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Induction of IFN-beta and the innate antiviral response in myeloid cells occurs through an IPS-1-dependent signal that does not require IRF-3 and IRF-7.

Daffis S, Suthar MS, Szretter KJ, Gale M, Diamond MS - PLoS Pathog. (2009)

Bottom Line: Ex vivo analysis showed complete ablation of the IFN-alpha response in DKO fibroblasts, macrophages, dendritic cells, and cortical neurons and a substantial decrease of the IFN-beta response in DKO fibroblasts and cortical neurons.However, pharmacological inhibition of NF-kappaB and ATF-2/c-Jun, the two other known components of the IFN-beta enhanceosome, strongly reduced IFN-beta gene transcription in the DKO dendritic cells.Overall, our experiments suggest that, unlike fibroblasts and cortical neurons, IFN-beta gene regulation after WNV infection in myeloid cells is IPS-1-dependent but does not require full occupancy of the IFN-beta enhanceosome by canonical constituent transcriptional factors.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, United States of America.

ABSTRACT
Interferon regulatory factors (IRF)-3 and IRF-7 are master transcriptional factors that regulate type I IFN gene (IFN-alpha/beta) induction and innate immune defenses after virus infection. Prior studies in mice with single deletions of the IRF-3 or IRF-7 genes showed increased vulnerability to West Nile virus (WNV) infection. Whereas mice and cells lacking IRF-7 showed reduced IFN-alpha levels after WNV infection, those lacking IRF-3 or IRF-7 had relatively normal IFN-b production. Here, we generated IRF-3(-/-)x IRF-7(-/-) double knockout (DKO) mice, analyzed WNV pathogenesis, IFN responses, and signaling of innate defenses. Compared to wild type mice, the DKO mice exhibited a blunted but not abrogated systemic IFN response and sustained uncontrolled WNV replication leading to rapid mortality. Ex vivo analysis showed complete ablation of the IFN-alpha response in DKO fibroblasts, macrophages, dendritic cells, and cortical neurons and a substantial decrease of the IFN-beta response in DKO fibroblasts and cortical neurons. In contrast, the IFN-beta response was minimally diminished in DKO macrophages and dendritic cells. However, pharmacological inhibition of NF-kappaB and ATF-2/c-Jun, the two other known components of the IFN-beta enhanceosome, strongly reduced IFN-beta gene transcription in the DKO dendritic cells. Finally, a genetic deficiency of IPS-1, an adaptor involved in RIG-I- and MDA5-mediated antiviral signaling, completely abolished the IFN-beta response after WNV infection. Overall, our experiments suggest that, unlike fibroblasts and cortical neurons, IFN-beta gene regulation after WNV infection in myeloid cells is IPS-1-dependent but does not require full occupancy of the IFN-beta enhanceosome by canonical constituent transcriptional factors.

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IRF-3 and IRF-7 restrict WNV infection by regulating the IFN-α/β response and ISG expression in primary cortical neurons.A. Primary cortical neurons generated from wild type or DKO mice were infected at an MOI of 0.001 and virus production was evaluated at the indicated times by plaque assay. Values are an average of triplicate samples generated from three independent experiments. Asterisks indicate values that are statistically significant (***, P<0.0001). B and C. Levels of (B) IFN-α and (C) IFN-β mRNA in WNV-infected cortical neurons were measured by qRT-PCR as described in the legend of Figure 3. D. Whole cell lysates were generated at the indicated times from wild type or DKO MEF that were uninfected (Un) or infected with WNV (W). Protein levels of ISG54, RIG-I, and MDA5 were examined by immunoblot analysis. The data is the average of at least three independent experiments performed in quadruplicate (***, P<0.0001).
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ppat-1000607-g004: IRF-3 and IRF-7 restrict WNV infection by regulating the IFN-α/β response and ISG expression in primary cortical neurons.A. Primary cortical neurons generated from wild type or DKO mice were infected at an MOI of 0.001 and virus production was evaluated at the indicated times by plaque assay. Values are an average of triplicate samples generated from three independent experiments. Asterisks indicate values that are statistically significant (***, P<0.0001). B and C. Levels of (B) IFN-α and (C) IFN-β mRNA in WNV-infected cortical neurons were measured by qRT-PCR as described in the legend of Figure 3. D. Whole cell lysates were generated at the indicated times from wild type or DKO MEF that were uninfected (Un) or infected with WNV (W). Protein levels of ISG54, RIG-I, and MDA5 were examined by immunoblot analysis. The data is the average of at least three independent experiments performed in quadruplicate (***, P<0.0001).

