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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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Efficiency of influenza A virus replication is dependent on SOCS3 expression levels.Wild type MEF and SOCS-3 knock-out MEF were infected with PR8 (MOI = 0.01) (A) or A/Victoria/3/75 (MOI = 0.001) (B) for the indicated times. In (C) A549 wt cells were transfected for 48 h with 150 nM human SOCS3 siRNA using Hiperfect according to manufacturers protocol and infected with PR8 (MOI = 0.001) for 9 h. In (D) the highly susceptible cell line HEK293 was transfected with either pSUPER empty vector or pSUPER-mSOCS-3 for 48 h. Subsequently cells were infected with PR8 (MOI = 0.001) for 9 h. For (A), (B), (C) and (D) progeny virus titers were determined from the supernatants of infected cells by means of plaque assay. To determine the effect of over expressed SOCS-3 on STAT1 phosphorylation (E) A549 cells were treated as in (D) and infected with PR8 (MOI = 5) and/or stimulated with human IFNβ (100 U/ml). Cells were lysed and cell extracts were analyzed for levels of phosphorylated STAT1 and over expressed mSOCS-3 using anti phospho-STAT1 and anti flag-antibody in Western blots. Effective of SOCS-3 knock down was determined by Western blot (C, right). Cells were treated as in (C, left) and total cells lysates were analyzed for endogenous SOCS-3 protein levels using anti-SOCS-3 antibody in Western blot. (F) Quantification of relative pSTAT1 band intensities in (E).
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ppat-1000196-g008: Efficiency of influenza A virus replication is dependent on SOCS3 expression levels.Wild type MEF and SOCS-3 knock-out MEF were infected with PR8 (MOI = 0.01) (A) or A/Victoria/3/75 (MOI = 0.001) (B) for the indicated times. In (C) A549 wt cells were transfected for 48 h with 150 nM human SOCS3 siRNA using Hiperfect according to manufacturers protocol and infected with PR8 (MOI = 0.001) for 9 h. In (D) the highly susceptible cell line HEK293 was transfected with either pSUPER empty vector or pSUPER-mSOCS-3 for 48 h. Subsequently cells were infected with PR8 (MOI = 0.001) for 9 h. For (A), (B), (C) and (D) progeny virus titers were determined from the supernatants of infected cells by means of plaque assay. To determine the effect of over expressed SOCS-3 on STAT1 phosphorylation (E) A549 cells were treated as in (D) and infected with PR8 (MOI = 5) and/or stimulated with human IFNβ (100 U/ml). Cells were lysed and cell extracts were analyzed for levels of phosphorylated STAT1 and over expressed mSOCS-3 using anti phospho-STAT1 and anti flag-antibody in Western blots. Effective of SOCS-3 knock down was determined by Western blot (C, right). Cells were treated as in (C, left) and total cells lysates were analyzed for endogenous SOCS-3 protein levels using anti-SOCS-3 antibody in Western blot. (F) Quantification of relative pSTAT1 band intensities in (E).

Mentions: The remaining question was, whether the elevated IFN-induced gene response in knock out cells might also affect propagation of influenza A viruses. Thus, both wild type and knock out cells were infected with PR8 (Figure 8A) or the strain A/Victoria/3/75 (H3N2) (Figure 8B). Virus titers were assessed at different time points post infection. Progeny virus titers from SOCS-3 knock out cells were significantly reduced compared to titers from infected wild type cells. To independently confirm these results and to verify that the observed effects are really due to the lack of SOCS-3, we used an siRNA approach to specifically knock down SOCS-3 mRNA in A549 cells. Cells were transfected with 150 nM siRNA for 48 h and SOCS-3 protein levels were compared to control transfected samples (Figure 8C, right). Subsequently, cells were infected and progeny virus titers were determined by plaque assay (Figure 8C, left). Similar to the results gained from infected knock out cells, knock down of SOCS-3 resulted in decreased virus titers.


