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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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SOCS-3 mRNA transcription is induced in an NF-κB dependent manner.A549 wt cells or A549 cells stably transduced with empty vector, dominant negative MKK6Ala or IKK2KD were either infected with PR8 for 3 hours (MOI = 5) (A and B) or with the influenza A virus mutant ΔNS1 and the corresponding isogenic wild type virus (G and H) [74] or transfected for 3 hours with RNA from infected or uninfected A549 cells (C–F). (E and F) A549 cells were treated with 40 µM of the NF-κB inhibitor BAY 11-7085 30 minutes prior transfection of RNA from infected (“viral RNA”) or uninfected A549 wt cells (“cellular RNA”). In all experiments shown total RNA from target cells was isolated and reverse transcribed. cDNA was subjected to quantitative real time PCR. mRNA levels of SOCS3 (A, C, E, G) or IFNβ (B, D, F, H) were assed by specific primers.
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ppat-1000196-g006: SOCS-3 mRNA transcription is induced in an NF-κB dependent manner.A549 wt cells or A549 cells stably transduced with empty vector, dominant negative MKK6Ala or IKK2KD were either infected with PR8 for 3 hours (MOI = 5) (A and B) or with the influenza A virus mutant ΔNS1 and the corresponding isogenic wild type virus (G and H) [74] or transfected for 3 hours with RNA from infected or uninfected A549 cells (C–F). (E and F) A549 cells were treated with 40 µM of the NF-κB inhibitor BAY 11-7085 30 minutes prior transfection of RNA from infected (“viral RNA”) or uninfected A549 wt cells (“cellular RNA”). In all experiments shown total RNA from target cells was isolated and reverse transcribed. cDNA was subjected to quantitative real time PCR. mRNA levels of SOCS3 (A, C, E, G) or IFNβ (B, D, F, H) were assed by specific primers.

Mentions: So far, our data suggest that influenza virus-induced transcriptional upregulation of the SOCS-3 gene is not mediated by the autoregulatory action of type I IFNs (Figure 4D and 4F) but is directly induced through accumulation of viral RNA during infection. This raises the question, which RNA-induced signaling pathways are responsible for SOCS-3 expression. The MKK/p38 mitogen activated protein kinase (MAPK) pathway [47]–[49] as well as the IκB kinase (IKK)/nuclear factor of κB (NF-κB) cascade [50]–[52] are both known to be activated by RNA or influenza virus infection and to be involved in the control of SOCS-3 expression. To assess whether the MKK6/p38- or the IKK/NF-κB-module is required for SOCS-3 gene induction, we generated A549 cell lines expressing dominant negative forms of either MKK6 (MKK6Ala) or IKK2 (IKK2KD) (Figure 6A to 6D). These mutants have been previously shown to efficiently block p38 or NF-κB signaling, respectively [52]–[54]. To monitor SOCS-3 gene induction, wild type, vector or mutant expressing cell lines were infected with PR8 (Figure 6A) or stimulated with RNA from virally infected or uninfected A549 cells (Figure 6C). Induction of IFNβ mRNA was monitored as a control (Figure 6B and 6D). While MKK6Ala expression did not result in significant reduction of SOCS-3 in either infected (Figure 6A) or RNA-stimulated cells (Figure 6C), transcription is markedly reduced in IKK2KD expressing cell lines. To obtain independent evidence for NF-κB dependence of SOCS-3 gene transcription, A549 wild type cells were incubated with the NF-κB specific inhibitor BAY 11-7085 prior to stimulation with RNA from virally infected or uninfected A549 cells (Figure 6E). Again, IFNβ mRNA levels were assessed for control purposes (Figure 6F). Both, SOCS-3 and IFNβ mRNA levels were strongly reduced in BAY 11-7085 treated cells. This indicates that virus-induced SOCS-3 expression strongly depends on IKK2 and NF-κB activation, while the MKK6/p38 appears not to play a prominent role.


