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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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Forced expression of influenza viral proteins does not result in impaired IFNβ-induced STAT1 and STAT2 phosphorylation.HEK293 cells were transfected with 500 ng plasmid DNA for expression of viral NP, M, NS, (A) PA, PB1 and PB2 (C) genes (see Table 1 for accession numbers of viral genes) using L2000 according to manufacturer's instructions. Note that the Pol II constructs in use also give rise to expression of second reading frames in the NS, M and PB1 genes (NS2, M2, PB1-F2). 48 h post transfection cells were stimulated with human IFNβ (500 U/ml) for 15 minutes. Total protein lysates were subjected to Western blot analysis using anti-phospho-STAT1, anti-phospho-STAT2, anti-STAT1 antibodies. Expression of influenza viral proteins was monitored with antibodies against NP, M1, NS1, PA, PB1 or PB2. (E) HEK293 cells were infected with the human influenza virus PR8 (H1N1) (MOI = 5) for the indicated time points and were subsequently stimulated for 15 min with either human IFNβ at a concentration of 100 U/ml. Cell lysates were subjected to Western blots as described. (B, D, F) Quantification of relative pSTAT1 and pSTAT2 band intensities in A, C and E using AIDA software and 2D densitometry (Fuji).
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ppat-1000196-g002: Forced expression of influenza viral proteins does not result in impaired IFNβ-induced STAT1 and STAT2 phosphorylation.HEK293 cells were transfected with 500 ng plasmid DNA for expression of viral NP, M, NS, (A) PA, PB1 and PB2 (C) genes (see Table 1 for accession numbers of viral genes) using L2000 according to manufacturer's instructions. Note that the Pol II constructs in use also give rise to expression of second reading frames in the NS, M and PB1 genes (NS2, M2, PB1-F2). 48 h post transfection cells were stimulated with human IFNβ (500 U/ml) for 15 minutes. Total protein lysates were subjected to Western blot analysis using anti-phospho-STAT1, anti-phospho-STAT2, anti-STAT1 antibodies. Expression of influenza viral proteins was monitored with antibodies against NP, M1, NS1, PA, PB1 or PB2. (E) HEK293 cells were infected with the human influenza virus PR8 (H1N1) (MOI = 5) for the indicated time points and were subsequently stimulated for 15 min with either human IFNβ at a concentration of 100 U/ml. Cell lysates were subjected to Western blots as described. (B, D, F) Quantification of relative pSTAT1 and pSTAT2 band intensities in A, C and E using AIDA software and 2D densitometry (Fuji).

Mentions: Phosphorylation of STAT1 and STAT2 by members of the JAK tyrosine kinase family is a prerequisite for activation of these transcription factors to drive type I IFN-induced gene expression. Therefore, we analyzed whether STAT phosphorylation patterns are altered in influenza A virus infected cells that were stimulated with IFN at different time points post infection (p.i.). The human alveolar epithelial cell line A549 was infected with the influenza A virus strain A/Puerto-Rico/8/34 (H1N1) (PR8) (Figure 1A). Cells were subsequently stimulated with IFNβ at given time-points p.i. and STAT phosphorylation was assessed in Western blots. Both STAT1 and STAT2 were readily phosphorylated upon cytokine stimulation in uninfected cells or in infected cells up to 4 h p.i. (Figure 1A). Furthermore, virus infection alone resulted in a significant induction of STAT phosphorylation 4–6 h p.i., presumably caused by virus-induced IFN expression. However, at later time points (6–10 h p.i.), in A549 cells both virus- and IFN-induced STAT1 and STAT2 phosphorylation was markedly reduced (Figure 1A). Similar patterns were observed upon stimulation of cells with IFNα or upon infection with other viruses, such as the human influenza virus A/Victora/3/75 (H3N2) (data not shown). In addition, this phenomenon could also be detected in other epithelial cells such as the human embryonic kidney cell line HEK293 (Figure 2E) or the human umbilical vein endothelial cells (HUVEC) (Figure S1B). Inhibition was not caused by indirect disturbing effects on cellular metabolism or enzyme activities due to ongoing virus replication, since IFNγ-induced STAT1 phosphorylation was not affected at all (Figure 1C). Finally, involvement of any auto- or paracrine action of virus-induced type I IFN could be ruled out, as the inhibitory effect was also observed in Vero cells lacking functional type I IFN genes (Figure 1E).