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)

Efficiency of influenza A virus replication is dependent on SOCS3 expression levels.Wild type MEF and SOCS-3 knock-out MEF were infected with PR8 (MOI = 0.01) (A) or A/Victoria/3/75 (MOI = 0.001) (B) for the indicated times. In (C) A549 wt cells were transfected for 48 h with 150 nM human SOCS3 siRNA using Hiperfect according to manufacturers protocol and infected with PR8 (MOI = 0.001) for 9 h. In (D) the highly susceptible cell line HEK293 was transfected with either pSUPER empty vector or pSUPER-mSOCS-3 for 48 h. Subsequently cells were infected with PR8 (MOI = 0.001) for 9 h. For (A), (B), (C) and (D) progeny virus titers were determined from the supernatants of infected cells by means of plaque assay. To determine the effect of over expressed SOCS-3 on STAT1 phosphorylation (E) A549 cells were treated as in (D) and infected with PR8 (MOI = 5) and/or stimulated with human IFNβ (100 U/ml). Cells were lysed and cell extracts were analyzed for levels of phosphorylated STAT1 and over expressed mSOCS-3 using anti phospho-STAT1 and anti flag-antibody in Western blots. Effective of SOCS-3 knock down was determined by Western blot (C, right). Cells were treated as in (C, left) and total cells lysates were analyzed for endogenous SOCS-3 protein levels using anti-SOCS-3 antibody in Western blot. (F) Quantification of relative pSTAT1 band intensities in (E).
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ppat-1000196-g008: Efficiency of influenza A virus replication is dependent on SOCS3 expression levels.Wild type MEF and SOCS-3 knock-out MEF were infected with PR8 (MOI = 0.01) (A) or A/Victoria/3/75 (MOI = 0.001) (B) for the indicated times. In (C) A549 wt cells were transfected for 48 h with 150 nM human SOCS3 siRNA using Hiperfect according to manufacturers protocol and infected with PR8 (MOI = 0.001) for 9 h. In (D) the highly susceptible cell line HEK293 was transfected with either pSUPER empty vector or pSUPER-mSOCS-3 for 48 h. Subsequently cells were infected with PR8 (MOI = 0.001) for 9 h. For (A), (B), (C) and (D) progeny virus titers were determined from the supernatants of infected cells by means of plaque assay. To determine the effect of over expressed SOCS-3 on STAT1 phosphorylation (E) A549 cells were treated as in (D) and infected with PR8 (MOI = 5) and/or stimulated with human IFNβ (100 U/ml). Cells were lysed and cell extracts were analyzed for levels of phosphorylated STAT1 and over expressed mSOCS-3 using anti phospho-STAT1 and anti flag-antibody in Western blots. Effective of SOCS-3 knock down was determined by Western blot (C, right). Cells were treated as in (C, left) and total cells lysates were analyzed for endogenous SOCS-3 protein levels using anti-SOCS-3 antibody in Western blot. (F) Quantification of relative pSTAT1 band intensities in (E).
Mentions: The remaining question was, whether the elevated IFN-induced gene response in knock out cells might also affect propagation of influenza A viruses. Thus, both wild type and knock out cells were infected with PR8 (Figure 8A) or the strain A/Victoria/3/75 (H3N2) (Figure 8B). Virus titers were assessed at different time points post infection. Progeny virus titers from SOCS-3 knock out cells were significantly reduced compared to titers from infected wild type cells. To independently confirm these results and to verify that the observed effects are really due to the lack of SOCS-3, we used an siRNA approach to specifically knock down SOCS-3 mRNA in A549 cells. Cells were transfected with 150 nM siRNA for 48 h and SOCS-3 protein levels were compared to control transfected samples (Figure 8C, right). Subsequently, cells were infected and progeny virus titers were determined by plaque assay (Figure 8C, left). Similar to the results gained from infected knock out cells, knock down of SOCS-3 resulted in decreased virus titers.

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