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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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Influenza A virus results in early SOCS-3 gene induction in an IFNβ-independent manner.A549 cells were infected with PR8 (MOI = 5) (A, B, C) or stimulated with 100 U/ml human IFNβ (D) for the indicated time points. (F) A549 cells stably expressing IFNAR II-1 mRNA specific shRNA or control empty vector were infected with PR8 (MOI = 5) for 3 hours. Cells were lysed and RNA was subjected to reverse transcription. cDNA was analyzed in quantitative real time PCR to assess mRNA amounts of IFNβ (B), SOCS-1 (A), SOCS-3, (A, D, F), OAS1 (D) or MxA (D). 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. To detect SOCS-3 protein expression (C) cells were infected for time points indicated or left uninfected. Total cell lysate was subjected to Western blot analysis using anti-SOCS-3 antibody. To allow better comparison of SOCS-3 protein expression and STAT1 phosphorylation phospho-STAT1 and STAT1 Western blots from figure 1A are shown again here. (E) To functionally test effective knock down of the IFNAR, A459 wild type, A549 vector control cells or A549 cells stably expressing IFNAR II-1specific shRNA were stimulated with human IFNβ (100 U/ml) for 15 min. Subsequently cells were lysed and levels of phospho-STAT1 were determined by Western blotting using specific antibodies. In addition, the relative pSTAT1 band intensities were quantified.
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ppat-1000196-g004: Influenza A virus results in early SOCS-3 gene induction in an IFNβ-independent manner.A549 cells were infected with PR8 (MOI = 5) (A, B, C) or stimulated with 100 U/ml human IFNβ (D) for the indicated time points. (F) A549 cells stably expressing IFNAR II-1 mRNA specific shRNA or control empty vector were infected with PR8 (MOI = 5) for 3 hours. Cells were lysed and RNA was subjected to reverse transcription. cDNA was analyzed in quantitative real time PCR to assess mRNA amounts of IFNβ (B), SOCS-1 (A), SOCS-3, (A, D, F), OAS1 (D) or MxA (D). 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. To detect SOCS-3 protein expression (C) cells were infected for time points indicated or left uninfected. Total cell lysate was subjected to Western blot analysis using anti-SOCS-3 antibody. To allow better comparison of SOCS-3 protein expression and STAT1 phosphorylation phospho-STAT1 and STAT1 Western blots from figure 1A are shown again here. (E) To functionally test effective knock down of the IFNAR, A459 wild type, A549 vector control cells or A549 cells stably expressing IFNAR II-1specific shRNA were stimulated with human IFNβ (100 U/ml) for 15 min. Subsequently cells were lysed and levels of phospho-STAT1 were determined by Western blotting using specific antibodies. In addition, the relative pSTAT1 band intensities were quantified.

Mentions: Phosphorylation of STATs in the IFNβ signaling cascade may not only be counter-regulated by phosphatases but also by other cellular factors, such as proteins of the suppressors of cytokine signaling (SOCS) family. Action of these proteins is mainly controlled on the level of transcriptional activation. SOCS proteins are described to have high affinity for JAK and STAT proteins and to inhibit the transmission of IFNα and IFNβ induced signaling [44],[45]. To examine whether expression of SOCS genes is induced in influenza virus infected cells, A549 cells were infected with PR8 for different time points. Subsequently total RNA was analyzed for the amount of SOCS-1 and SOCS-3 mRNA by means of quantitative real time-PCR (qRT-PCR). The mRNA levels of SOCS-1 and SOCS-3 differed notably in the time course (Figure 4A). While SOCS-3 mRNA is strongly and transiently elevated in the early phases of infection, SOCS-1 gene transcription is only significantly induced 15 h p.i.. Elevated SOCS-3 mRNA levels were also observed in other host cell types, such as HUVEC starting 3 h p.i. (Figure S1A). Although elevation of SOCS-3 mRNA levels in infected cells was rather transient, there appears to be a robust induction on protein level (Figure 4C). First detected at 4 h p.i., SOCS-3 protein levels increased and stayed on a high level throughout the observation period. Strikingly, expression kinetics of the SOCS-3 protein perfectly matched the kinetics of virus-induced inhibition of STAT1 phosphorylation (Figure 4C), indicating that both processes are functionally linked.


