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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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A model for detection of WNV and IFN-α/β gene activation in MEF.(1). The host through recognition of an as yet undefined viral RNA PAMP in the cytoplasm detects WNV. RIG-I acts as the primary PRR sensor for WNV during the early stages of infection. RIG-I activation promotes association with IPS-1, which leads to recruitment of TRAF3 and TBK1, and phosphorylation of IRF-3. NF-κB and ATF-2/c-Jun and the small amounts of constitutively expressed IRF-7 may also be activated via this IPS-1-dependent pathway. IRF-3, IRF-7 NF-κB, ATF-2/c-Jun translocate to the nucleus, bind the IFN-β gene promoter and promote transcription. Secretion of IFN-β by infected cells during this early phase results in autocrine and paracrine type I IFN signaling through binding of the IFN-αβR. (2). Activation of IFN-αβR results in phosphorylation of JAK1 and Tyk2, which activate STAT1 and STAT2 leading to formation of the heterotrimer ISGF3 (STAT1, STAT2 and IRF-9). Nuclear translocation and promoter binding of ISGF3 upregulates hundreds of different ISG, including IRF-7. (3). During a later phase of infection, detection of WNV in MEF also relies on MDA5 and PKR. Recruitment of TRAF3 and TRAF6 activates IRF-3 and IRF-7. NF-κB and ATF-2/c-Jun are also activated via an as yet undefined mechanism. Subsequently, IRF-3, IRF-7, NF-κB, and ATF-2/c-Jun translocate to the nucleus, bind the IFN-β gene promoter and induce optimal transcription. Induction of IFN-α genes occurs through TRAF6 and the transcriptional activation of IRF-7.
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ppat-1000607-g012: A model for detection of WNV and IFN-α/β gene activation in MEF.(1). The host through recognition of an as yet undefined viral RNA PAMP in the cytoplasm detects WNV. RIG-I acts as the primary PRR sensor for WNV during the early stages of infection. RIG-I activation promotes association with IPS-1, which leads to recruitment of TRAF3 and TBK1, and phosphorylation of IRF-3. NF-κB and ATF-2/c-Jun and the small amounts of constitutively expressed IRF-7 may also be activated via this IPS-1-dependent pathway. IRF-3, IRF-7 NF-κB, ATF-2/c-Jun translocate to the nucleus, bind the IFN-β gene promoter and promote transcription. Secretion of IFN-β by infected cells during this early phase results in autocrine and paracrine type I IFN signaling through binding of the IFN-αβR. (2). Activation of IFN-αβR results in phosphorylation of JAK1 and Tyk2, which activate STAT1 and STAT2 leading to formation of the heterotrimer ISGF3 (STAT1, STAT2 and IRF-9). Nuclear translocation and promoter binding of ISGF3 upregulates hundreds of different ISG, including IRF-7. (3). During a later phase of infection, detection of WNV in MEF also relies on MDA5 and PKR. Recruitment of TRAF3 and TRAF6 activates IRF-3 and IRF-7. NF-κB and ATF-2/c-Jun are also activated via an as yet undefined mechanism. Subsequently, IRF-3, IRF-7, NF-κB, and ATF-2/c-Jun translocate to the nucleus, bind the IFN-β gene promoter and induce optimal transcription. Induction of IFN-α genes occurs through TRAF6 and the transcriptional activation of IRF-7.

Mentions: Further dissection of the IPS-1-dependent signaling pathway in MEF showed a differential role of TRAF3 and TRAF6 in regulating IFN-β responses. Based on studies with deficient MEF, TRAF3 contributes dominantly to the early phase of IFN-β production, likely by recruiting TBK1. Consistent with this, others have shown that TBK1−/− MEF infected with Sendai virus have a reduced IFN-β response [69]. These results also agree with our unpublished data in IRF-3−/− MEF; a deficiency of IRF-3, which is activated primarily by TRAF3 [49], results in a blunted IFN-α/β response. In contrast to TRAF3, TRAF6 had a more dominant function in sustaining the type I IFN positive feedback. Thus, after WNV infection, the type I IFN amplification loop appears mediated by signals downstream of the IFN-αβR receptor, which may include induction and/or activation of IRF-7 and TRAF6. Since TRAF3 and TRAF6 activate IRF-3, NF-κB, and p38 in MEF [49],[52], induction of the late phase of type I IFN may require these signaling adaptor molecules to activate the four components (IRF-3, IRF-7, NF-κB and ATF-2/c-Jun) of the enhanceosome. Based on the data presented here and elsewhere [16], we propose a model for host detection of WNV, signaling through IPS-1 and key adaptor molecules, and transcriptional activation of the IFN-α and β genes at early and late times after infection of MEF (Fig 12).


