient cells would also lead to enhanced expression of ISGs, total RNA was isolated at different time points p.i. from infected wild type and knock out cells and monitored for induction of SP110, IRF-1 and OAS1 (Figure 7E, 7F and 7G). These genes are described as type I IFN-induced genes [18]. Indeed mRNA levels of all three representative ISGs were elevated in SOCS-3 knock out versus wild type cells at almost every time point during the course of infection. This indicates that enhanced STAT1 phosphorylation and activation in SOCS-3 deficient cells results in elevated expression of ISGs.

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