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)

Influenza A virus results in early SOCS-3 gene induction in an IFNβ-independent manner.A549 cells were infected with PR8 (MOI = 5) (A, B, C) or stimulated with 100 U/ml human IFNβ (D) for the indicated time points. (F) A549 cells stably expressing IFNAR II-1 mRNA specific shRNA or control empty vector were infected with PR8 (MOI = 5) for 3 hours. Cells were lysed and RNA was subjected to reverse transcription. cDNA was analyzed in quantitative real time PCR to assess mRNA amounts of IFNβ (B), SOCS-1 (A), SOCS-3, (A, D, F), OAS1 (D) or MxA (D). 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. To detect SOCS-3 protein expression (C) cells were infected for time points indicated or left uninfected. Total cell lysate was subjected to Western blot analysis using anti-SOCS-3 antibody. To allow better comparison of SOCS-3 protein expression and STAT1 phosphorylation phospho-STAT1 and STAT1 Western blots from figure 1A are shown again here. (E) To functionally test effective knock down of the IFNAR, A459 wild type, A549 vector control cells or A549 cells stably expressing IFNAR II-1specific shRNA were stimulated with human IFNβ (100 U/ml) for 15 min. Subsequently cells were lysed and levels of phospho-STAT1 were determined by Western blotting using specific antibodies. In addition, the relative pSTAT1 band intensities were quantified.
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getmorefigures.php?uid=PMC2572141&req=5

ppat-1000196-g004: Influenza A virus results in early SOCS-3 gene induction in an IFNβ-independent manner.A549 cells were infected with PR8 (MOI = 5) (A, B, C) or stimulated with 100 U/ml human IFNβ (D) for the indicated time points. (F) A549 cells stably expressing IFNAR II-1 mRNA specific shRNA or control empty vector were infected with PR8 (MOI = 5) for 3 hours. Cells were lysed and RNA was subjected to reverse transcription. cDNA was analyzed in quantitative real time PCR to assess mRNA amounts of IFNβ (B), SOCS-1 (A), SOCS-3, (A, D, F), OAS1 (D) or MxA (D). 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. To detect SOCS-3 protein expression (C) cells were infected for time points indicated or left uninfected. Total cell lysate was subjected to Western blot analysis using anti-SOCS-3 antibody. To allow better comparison of SOCS-3 protein expression and STAT1 phosphorylation phospho-STAT1 and STAT1 Western blots from figure 1A are shown again here. (E) To functionally test effective knock down of the IFNAR, A459 wild type, A549 vector control cells or A549 cells stably expressing IFNAR II-1specific shRNA were stimulated with human IFNβ (100 U/ml) for 15 min. Subsequently cells were lysed and levels of phospho-STAT1 were determined by Western blotting using specific antibodies. In addition, the relative pSTAT1 band intensities were quantified.
Mentions: Phosphorylation of STATs in the IFNβ signaling cascade may not only be counter-regulated by phosphatases but also by other cellular factors, such as proteins of the suppressors of cytokine signaling (SOCS) family. Action of these proteins is mainly controlled on the level of transcriptional activation. SOCS proteins are described to have high affinity for JAK and STAT proteins and to inhibit the transmission of IFNα and IFNβ induced signaling [44],[45]. To examine whether expression of SOCS genes is induced in influenza virus infected cells, A549 cells were infected with PR8 for different time points. Subsequently total RNA was analyzed for the amount of SOCS-1 and SOCS-3 mRNA by means of quantitative real time-PCR (qRT-PCR). The mRNA levels of SOCS-1 and SOCS-3 differed notably in the time course (Figure 4A). While SOCS-3 mRNA is strongly and transiently elevated in the early phases of infection, SOCS-1 gene transcription is only significantly induced 15 h p.i.. Elevated SOCS-3 mRNA levels were also observed in other host cell types, such as HUVEC starting 3 h p.i. (Figure S1A). Although elevation of SOCS-3 mRNA levels in infected cells was rather transient, there appears to be a robust induction on protein level (Figure 4C). First detected at 4 h p.i., SOCS-3 protein levels increased and stayed on a high level throughout the observation period. Strikingly, expression kinetics of the SOCS-3 protein perfectly matched the kinetics of virus-induced inhibition of STAT1 phosphorylation (Figure 4C), indicating that both processes are functionally linked.

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