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Optineurin regulates the interferon response in a cell cycle-dependent manner.

Génin P, Cuvelier F, Lambin S, Côrte-Real Filipe J, Autrusseau E, Laurent C, Laplantine E, Weil R - PLoS Pathog. (2015)

Bottom Line: Viral invasion into a host is initially recognized by the innate immune system, mainly through activation of the intracellular cytosolic signaling pathway and coordinated activation of interferon regulatory factor 3 (IRF3) and nuclear factor kappa B (NF-κB) transcription factors that promote type I interferon gene induction.The TANK-binding Kinase 1 (TBK1) phosphorylates and activates IRF3.As a result, we observed, at this cell division stage, an increased activity and phosphorylation of TBK1 that lead to its relocalization to mitochondria and to enhanced interferon production, suggesting that this process, which relies on Optn function, might be of major importance to mount a preventive antiviral response during mitosis.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Signalisation et Pathogenèse, CNRS UMR3691, Institut Pasteur, Paris, France.

ABSTRACT
Viral invasion into a host is initially recognized by the innate immune system, mainly through activation of the intracellular cytosolic signaling pathway and coordinated activation of interferon regulatory factor 3 (IRF3) and nuclear factor kappa B (NF-κB) transcription factors that promote type I interferon gene induction. The TANK-binding Kinase 1 (TBK1) phosphorylates and activates IRF3. Here, we show that Optineurin (Optn) dampens the antiviral innate immune response by targeting the deubiquitinating enzyme CYLD to TBK1 in order to inhibit its enzymatic activity. Importantly, we found that this regulatory mechanism is abolished at the G2/M phase as a consequence of the nuclear translocation of CYLD and Optn. As a result, we observed, at this cell division stage, an increased activity and phosphorylation of TBK1 that lead to its relocalization to mitochondria and to enhanced interferon production, suggesting that this process, which relies on Optn function, might be of major importance to mount a preventive antiviral response during mitosis.

No MeSH data available.


Related in: MedlinePlus

Optn negative effect on the virus-induced interferon pathway requires its phosphorylation and ubiquitin-binding activity.(A) Phosphorylation status of Optn (on S177) and IRF3 (on S396) were determined by Western blot in uninfected HeLa cells and at different time after infection by Sendai virus. (B) Total cell lysates from HEK293T infected for 6h with SeV in the presence or absence of the TBK1-specific inhibitor BX795 were analyzed by Western blot using anti-pS177 Optn, anti-Optn, anti-pS396 IRF3, anti-IRF3 and anti-tubulin antibodies. (C) Total cell lysates extracted from HEK293T cells expressing either VSV-Optn (Optn, left panels) or VSV-Optn S162-170-171-173-174-177A (Optn-S6A, right panels) were transfected with empty vector (-) or with TBK1-expressing plasmid (TBK1) and subjected to two-dimensional gel electrophoresis. Optn isoforms were analyzed by immunoblotting with an anti-VSV antiserum. As control, lysates were pre-treated with lambda phosphatase (λ-Phos.) at 30°C. (D) HEK293T cells were transiently transfected with Flag-TBK1. TBK1 kinase assays were carried out using Flag immunoprecipitates as enzyme and GST-Optn wt, GST-Optn S177A, GST-Optn S171-173A or GST-Optn S171-173-177A as substrates. (E) IFN-B mRNA levels were determined by RT-QPCR in control HeLa cells, Optn-deficient cells and deficient cells reconstituted with wt, D474N- or S177A-mutated forms of Optn following infection by SeV or stimulation with dsRNA (pIC) or dsDNA (poly(dA):(dT)) and presented as mRNA transcripts levels related to the mRNA of 18S (set at 100). Mean ± SD values of expression levels are shown.
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ppat.1004877.g001: Optn negative effect on the virus-induced interferon pathway requires its phosphorylation and ubiquitin-binding activity.(A) Phosphorylation status of Optn (on S177) and IRF3 (on S396) were determined by Western blot in uninfected HeLa cells and at different time after infection by Sendai virus. (B) Total cell lysates from HEK293T infected for 6h with SeV in the presence or absence of the TBK1-specific inhibitor BX795 were analyzed by Western blot using anti-pS177 Optn, anti-Optn, anti-pS396 IRF3, anti-IRF3 and anti-tubulin antibodies. (C) Total cell lysates extracted from HEK293T cells expressing either VSV-Optn (Optn, left panels) or VSV-Optn S162-170-171-173-174-177A (Optn-S6A, right panels) were transfected with empty vector (-) or with TBK1-expressing plasmid (TBK1) and subjected to two-dimensional gel electrophoresis. Optn isoforms were analyzed by immunoblotting with an anti-VSV antiserum. As control, lysates were pre-treated with lambda phosphatase (λ-Phos.) at 30°C. (D) HEK293T cells were transiently transfected with Flag-TBK1. TBK1 kinase assays were carried out using Flag immunoprecipitates as enzyme and GST-Optn wt, GST-Optn S177A, GST-Optn S171-173A or GST-Optn S171-173-177A as substrates. (E) IFN-B mRNA levels were determined by RT-QPCR in control HeLa cells, Optn-deficient cells and deficient cells reconstituted with wt, D474N- or S177A-mutated forms of Optn following infection by SeV or stimulation with dsRNA (pIC) or dsDNA (poly(dA):(dT)) and presented as mRNA transcripts levels related to the mRNA of 18S (set at 100). Mean ± SD values of expression levels are shown.

