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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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Viral 5′ triphosphate RNA efficiently induces SOCS-3 expression.Total RNA from infected or uninfected A549 cells was isolated and used for transfection of native A549 cells with L2000 according to manufacturer's instructions (A–G). Transfection of RNA from infected cells (“viral RNA”) serves as a mimic for vRNA accumulation in infected cells while total cellular RNA from uninfected cells (“cellular RNA”) was used as a control. In (E and F) different amounts of poly (I:C) or RNA from infected or uninfected cells treated with phosphatase (CIAP) as indicated were transfected using L2000. In (G) viral RNA transfected cells were additionally treated with DMSO (solvent) or the protein synthesis inhibitor anisomycin (aniso.) at the concentrations indicated. (A, B, E, F, G) Cells were lysed 3 h post transfection and total RNA was reverse transcribed. cDNA was analyzed in quantitative real time PCR to assess amounts of SOCS-1 (A), SOCS-3 (A, E, G) and IFNβ (B, F) mRNA levels. Equivalent amounts of mRNA were normalized to GAPDH mRNA levels and calculated as n-fold of untreated cells, arbitrarily set as 1. In (C) cells were treated as in (A) and (B) and monitored for phospho-STAT1 and phospho-STAT2 levels in Western blot analysis. (D) Quantification of relative phospho-STAT1 and phospho-STAT2 band intensities in (C).
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ppat-1000196-g005: Viral 5′ triphosphate RNA efficiently induces SOCS-3 expression.Total RNA from infected or uninfected A549 cells was isolated and used for transfection of native A549 cells with L2000 according to manufacturer's instructions (A–G). Transfection of RNA from infected cells (“viral RNA”) serves as a mimic for vRNA accumulation in infected cells while total cellular RNA from uninfected cells (“cellular RNA”) was used as a control. In (E and F) different amounts of poly (I:C) or RNA from infected or uninfected cells treated with phosphatase (CIAP) as indicated were transfected using L2000. In (G) viral RNA transfected cells were additionally treated with DMSO (solvent) or the protein synthesis inhibitor anisomycin (aniso.) at the concentrations indicated. (A, B, E, F, G) Cells were lysed 3 h post transfection and total RNA was reverse transcribed. cDNA was analyzed in quantitative real time PCR to assess amounts of SOCS-1 (A), SOCS-3 (A, E, G) and IFNβ (B, F) mRNA levels. Equivalent amounts of mRNA were normalized to GAPDH mRNA levels and calculated as n-fold of untreated cells, arbitrarily set as 1. In (C) cells were treated as in (A) and (B) and monitored for phospho-STAT1 and phospho-STAT2 levels in Western blot analysis. (D) Quantification of relative phospho-STAT1 and phospho-STAT2 band intensities in (C).

Mentions: Since accumulation of viral RNA in infected cells is a potent inducer of antiviral gene expression we investigated its ability to induce SOCS-3 gene transcription. As a source for viral RNA, A549 cells were infected with influenza A virus for 10 h and total RNA from these cells was isolated. RNA from uninfected A549 cells served as a negative control. Different amounts of these RNAs were used for stimulation of A549 cells for 3 h (Figures 5A, 5B and 5C). Transfection of RNA from uninfected cells did not result in an increase of SOCS-1 or SOCS-3 gene transcription (Figure 5A) or IFNβ induction as a control (Figure 5B). However, transfection of RNA from virally infected cells led to strongly elevated SOCS-3 mRNA amounts while SOCS-1 mRNA is only induced weakly (Figure 5A). This dose dependent induction of SOCS-3 by stimulation with increasing amounts of RNA from infected cells corresponds with a gradual decrease in the ability of this RNA to induce or potentiate STAT1/2 phosphorylation (Figure 5C).


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)

Viral 5′ triphosphate RNA efficiently induces SOCS-3 expression.Total RNA from infected or uninfected A549 cells was isolated and used for transfection of native A549 cells with L2000 according to manufacturer's instructions (A–G). Transfection of RNA from infected cells (“viral RNA”) serves as a mimic for vRNA accumulation in infected cells while total cellular RNA from uninfected cells (“cellular RNA”) was used as a control. In (E and F) different amounts of poly (I:C) or RNA from infected or uninfected cells treated with phosphatase (CIAP) as indicated were transfected using L2000. In (G) viral RNA transfected cells were additionally treated with DMSO (solvent) or the protein synthesis inhibitor anisomycin (aniso.) at the concentrations indicated. (A, B, E, F, G) Cells were lysed 3 h post transfection and total RNA was reverse transcribed. cDNA was analyzed in quantitative real time PCR to assess amounts of SOCS-1 (A), SOCS-3 (A, E, G) and IFNβ (B, F) mRNA levels. Equivalent amounts of mRNA were normalized to GAPDH mRNA levels and calculated as n-fold of untreated cells, arbitrarily set as 1. In (C) cells were treated as in (A) and (B) and monitored for phospho-STAT1 and phospho-STAT2 levels in Western blot analysis. (D) Quantification of relative phospho-STAT1 and phospho-STAT2 band intensities in (C).
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ppat-1000196-g005: Viral 5′ triphosphate RNA efficiently induces SOCS-3 expression.Total RNA from infected or uninfected A549 cells was isolated and used for transfection of native A549 cells with L2000 according to manufacturer's instructions (A–G). Transfection of RNA from infected cells (“viral RNA”) serves as a mimic for vRNA accumulation in infected cells while total cellular RNA from uninfected cells (“cellular RNA”) was used as a control. In (E and F) different amounts of poly (I:C) or RNA from infected or uninfected cells treated with phosphatase (CIAP) as indicated were transfected using L2000. In (G) viral RNA transfected cells were additionally treated with DMSO (solvent) or the protein synthesis inhibitor anisomycin (aniso.) at the concentrations indicated. (A, B, E, F, G) Cells were lysed 3 h post transfection and total RNA was reverse transcribed. cDNA was analyzed in quantitative real time PCR to assess amounts of SOCS-1 (A), SOCS-3 (A, E, G) and IFNβ (B, F) mRNA levels. Equivalent amounts of mRNA were normalized to GAPDH mRNA levels and calculated as n-fold of untreated cells, arbitrarily set as 1. In (C) cells were treated as in (A) and (B) and monitored for phospho-STAT1 and phospho-STAT2 levels in Western blot analysis. (D) Quantification of relative phospho-STAT1 and phospho-STAT2 band intensities in (C).
Mentions: Since accumulation of viral RNA in infected cells is a potent inducer of antiviral gene expression we investigated its ability to induce SOCS-3 gene transcription. As a source for viral RNA, A549 cells were infected with influenza A virus for 10 h and total RNA from these cells was isolated. RNA from uninfected A549 cells served as a negative control. Different amounts of these RNAs were used for stimulation of A549 cells for 3 h (Figures 5A, 5B and 5C). Transfection of RNA from uninfected cells did not result in an increase of SOCS-1 or SOCS-3 gene transcription (Figure 5A) or IFNβ induction as a control (Figure 5B). However, transfection of RNA from virally infected cells led to strongly elevated SOCS-3 mRNA amounts while SOCS-1 mRNA is only induced weakly (Figure 5A). This dose dependent induction of SOCS-3 by stimulation with increasing amounts of RNA from infected cells corresponds with a gradual decrease in the ability of this RNA to induce or potentiate STAT1/2 phosphorylation (Figure 5C).

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