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DEAF1 is a Pellino1-interacting protein required for interferon production by Sendai virus and double-stranded RNA.

Ordureau A, Enesa K, Nanda S, Le Francois B, Peggie M, Prescott A, Albert PR, Cohen P - J. Biol. Chem. (2013)

Bottom Line: The interaction is independent of the E3 ligase activity of Pellino1, but weakened by the phosphorylation of Pellino1.DEAF1 is also needed for TLR3-dependent IFNβ production.Taken together, our results identify DEAF1 as a novel component of the signal transduction network by which dsRNA of viral origin stimulates IFNβ production.

View Article: PubMed Central - PubMed

Affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, Sir James Black Centre, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom.

ABSTRACT
Double-stranded (ds) RNA of viral origin, a ligand for Melanoma Differentiation-associated gene 5 (MDA5) and Toll-Like Receptor 3 (TLR3), induces the TANK-Binding Kinase 1 (TBK1)-dependent phosphorylation and activation of Interferon Regulatory Factor 3 (IRF3) and the E3 ubiquitin ligase Pellino1, which are required for interferon β (IFNβ) gene transcription. Here, we report that Pellino1 interacts with the transcription factor Deformed Epidermal Autoregulatory Factor 1 (DEAF1). The interaction is independent of the E3 ligase activity of Pellino1, but weakened by the phosphorylation of Pellino1. We show that DEAF1 binds to the IFNβ promoter and to IRF3 and IRF7, that it is required for the transcription of the IFNβ gene and IFNβ secretion in MEFs infected with Sendai virus or transfected with poly(I:C). DEAF1 is also needed for TLR3-dependent IFNβ production. Taken together, our results identify DEAF1 as a novel component of the signal transduction network by which dsRNA of viral origin stimulates IFNβ production.

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The production of IFNβ in Sendai virus infected MEFs is reduced in DEAF1−/− mice.A–I, immortalized MEFs from wild type mice (black bars, black circles in B) or DEAF1−/− mice (white bars, white squares in B) were infected with Sendai virus (100 HA/ml) for the times indicated. Total RNA was extracted and analyzed by qRT-PCR to measure the mRNA encoding IFNβ, IRF7, IFNα4, IFNα6, CXCL10, IκBα, IL-12p40, and the Sendai virus Pgene. The results show fold increase in mRNA relative to the values measured in unstimulated MEFs (± S.D. for triplicate determinations). In panel B, the concentration of IFNβ in the culture medium was measured by ELISA. The results are shown ± S.D. for triplicate determinations. Similar results were obtained in four independent experiments using MEFs immortalized from two different wild type and two different DEAF1−/− mice.
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Figure 2: The production of IFNβ in Sendai virus infected MEFs is reduced in DEAF1−/− mice.A–I, immortalized MEFs from wild type mice (black bars, black circles in B) or DEAF1−/− mice (white bars, white squares in B) were infected with Sendai virus (100 HA/ml) for the times indicated. Total RNA was extracted and analyzed by qRT-PCR to measure the mRNA encoding IFNβ, IRF7, IFNα4, IFNα6, CXCL10, IκBα, IL-12p40, and the Sendai virus Pgene. The results show fold increase in mRNA relative to the values measured in unstimulated MEFs (± S.D. for triplicate determinations). In panel B, the concentration of IFNβ in the culture medium was measured by ELISA. The results are shown ± S.D. for triplicate determinations. Similar results were obtained in four independent experiments using MEFs immortalized from two different wild type and two different DEAF1−/− mice.

Mentions: The MEFs were infected with Sendai virus containing defective-interfering genomes, which strongly stimulate IFNβ production in these cells (21, 22). The dsRNA formed during the replication of these genomes is thought to stimulate IFNβ mainly by activating the MDA5-MAVS-TBK1-IRF3 pathway (23). The production of IFNβ mRNA (Fig. 2A) and IFNβ secretion (Fig. 2B) induced by infection with Sendai virus was reduced considerably in MEFs from DEAF1−/− mice compared with wild type controls. The mRNAs encoding IFNβ-dependent genes, such as IRF7 (Fig. 2C), IFNα4 (Fig. 2D), and IFNα6 (Fig. 2E), as well as CXCL10 (Fig. 2F) an established marker of viral infection, were also suppressed in DEAF1−/− MEFs. In contrast, the mRNAs encoding IκBα (Fig. 2G) or the pro-inflammatory cytokine IL-12p70 (Fig. 2H), which are controlled by the transcription factors NFκB (24) and IRF5 (25), respectively, were little affected. The expression of the Sendai virus nucleocapsid protein was not decreased in MEFs from the knock-in mice (Fig. 2I), indicating that the decreased production of IFNβ in DEAF1−/− MEFs was not due to the failure of the virus to infect the MEFs. Moreover, the IL-1-stimulated activation of p38α MAP kinase, the IKKβ-dependent phosphorylation of p105/NFκB1 or degradation of IκBα (supplemental Fig. S2A), or the IL-1-stimulated production of the mRNA encoding IRF1 (supplemental Fig. S2B) and CXCL10 (supplemental Fig. S2C) were unaffected in DEAF1−/− MEFs. Indeed, the production of IκBα mRNA (supplemental Fig. S2D) and IL-6 mRNA (supplemental Fig. S2E) in DEAF1−/− MEFs was increased slightly. Thus the reduced production of IFNs and IFN-regulated genes in DEAF1−/− MEFs was specific and did not result from a general loss in the transcription of many genes.