Mentions: To evaluate the combined roles of IRF-3 and IRF-7 in control of viral replication and the IFN response in neuronal cells, we assessed WNV infection of primary cortical neurons isolated from DKO mice. Analysis of viral growth kinetics confirmed that IRF-3 and IRF-7 restrict WNV replication as a ∼5 to 6-fold increase (P<0.0001) in viral titer was observed at 24 h and 48 h compared to wild type cells (Fig 4A). Somewhat surprisingly, the DKO neurons did not show increased replication relative to cells lacking either IRF-3 or IRF-7 (data not shown and [21],[22]). The relatively modest replication phenotype in the absence of IRF-3 and IRF-7 is consistent with only a small IFN-dependent antiviral effect in these cells: IFN-α or -β pre-treatment inhibits WNV infection in cortical neurons a maximum of 5 to 8-fold [13]. Analysis of the IFN response of WNV-infected DKO neurons showed a complete ablation of the IFN-α gene induction confirming results with the IRF-7−/− cortical neurons [22] (Fig 4B). In contrast, and unlike that observed with MEF or other primary myeloid cells (see below), induction of IFN-β mRNA in DKO cortical neurons was also entirely abolished (Fig 4C). Consistent with this, analysis of ISG in DKO neurons showed a complete loss of induction of ISG54, RIG-I and MDA5 following WNV infection (Fig 4D). Thus, in cortical neurons coordinate signals through the transcriptional regulators IRF-3 and IRF-7 are required for IFN-α and IFN-β gene induction after WNV infection.


Induction of IFN-beta and the innate antiviral response in myeloid cells occurs through an IPS-1-dependent signal that does not require IRF-3 and IRF-7.

Daffis S, Suthar MS, Szretter KJ, Gale M, Diamond MS - PLoS Pathog. (2009)

IRF-3 and IRF-7 restrict WNV infection by regulating the IFN-α/β response and ISG expression in primary cortical neurons.A. Primary cortical neurons generated from wild type or DKO mice were infected at an MOI of 0.001 and virus production was evaluated at the indicated times by plaque assay. Values are an average of triplicate samples generated from three independent experiments. Asterisks indicate values that are statistically significant (***, P<0.0001). B and C. Levels of (B) IFN-α and (C) IFN-β mRNA in WNV-infected cortical neurons were measured by qRT-PCR as described in the legend of Figure 3. D. Whole cell lysates were generated at the indicated times from wild type or DKO MEF that were uninfected (Un) or infected with WNV (W). Protein levels of ISG54, RIG-I, and MDA5 were examined by immunoblot analysis. The data is the average of at least three independent experiments performed in quadruplicate (***, P<0.0001).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2747008&req=5

