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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

The IFN/ISG signaling pathway is induced in G2/M synchronized cells.(A) IFN-B mRNA levels were determined by RT-QPCR as described in Fig 1E, in HeLa cells transfected with non-target siRNAs (white bars) or with Optn-specific siRNAs (dark bars) and left unsynchronized (AS), blocked in G2/M phases by RO-336 treatment (RO) or blocked in G1/S transition by double thymidine block and release for the time indicated (in hours). Mean ± SD values of expression levels are presented. Paired t-test was used to determine the significance of the IFN-B level difference between asynchronous and synchronized cells. ** p values < 0.01, *** p values < 0.001. Average % of cells in G2/M determined by PI staining/FACS analysis for both siNT- and siOptn-transfected cells is shown. (B) IFN-β protein levels were determined by ELISA in the supernatants of HeLa cells left untreated (NI), synchronized in G2/M by RO-3306 (RO) or stimulated by poly(I:C) (pIC). Mean ± SD values of expression levels relative to 104 cells are presented. Mean ± SD values of the induction folds corresponding to the ratio of the IFN-β protein level observed in RO-treated cells to that observed in control cells, is shown. * p values < 0.05. (C) IFN-B mRNA levels were determined by RT-QPCR in HeLa cells transfected with poly(I):(C) (pIC) and left unsynchronized (-), blocked in G2/M phases by RO-336 treatment (RO) as described in (A). Mean ± SD values of the induction folds corresponding to the ratio of the IFN-B expression level observed in RO-treated cells to that observed in control cells, is shown. *** p values < 0.001. (D) IFN-B mRNA levels were determined by RT-QPCR as described in (A) in control or stably Optn deficient HeLa cells transfected left unsynchronized (AS) or blocked in G2/M phases by RO-336 treatment (RO). The % of cells in G2/M determined in each condition by PI staining/FACS analysis is shown. (E) ISG56 mRNA levels were determined by RT-QPCR as described in (A), in HeLa cells transfected with non-target siRNAs or with Optn-specific siRNAs and left unsynchronized (AS), blocked in G2/M phases by RO-336 treatment (RO) or blocked in G1/S transition by double thymidine block and release for the time indicated (in hours). Mean ± SD values of expression levels, significance and average % of cells in G2/M are calculated as in (A). (F) Western blot analyses of extracts from asynchronous and RO-treated cells before and after different time (in hours) of SeV-infection using Hemagglutinin-Neuraminidase Protein of the Sendai virus (SeV-HN) and anti-actin-α antibodies. (G) Quantification of the signals obtained in (F) is presented as a ratio of SeV-HN protein levels versus actin levels. Paired t-test was used to determine the significance of the difference between the SeV-HN protein levels observed in asynchronous versus G2/M-treated cells after infection. * p values < 0.05, ** p values < 0.01, *** p values < 0.001.