Mentions: Since it was previously reported that TBK1 phosphorylates Optn on Serine 177 (S177) in response to LPS [14,35], we determined the S177 phosphorylation status of Optn in SeV-infected cells by Western blot analysis using a phospho-specific antibody (pS177) (S2A Fig, [38]). We observed basal phosphorylation of Optn S177 that was enhanced in response to virus infection, with a kinetic similar to that of IRF3 (on Serine 396) that reflects its activation (Fig 1A). As for IRF3 phosphorylation, virus-induced phosphorylation of Optn at S177 was abrogated in the presence of TBK1 inhibitor BX795 (Fig 1B). Consistent with the presence of several consensus phosphorylation sites for TBK1 in the insert region of Optn (see S2A Fig), multiple phosphorylated forms of Optn were separated in two-dimensional gel electrophoresis when VSV-tagged Optn was co-expressed with TBK1 in HEK293T cells (Fig 1C). These post-translational modified forms disappeared after treatment with λ-phosphatase or after substitutions of the Serine residues present in the consensus motif for TBK1 by Alanine residues. Kinase assays performed using immunoprecipitated TBK1 (as source of kinase) and mutants of Optn on multiple Serine (as substrates) further indicated that S177 was the preferential site of phosphorylation by TBK1 (Fig 1D). The inhibitory effect of wt Optn on virus-induced IFN-B gene expression was abolished by the S177A substitution and by mutation in its ubiquitin-binding domain (D474N), but was not affected by the pathogenic E50K mutation (Figs 1E, left panel and S2B–S2C). Similar results were obtained when HeLa cells were transfected with dsRNA or dsDNA (Figs 1E, middle and right panels, respectively and S2B). These data indicated that the inhibitory function of Optn on stimulated IFN-B expression requires integrity of its ubiquitin-binding domain (consistent with previous results [36]) and of its main phosphorylation site.


Optineurin regulates the interferon response in a cell cycle-dependent manner.

Génin P, Cuvelier F, Lambin S, Côrte-Real Filipe J, Autrusseau E, Laurent C, Laplantine E, Weil R - PLoS Pathog. (2015)