DEAF1 is a Pellino1-interacting protein required for interferon production by Sendai virus and double-stranded RNA.

Ordureau A, Enesa K, Nanda S, Le Francois B, Peggie M, Prescott A, Albert PR, Cohen P - J. Biol. Chem. (2013)

The production of IFNβ in Sendai virus infected MEFs is reduced in DEAF1−/− mice.A–I, immortalized MEFs from wild type mice (black bars, black circles in B) or DEAF1−/− mice (white bars, white squares in B) were infected with Sendai virus (100 HA/ml) for the times indicated. Total RNA was extracted and analyzed by qRT-PCR to measure the mRNA encoding IFNβ, IRF7, IFNα4, IFNα6, CXCL10, IκBα, IL-12p40, and the Sendai virus Pgene. The results show fold increase in mRNA relative to the values measured in unstimulated MEFs (± S.D. for triplicate determinations). In panel B, the concentration of IFNβ in the culture medium was measured by ELISA. The results are shown ± S.D. for triplicate determinations. Similar results were obtained in four independent experiments using MEFs immortalized from two different wild type and two different DEAF1−/− mice.
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Figure 2: The production of IFNβ in Sendai virus infected MEFs is reduced in DEAF1−/− mice.A–I, immortalized MEFs from wild type mice (black bars, black circles in B) or DEAF1−/− mice (white bars, white squares in B) were infected with Sendai virus (100 HA/ml) for the times indicated. Total RNA was extracted and analyzed by qRT-PCR to measure the mRNA encoding IFNβ, IRF7, IFNα4, IFNα6, CXCL10, IκBα, IL-12p40, and the Sendai virus Pgene. The results show fold increase in mRNA relative to the values measured in unstimulated MEFs (± S.D. for triplicate determinations). In panel B, the concentration of IFNβ in the culture medium was measured by ELISA. The results are shown ± S.D. for triplicate determinations. Similar results were obtained in four independent experiments using MEFs immortalized from two different wild type and two different DEAF1−/− mice.
Mentions: The MEFs were infected with Sendai virus containing defective-interfering genomes, which strongly stimulate IFNβ production in these cells (21, 22). The dsRNA formed during the replication of these genomes is thought to stimulate IFNβ mainly by activating the MDA5-MAVS-TBK1-IRF3 pathway (23). The production of IFNβ mRNA (Fig. 2A) and IFNβ secretion (Fig. 2B) induced by infection with Sendai virus was reduced considerably in MEFs from DEAF1−/− mice compared with wild type controls. The mRNAs encoding IFNβ-dependent genes, such as IRF7 (Fig. 2C), IFNα4 (Fig. 2D), and IFNα6 (Fig. 2E), as well as CXCL10 (Fig. 2F) an established marker of viral infection, were also suppressed in DEAF1−/− MEFs. In contrast, the mRNAs encoding IκBα (Fig. 2G) or the pro-inflammatory cytokine IL-12p70 (Fig. 2H), which are controlled by the transcription factors NFκB (24) and IRF5 (25), respectively, were little affected. The expression of the Sendai virus nucleocapsid protein was not decreased in MEFs from the knock-in mice (Fig. 2I), indicating that the decreased production of IFNβ in DEAF1−/− MEFs was not due to the failure of the virus to infect the MEFs. Moreover, the IL-1-stimulated activation of p38α MAP kinase, the IKKβ-dependent phosphorylation of p105/NFκB1 or degradation of IκBα (supplemental Fig. S2A), or the IL-1-stimulated production of the mRNA encoding IRF1 (supplemental Fig. S2B) and CXCL10 (supplemental Fig. S2C) were unaffected in DEAF1−/− MEFs. Indeed, the production of IκBα mRNA (supplemental Fig. S2D) and IL-6 mRNA (supplemental Fig. S2E) in DEAF1−/− MEFs was increased slightly. Thus the reduced production of IFNs and IFN-regulated genes in DEAF1−/− MEFs was specific and did not result from a general loss in the transcription of many genes.

Bottom Line: The interaction is independent of the E3 ligase activity of Pellino1, but weakened by the phosphorylation of Pellino1.DEAF1 is also needed for TLR3-dependent IFNβ production.Taken together, our results identify DEAF1 as a novel component of the signal transduction network by which dsRNA of viral origin stimulates IFNβ production.

View Article: PubMed Central - PubMed

Affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, Sir James Black Centre, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom.

ABSTRACT
Double-stranded (ds) RNA of viral origin, a ligand for Melanoma Differentiation-associated gene 5 (MDA5) and Toll-Like Receptor 3 (TLR3), induces the TANK-Binding Kinase 1 (TBK1)-dependent phosphorylation and activation of Interferon Regulatory Factor 3 (IRF3) and the E3 ubiquitin ligase Pellino1, which are required for interferon β (IFNβ) gene transcription. Here, we report that Pellino1 interacts with the transcription factor Deformed Epidermal Autoregulatory Factor 1 (DEAF1). The interaction is independent of the E3 ligase activity of Pellino1, but weakened by the phosphorylation of Pellino1. We show that DEAF1 binds to the IFNβ promoter and to IRF3 and IRF7, that it is required for the transcription of the IFNβ gene and IFNβ secretion in MEFs infected with Sendai virus or transfected with poly(I:C). DEAF1 is also needed for TLR3-dependent IFNβ production. Taken together, our results identify DEAF1 as a novel component of the signal transduction network by which dsRNA of viral origin stimulates IFNβ production.

Show MeSH
Related in: MedlinePlus