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ppat.1004877.g007: The IFN/ISG signaling pathway is induced in G2/M synchronized cells.(A) IFN-B mRNA levels were determined by RT-QPCR as described in Fig 1E, in HeLa cells transfected with non-target siRNAs (white bars) or with Optn-specific siRNAs (dark bars) and left unsynchronized (AS), blocked in G2/M phases by RO-336 treatment (RO) or blocked in G1/S transition by double thymidine block and release for the time indicated (in hours). Mean ± SD values of expression levels are presented. Paired t-test was used to determine the significance of the IFN-B level difference between asynchronous and synchronized cells. ** p values < 0.01, *** p values < 0.001. Average % of cells in G2/M determined by PI staining/FACS analysis for both siNT- and siOptn-transfected cells is shown. (B) IFN-β protein levels were determined by ELISA in the supernatants of HeLa cells left untreated (NI), synchronized in G2/M by RO-3306 (RO) or stimulated by poly(I:C) (pIC). Mean ± SD values of expression levels relative to 104 cells are presented. Mean ± SD values of the induction folds corresponding to the ratio of the IFN-β protein level observed in RO-treated cells to that observed in control cells, is shown. * p values < 0.05. (C) IFN-B mRNA levels were determined by RT-QPCR in HeLa cells transfected with poly(I):(C) (pIC) and left unsynchronized (-), blocked in G2/M phases by RO-336 treatment (RO) as described in (A). Mean ± SD values of the induction folds corresponding to the ratio of the IFN-B expression level observed in RO-treated cells to that observed in control cells, is shown. *** p values < 0.001. (D) IFN-B mRNA levels were determined by RT-QPCR as described in (A) in control or stably Optn deficient HeLa cells transfected left unsynchronized (AS) or blocked in G2/M phases by RO-336 treatment (RO). The % of cells in G2/M determined in each condition by PI staining/FACS analysis is shown. (E) ISG56 mRNA levels were determined by RT-QPCR as described in (A), in HeLa cells transfected with non-target siRNAs or with Optn-specific siRNAs and left unsynchronized (AS), blocked in G2/M phases by RO-336 treatment (RO) or blocked in G1/S transition by double thymidine block and release for the time indicated (in hours). Mean ± SD values of expression levels, significance and average % of cells in G2/M are calculated as in (A). (F) Western blot analyses of extracts from asynchronous and RO-treated cells before and after different time (in hours) of SeV-infection using Hemagglutinin-Neuraminidase Protein of the Sendai virus (SeV-HN) and anti-actin-α antibodies. (G) Quantification of the signals obtained in (F) is presented as a ratio of SeV-HN protein levels versus actin levels. Paired t-test was used to determine the significance of the difference between the SeV-HN protein levels observed in asynchronous versus G2/M-treated cells after infection. * p values < 0.05, ** p values < 0.01, *** p values < 0.001.

Mentions: Enhancement of TBK1 activity in G2/M synchronized cells should lead to increased IFN-B gene expression at this cell cycle phase independently of viral infection. Indeed, the basal expression level of IFN-B, that was barely detectable by RT-QPCR in asynchronous (AS) cells, was 7-fold increased when more than 50% of cells were blocked at G2/M phase following RO treatment (Figs 7A and S7A and S7B). Similarly, the enhancement of IFN-B expression observed following cell synchronization by thymidine block and release was correlated with the percentage of cells in G2/M phase. Accordingly, ELISA experiments indicated a 3- to 4-fold increase in the RO-induced IFN-β protein level, although this amount remains 10-fold lower than the IFN-β production following poly(I:C) stimulation (Fig 7B). This enhancement was linked to activation of the TBK1/IRF3 pathway as depletion of TBK1 resulted in strong inhibition of the RO-induced IFN-B gene expression, as for poly(I:C) stimulation (S7C and S7D Fig). The dsRNA-induced levels of IFN-B transcription was also increased in G2/M-arrested cells, since an almost 2-fold higher level of IFN-B gene expression was observed in RO-treated cells compared to unsynchronized cells following dsRNA stimulation (Fig 7C). Such a cell cycle-dependent regulation of the IFN-B gene expression was not observed in Optn-depleted cells (after siRNAs transfection), in which IFN-B transcription levels were high and unregulated regardless of the cell cycle stage (Fig 7A). Similar results were obtained in HeLa cells stably depleted for Optn and synchronized at the G2/M transition following RO treatment (Fig 7D).