Optn negative effect on the virus-induced interferon pathway requires its phosphorylation and ubiquitin-binding activity.(A) Phosphorylation status of Optn (on S177) and IRF3 (on S396) were determined by Western blot in uninfected HeLa cells and at different time after infection by Sendai virus. (B) Total cell lysates from HEK293T infected for 6h with SeV in the presence or absence of the TBK1-specific inhibitor BX795 were analyzed by Western blot using anti-pS177 Optn, anti-Optn, anti-pS396 IRF3, anti-IRF3 and anti-tubulin antibodies. (C) Total cell lysates extracted from HEK293T cells expressing either VSV-Optn (Optn, left panels) or VSV-Optn S162-170-171-173-174-177A (Optn-S6A, right panels) were transfected with empty vector (-) or with TBK1-expressing plasmid (TBK1) and subjected to two-dimensional gel electrophoresis. Optn isoforms were analyzed by immunoblotting with an anti-VSV antiserum. As control, lysates were pre-treated with lambda phosphatase (λ-Phos.) at 30°C. (D) HEK293T cells were transiently transfected with Flag-TBK1. TBK1 kinase assays were carried out using Flag immunoprecipitates as enzyme and GST-Optn wt, GST-Optn S177A, GST-Optn S171-173A or GST-Optn S171-173-177A as substrates. (E) IFN-B mRNA levels were determined by RT-QPCR in control HeLa cells, Optn-deficient cells and deficient cells reconstituted with wt, D474N- or S177A-mutated forms of Optn following infection by SeV or stimulation with dsRNA (pIC) or dsDNA (poly(dA):(dT)) and presented as mRNA transcripts levels related to the mRNA of 18S (set at 100). Mean ± SD values of expression levels are shown.
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ppat.1004877.g001: Optn negative effect on the virus-induced interferon pathway requires its phosphorylation and ubiquitin-binding activity.(A) Phosphorylation status of Optn (on S177) and IRF3 (on S396) were determined by Western blot in uninfected HeLa cells and at different time after infection by Sendai virus. (B) Total cell lysates from HEK293T infected for 6h with SeV in the presence or absence of the TBK1-specific inhibitor BX795 were analyzed by Western blot using anti-pS177 Optn, anti-Optn, anti-pS396 IRF3, anti-IRF3 and anti-tubulin antibodies. (C) Total cell lysates extracted from HEK293T cells expressing either VSV-Optn (Optn, left panels) or VSV-Optn S162-170-171-173-174-177A (Optn-S6A, right panels) were transfected with empty vector (-) or with TBK1-expressing plasmid (TBK1) and subjected to two-dimensional gel electrophoresis. Optn isoforms were analyzed by immunoblotting with an anti-VSV antiserum. As control, lysates were pre-treated with lambda phosphatase (λ-Phos.) at 30°C. (D) HEK293T cells were transiently transfected with Flag-TBK1. TBK1 kinase assays were carried out using Flag immunoprecipitates as enzyme and GST-Optn wt, GST-Optn S177A, GST-Optn S171-173A or GST-Optn S171-173-177A as substrates. (E) IFN-B mRNA levels were determined by RT-QPCR in control HeLa cells, Optn-deficient cells and deficient cells reconstituted with wt, D474N- or S177A-mutated forms of Optn following infection by SeV or stimulation with dsRNA (pIC) or dsDNA (poly(dA):(dT)) and presented as mRNA transcripts levels related to the mRNA of 18S (set at 100). Mean ± SD values of expression levels are shown.
Mentions: Since it was previously reported that TBK1 phosphorylates Optn on Serine 177 (S177) in response to LPS [14,35], we determined the S177 phosphorylation status of Optn in SeV-infected cells by Western blot analysis using a phospho-specific antibody (pS177) (S2A Fig, [38]). We observed basal phosphorylation of Optn S177 that was enhanced in response to virus infection, with a kinetic similar to that of IRF3 (on Serine 396) that reflects its activation (Fig 1A). As for IRF3 phosphorylation, virus-induced phosphorylation of Optn at S177 was abrogated in the presence of TBK1 inhibitor BX795 (Fig 1B). Consistent with the presence of several consensus phosphorylation sites for TBK1 in the insert region of Optn (see S2A Fig), multiple phosphorylated forms of Optn were separated in two-dimensional gel electrophoresis when VSV-tagged Optn was co-expressed with TBK1 in HEK293T cells (Fig 1C). These post-translational modified forms disappeared after treatment with λ-phosphatase or after substitutions of the Serine residues present in the consensus motif for TBK1 by Alanine residues. Kinase assays performed using immunoprecipitated TBK1 (as source of kinase) and mutants of Optn on multiple Serine (as substrates) further indicated that S177 was the preferential site of phosphorylation by TBK1 (Fig 1D). The inhibitory effect of wt Optn on virus-induced IFN-B gene expression was abolished by the S177A substitution and by mutation in its ubiquitin-binding domain (D474N), but was not affected by the pathogenic E50K mutation (Figs 1E, left panel and S2B–S2C). Similar results were obtained when HeLa cells were transfected with dsRNA or dsDNA (Figs 1E, middle and right panels, respectively and S2B). These data indicated that the inhibitory function of Optn on stimulated IFN-B expression requires integrity of its ubiquitin-binding domain (consistent with previous results [36]) and of its main phosphorylation site.

Bottom Line: Viral invasion into a host is initially recognized by the innate immune system, mainly through activation of the intracellular cytosolic signaling pathway and coordinated activation of interferon regulatory factor 3 (IRF3) and nuclear factor kappa B (NF-κB) transcription factors that promote type I interferon gene induction.The TANK-binding Kinase 1 (TBK1) phosphorylates and activates IRF3.As a result, we observed, at this cell division stage, an increased activity and phosphorylation of TBK1 that lead to its relocalization to mitochondria and to enhanced interferon production, suggesting that this process, which relies on Optn function, might be of major importance to mount a preventive antiviral response during mitosis.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Signalisation et Pathogenèse, CNRS UMR3691, Institut Pasteur, Paris, France.

ABSTRACT
Viral invasion into a host is initially recognized by the innate immune system, mainly through activation of the intracellular cytosolic signaling pathway and coordinated activation of interferon regulatory factor 3 (IRF3) and nuclear factor kappa B (NF-κB) transcription factors that promote type I interferon gene induction. The TANK-binding Kinase 1 (TBK1) phosphorylates and activates IRF3. Here, we show that Optineurin (Optn) dampens the antiviral innate immune response by targeting the deubiquitinating enzyme CYLD to TBK1 in order to inhibit its enzymatic activity. Importantly, we found that this regulatory mechanism is abolished at the G2/M phase as a consequence of the nuclear translocation of CYLD and Optn. As a result, we observed, at this cell division stage, an increased activity and phosphorylation of TBK1 that lead to its relocalization to mitochondria and to enhanced interferon production, suggesting that this process, which relies on Optn function, might be of major importance to mount a preventive antiviral response during mitosis.

No MeSH data available.


Related in: MedlinePlus