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)

A model for detection of WNV and IFN-α/β gene activation in MEF.(1). The host through recognition of an as yet undefined viral RNA PAMP in the cytoplasm detects WNV. RIG-I acts as the primary PRR sensor for WNV during the early stages of infection. RIG-I activation promotes association with IPS-1, which leads to recruitment of TRAF3 and TBK1, and phosphorylation of IRF-3. NF-κB and ATF-2/c-Jun and the small amounts of constitutively expressed IRF-7 may also be activated via this IPS-1-dependent pathway. IRF-3, IRF-7 NF-κB, ATF-2/c-Jun translocate to the nucleus, bind the IFN-β gene promoter and promote transcription. Secretion of IFN-β by infected cells during this early phase results in autocrine and paracrine type I IFN signaling through binding of the IFN-αβR. (2). Activation of IFN-αβR results in phosphorylation of JAK1 and Tyk2, which activate STAT1 and STAT2 leading to formation of the heterotrimer ISGF3 (STAT1, STAT2 and IRF-9). Nuclear translocation and promoter binding of ISGF3 upregulates hundreds of different ISG, including IRF-7. (3). During a later phase of infection, detection of WNV in MEF also relies on MDA5 and PKR. Recruitment of TRAF3 and TRAF6 activates IRF-3 and IRF-7. NF-κB and ATF-2/c-Jun are also activated via an as yet undefined mechanism. Subsequently, IRF-3, IRF-7, NF-κB, and ATF-2/c-Jun translocate to the nucleus, bind the IFN-β gene promoter and induce optimal transcription. Induction of IFN-α genes occurs through TRAF6 and the transcriptional activation of IRF-7.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2747008&req=5

ppat-1000607-g012: A model for detection of WNV and IFN-α/β gene activation in MEF.(1). The host through recognition of an as yet undefined viral RNA PAMP in the cytoplasm detects WNV. RIG-I acts as the primary PRR sensor for WNV during the early stages of infection. RIG-I activation promotes association with IPS-1, which leads to recruitment of TRAF3 and TBK1, and phosphorylation of IRF-3. NF-κB and ATF-2/c-Jun and the small amounts of constitutively expressed IRF-7 may also be activated via this IPS-1-dependent pathway. IRF-3, IRF-7 NF-κB, ATF-2/c-Jun translocate to the nucleus, bind the IFN-β gene promoter and promote transcription. Secretion of IFN-β by infected cells during this early phase results in autocrine and paracrine type I IFN signaling through binding of the IFN-αβR. (2). Activation of IFN-αβR results in phosphorylation of JAK1 and Tyk2, which activate STAT1 and STAT2 leading to formation of the heterotrimer ISGF3 (STAT1, STAT2 and IRF-9). Nuclear translocation and promoter binding of ISGF3 upregulates hundreds of different ISG, including IRF-7. (3). During a later phase of infection, detection of WNV in MEF also relies on MDA5 and PKR. Recruitment of TRAF3 and TRAF6 activates IRF-3 and IRF-7. NF-κB and ATF-2/c-Jun are also activated via an as yet undefined mechanism. Subsequently, IRF-3, IRF-7, NF-κB, and ATF-2/c-Jun translocate to the nucleus, bind the IFN-β gene promoter and induce optimal transcription. Induction of IFN-α genes occurs through TRAF6 and the transcriptional activation of IRF-7.
Mentions: Further dissection of the IPS-1-dependent signaling pathway in MEF showed a differential role of TRAF3 and TRAF6 in regulating IFN-β responses. Based on studies with deficient MEF, TRAF3 contributes dominantly to the early phase of IFN-β production, likely by recruiting TBK1. Consistent with this, others have shown that TBK1−/− MEF infected with Sendai virus have a reduced IFN-β response [69]. These results also agree with our unpublished data in IRF-3−/− MEF; a deficiency of IRF-3, which is activated primarily by TRAF3 [49], results in a blunted IFN-α/β response. In contrast to TRAF3, TRAF6 had a more dominant function in sustaining the type I IFN positive feedback. Thus, after WNV infection, the type I IFN amplification loop appears mediated by signals downstream of the IFN-αβR receptor, which may include induction and/or activation of IRF-7 and TRAF6. Since TRAF3 and TRAF6 activate IRF-3, NF-κB, and p38 in MEF [49],[52], induction of the late phase of type I IFN may require these signaling adaptor molecules to activate the four components (IRF-3, IRF-7, NF-κB and ATF-2/c-Jun) of the enhanceosome. Based on the data presented here and elsewhere [16], we propose a model for host detection of WNV, signaling through IPS-1 and key adaptor molecules, and transcriptional activation of the IFN-α and β genes at early and late times after infection of MEF (Fig 12).

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