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)

SOCS-3 mRNA transcription is induced in an NF-κB dependent manner.A549 wt cells or A549 cells stably transduced with empty vector, dominant negative MKK6Ala or IKK2KD were either infected with PR8 for 3 hours (MOI = 5) (A and B) or with the influenza A virus mutant ΔNS1 and the corresponding isogenic wild type virus (G and H) [74] or transfected for 3 hours with RNA from infected or uninfected A549 cells (C–F). (E and F) A549 cells were treated with 40 µM of the NF-κB inhibitor BAY 11-7085 30 minutes prior transfection of RNA from infected (“viral RNA”) or uninfected A549 wt cells (“cellular RNA”). In all experiments shown total RNA from target cells was isolated and reverse transcribed. cDNA was subjected to quantitative real time PCR. mRNA levels of SOCS3 (A, C, E, G) or IFNβ (B, D, F, H) were assed by specific primers.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2572141&req=5

ppat-1000196-g006: SOCS-3 mRNA transcription is induced in an NF-κB dependent manner.A549 wt cells or A549 cells stably transduced with empty vector, dominant negative MKK6Ala or IKK2KD were either infected with PR8 for 3 hours (MOI = 5) (A and B) or with the influenza A virus mutant ΔNS1 and the corresponding isogenic wild type virus (G and H) [74] or transfected for 3 hours with RNA from infected or uninfected A549 cells (C–F). (E and F) A549 cells were treated with 40 µM of the NF-κB inhibitor BAY 11-7085 30 minutes prior transfection of RNA from infected (“viral RNA”) or uninfected A549 wt cells (“cellular RNA”). In all experiments shown total RNA from target cells was isolated and reverse transcribed. cDNA was subjected to quantitative real time PCR. mRNA levels of SOCS3 (A, C, E, G) or IFNβ (B, D, F, H) were assed by specific primers.
Mentions: So far, our data suggest that influenza virus-induced transcriptional upregulation of the SOCS-3 gene is not mediated by the autoregulatory action of type I IFNs (Figure 4D and 4F) but is directly induced through accumulation of viral RNA during infection. This raises the question, which RNA-induced signaling pathways are responsible for SOCS-3 expression. The MKK/p38 mitogen activated protein kinase (MAPK) pathway [47]–[49] as well as the IκB kinase (IKK)/nuclear factor of κB (NF-κB) cascade [50]–[52] are both known to be activated by RNA or influenza virus infection and to be involved in the control of SOCS-3 expression. To assess whether the MKK6/p38- or the IKK/NF-κB-module is required for SOCS-3 gene induction, we generated A549 cell lines expressing dominant negative forms of either MKK6 (MKK6Ala) or IKK2 (IKK2KD) (Figure 6A to 6D). These mutants have been previously shown to efficiently block p38 or NF-κB signaling, respectively [52]–[54]. To monitor SOCS-3 gene induction, wild type, vector or mutant expressing cell lines were infected with PR8 (Figure 6A) or stimulated with RNA from virally infected or uninfected A549 cells (Figure 6C). Induction of IFNβ mRNA was monitored as a control (Figure 6B and 6D). While MKK6Ala expression did not result in significant reduction of SOCS-3 in either infected (Figure 6A) or RNA-stimulated cells (Figure 6C), transcription is markedly reduced in IKK2KD expressing cell lines. To obtain independent evidence for NF-κB dependence of SOCS-3 gene transcription, A549 wild type cells were incubated with the NF-κB specific inhibitor BAY 11-7085 prior to stimulation with RNA from virally infected or uninfected A549 cells (Figure 6E). Again, IFNβ mRNA levels were assessed for control purposes (Figure 6F). Both, SOCS-3 and IFNβ mRNA levels were strongly reduced in BAY 11-7085 treated cells. This indicates that virus-induced SOCS-3 expression strongly depends on IKK2 and NF-κB activation, while the MKK6/p38 appears not to play a prominent role.

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