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)

Forced expression of influenza viral proteins does not result in impaired IFNβ-induced STAT1 and STAT2 phosphorylation.HEK293 cells were transfected with 500 ng plasmid DNA for expression of viral NP, M, NS, (A) PA, PB1 and PB2 (C) genes (see Table 1 for accession numbers of viral genes) using L2000 according to manufacturer's instructions. Note that the Pol II constructs in use also give rise to expression of second reading frames in the NS, M and PB1 genes (NS2, M2, PB1-F2). 48 h post transfection cells were stimulated with human IFNβ (500 U/ml) for 15 minutes. Total protein lysates were subjected to Western blot analysis using anti-phospho-STAT1, anti-phospho-STAT2, anti-STAT1 antibodies. Expression of influenza viral proteins was monitored with antibodies against NP, M1, NS1, PA, PB1 or PB2. (E) HEK293 cells were infected with the human influenza virus PR8 (H1N1) (MOI = 5) for the indicated time points and were subsequently stimulated for 15 min with either human IFNβ at a concentration of 100 U/ml. Cell lysates were subjected to Western blots as described. (B, D, F) Quantification of relative pSTAT1 and pSTAT2 band intensities in A, C and E using AIDA software and 2D densitometry (Fuji).
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getmorefigures.php?uid=PMC2572141&req=5

ppat-1000196-g002: Forced expression of influenza viral proteins does not result in impaired IFNβ-induced STAT1 and STAT2 phosphorylation.HEK293 cells were transfected with 500 ng plasmid DNA for expression of viral NP, M, NS, (A) PA, PB1 and PB2 (C) genes (see Table 1 for accession numbers of viral genes) using L2000 according to manufacturer's instructions. Note that the Pol II constructs in use also give rise to expression of second reading frames in the NS, M and PB1 genes (NS2, M2, PB1-F2). 48 h post transfection cells were stimulated with human IFNβ (500 U/ml) for 15 minutes. Total protein lysates were subjected to Western blot analysis using anti-phospho-STAT1, anti-phospho-STAT2, anti-STAT1 antibodies. Expression of influenza viral proteins was monitored with antibodies against NP, M1, NS1, PA, PB1 or PB2. (E) HEK293 cells were infected with the human influenza virus PR8 (H1N1) (MOI = 5) for the indicated time points and were subsequently stimulated for 15 min with either human IFNβ at a concentration of 100 U/ml. Cell lysates were subjected to Western blots as described. (B, D, F) Quantification of relative pSTAT1 and pSTAT2 band intensities in A, C and E using AIDA software and 2D densitometry (Fuji).
Mentions: Phosphorylation of STAT1 and STAT2 by members of the JAK tyrosine kinase family is a prerequisite for activation of these transcription factors to drive type I IFN-induced gene expression. Therefore, we analyzed whether STAT phosphorylation patterns are altered in influenza A virus infected cells that were stimulated with IFN at different time points post infection (p.i.). The human alveolar epithelial cell line A549 was infected with the influenza A virus strain A/Puerto-Rico/8/34 (H1N1) (PR8) (Figure 1A). Cells were subsequently stimulated with IFNβ at given time-points p.i. and STAT phosphorylation was assessed in Western blots. Both STAT1 and STAT2 were readily phosphorylated upon cytokine stimulation in uninfected cells or in infected cells up to 4 h p.i. (Figure 1A). Furthermore, virus infection alone resulted in a significant induction of STAT phosphorylation 4–6 h p.i., presumably caused by virus-induced IFN expression. However, at later time points (6–10 h p.i.), in A549 cells both virus- and IFN-induced STAT1 and STAT2 phosphorylation was markedly reduced (Figure 1A). Similar patterns were observed upon stimulation of cells with IFNα or upon infection with other viruses, such as the human influenza virus A/Victora/3/75 (H3N2) (data not shown). In addition, this phenomenon could also be detected in other epithelial cells such as the human embryonic kidney cell line HEK293 (Figure 2E) or the human umbilical vein endothelial cells (HUVEC) (Figure S1B). Inhibition was not caused by indirect disturbing effects on cellular metabolism or enzyme activities due to ongoing virus replication, since IFNγ-induced STAT1 phosphorylation was not affected at all (Figure 1C). Finally, involvement of any auto- or paracrine action of virus-induced type I IFN could be ruled out, as the inhibitory effect was also observed in Vero cells lacking functional type I IFN genes (Figure 1E).

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