ppat-1000607-g004: IRF-3 and IRF-7 restrict WNV infection by regulating the IFN-α/β response and ISG expression in primary cortical neurons.A. Primary cortical neurons generated from wild type or DKO mice were infected at an MOI of 0.001 and virus production was evaluated at the indicated times by plaque assay. Values are an average of triplicate samples generated from three independent experiments. Asterisks indicate values that are statistically significant (***, P<0.0001). B and C. Levels of (B) IFN-α and (C) IFN-β mRNA in WNV-infected cortical neurons were measured by qRT-PCR as described in the legend of Figure 3. D. Whole cell lysates were generated at the indicated times from wild type or DKO MEF that were uninfected (Un) or infected with WNV (W). Protein levels of ISG54, RIG-I, and MDA5 were examined by immunoblot analysis. The data is the average of at least three independent experiments performed in quadruplicate (***, P<0.0001).
Mentions: To evaluate the combined roles of IRF-3 and IRF-7 in control of viral replication and the IFN response in neuronal cells, we assessed WNV infection of primary cortical neurons isolated from DKO mice. Analysis of viral growth kinetics confirmed that IRF-3 and IRF-7 restrict WNV replication as a ∼5 to 6-fold increase (P<0.0001) in viral titer was observed at 24 h and 48 h compared to wild type cells (Fig 4A). Somewhat surprisingly, the DKO neurons did not show increased replication relative to cells lacking either IRF-3 or IRF-7 (data not shown and [21],[22]). The relatively modest replication phenotype in the absence of IRF-3 and IRF-7 is consistent with only a small IFN-dependent antiviral effect in these cells: IFN-α or -β pre-treatment inhibits WNV infection in cortical neurons a maximum of 5 to 8-fold [13]. Analysis of the IFN response of WNV-infected DKO neurons showed a complete ablation of the IFN-α gene induction confirming results with the IRF-7−/− cortical neurons [22] (Fig 4B). In contrast, and unlike that observed with MEF or other primary myeloid cells (see below), induction of IFN-β mRNA in DKO cortical neurons was also entirely abolished (Fig 4C). Consistent with this, analysis of ISG in DKO neurons showed a complete loss of induction of ISG54, RIG-I and MDA5 following WNV infection (Fig 4D). Thus, in cortical neurons coordinate signals through the transcriptional regulators IRF-3 and IRF-7 are required for IFN-α and IFN-β gene induction after WNV infection.

Bottom Line: Ex vivo analysis showed complete ablation of the IFN-alpha response in DKO fibroblasts, macrophages, dendritic cells, and cortical neurons and a substantial decrease of the IFN-beta response in DKO fibroblasts and cortical neurons.However, pharmacological inhibition of NF-kappaB and ATF-2/c-Jun, the two other known components of the IFN-beta enhanceosome, strongly reduced IFN-beta gene transcription in the DKO dendritic cells.Overall, our experiments suggest that, unlike fibroblasts and cortical neurons, IFN-beta gene regulation after WNV infection in myeloid cells is IPS-1-dependent but does not require full occupancy of the IFN-beta enhanceosome by canonical constituent transcriptional factors.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, United States of America.

ABSTRACT
Interferon regulatory factors (IRF)-3 and IRF-7 are master transcriptional factors that regulate type I IFN gene (IFN-alpha/beta) induction and innate immune defenses after virus infection. Prior studies in mice with single deletions of the IRF-3 or IRF-7 genes showed increased vulnerability to West Nile virus (WNV) infection. Whereas mice and cells lacking IRF-7 showed reduced IFN-alpha levels after WNV infection, those lacking IRF-3 or IRF-7 had relatively normal IFN-b production. Here, we generated IRF-3(-/-)x IRF-7(-/-) double knockout (DKO) mice, analyzed WNV pathogenesis, IFN responses, and signaling of innate defenses. Compared to wild type mice, the DKO mice exhibited a blunted but not abrogated systemic IFN response and sustained uncontrolled WNV replication leading to rapid mortality. Ex vivo analysis showed complete ablation of the IFN-alpha response in DKO fibroblasts, macrophages, dendritic cells, and cortical neurons and a substantial decrease of the IFN-beta response in DKO fibroblasts and cortical neurons. In contrast, the IFN-beta response was minimally diminished in DKO macrophages and dendritic cells. However, pharmacological inhibition of NF-kappaB and ATF-2/c-Jun, the two other known components of the IFN-beta enhanceosome, strongly reduced IFN-beta gene transcription in the DKO dendritic cells. Finally, a genetic deficiency of IPS-1, an adaptor involved in RIG-I- and MDA5-mediated antiviral signaling, completely abolished the IFN-beta response after WNV infection. Overall, our experiments suggest that, unlike fibroblasts and cortical neurons, IFN-beta gene regulation after WNV infection in myeloid cells is IPS-1-dependent but does not require full occupancy of the IFN-beta enhanceosome by canonical constituent transcriptional factors.

Show MeSH
Related in: MedlinePlus