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)

The IFN/ISG signaling pathway is induced in G2/M synchronized cells.(A) IFN-B mRNA levels were determined by RT-QPCR as described in Fig 1E, in HeLa cells transfected with non-target siRNAs (white bars) or with Optn-specific siRNAs (dark bars) and left unsynchronized (AS), blocked in G2/M phases by RO-336 treatment (RO) or blocked in G1/S transition by double thymidine block and release for the time indicated (in hours). Mean ± SD values of expression levels are presented. Paired t-test was used to determine the significance of the IFN-B level difference between asynchronous and synchronized cells. ** p values < 0.01, *** p values < 0.001. Average % of cells in G2/M determined by PI staining/FACS analysis for both siNT- and siOptn-transfected cells is shown. (B) IFN-β protein levels were determined by ELISA in the supernatants of HeLa cells left untreated (NI), synchronized in G2/M by RO-3306 (RO) or stimulated by poly(I:C) (pIC). Mean ± SD values of expression levels relative to 104 cells are presented. Mean ± SD values of the induction folds corresponding to the ratio of the IFN-β protein level observed in RO-treated cells to that observed in control cells, is shown. * p values < 0.05. (C) IFN-B mRNA levels were determined by RT-QPCR in HeLa cells transfected with poly(I):(C) (pIC) and left unsynchronized (-), blocked in G2/M phases by RO-336 treatment (RO) as described in (A). Mean ± SD values of the induction folds corresponding to the ratio of the IFN-B expression level observed in RO-treated cells to that observed in control cells, is shown. *** p values < 0.001. (D) IFN-B mRNA levels were determined by RT-QPCR as described in (A) in control or stably Optn deficient HeLa cells transfected left unsynchronized (AS) or blocked in G2/M phases by RO-336 treatment (RO). The % of cells in G2/M determined in each condition by PI staining/FACS analysis is shown. (E) ISG56 mRNA levels were determined by RT-QPCR as described in (A), in HeLa cells transfected with non-target siRNAs or with Optn-specific siRNAs and left unsynchronized (AS), blocked in G2/M phases by RO-336 treatment (RO) or blocked in G1/S transition by double thymidine block and release for the time indicated (in hours). Mean ± SD values of expression levels, significance and average % of cells in G2/M are calculated as in (A). (F) Western blot analyses of extracts from asynchronous and RO-treated cells before and after different time (in hours) of SeV-infection using Hemagglutinin-Neuraminidase Protein of the Sendai virus (SeV-HN) and anti-actin-α antibodies. (G) Quantification of the signals obtained in (F) is presented as a ratio of SeV-HN protein levels versus actin levels. Paired t-test was used to determine the significance of the difference between the SeV-HN protein levels observed in asynchronous versus G2/M-treated cells after infection. * p values < 0.05, ** p values < 0.01, *** p values < 0.001.
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ppat.1004877.g007: The IFN/ISG signaling pathway is induced in G2/M synchronized cells.(A) IFN-B mRNA levels were determined by RT-QPCR as described in Fig 1E, in HeLa cells transfected with non-target siRNAs (white bars) or with Optn-specific siRNAs (dark bars) and left unsynchronized (AS), blocked in G2/M phases by RO-336 treatment (RO) or blocked in G1/S transition by double thymidine block and release for the time indicated (in hours). Mean ± SD values of expression levels are presented. Paired t-test was used to determine the significance of the IFN-B level difference between asynchronous and synchronized cells. ** p values < 0.01, *** p values < 0.001. Average % of cells in G2/M determined by PI staining/FACS analysis for both siNT- and siOptn-transfected cells is shown. (B) IFN-β protein levels were determined by ELISA in the supernatants of HeLa cells left untreated (NI), synchronized in G2/M by RO-3306 (RO) or stimulated by poly(I:C) (pIC). Mean ± SD values of expression levels relative to 104 cells are presented. Mean ± SD values of the induction folds corresponding to the ratio of the IFN-β protein level observed in RO-treated cells to that observed in control cells, is shown. * p values < 0.05. (C) IFN-B mRNA levels were determined by RT-QPCR in HeLa cells transfected with poly(I):(C) (pIC) and left unsynchronized (-), blocked in G2/M phases by RO-336 treatment (RO) as described in (A). Mean ± SD values of the induction folds corresponding to the ratio of the IFN-B expression level observed in RO-treated cells to that observed in control cells, is shown. *** p values < 0.001. (D) IFN-B mRNA levels were determined by RT-QPCR as described in (A) in control or stably Optn deficient HeLa cells transfected left unsynchronized (AS) or blocked in G2/M phases by RO-336 treatment (RO). The % of cells in G2/M determined in each condition by PI staining/FACS analysis is shown. (E) ISG56 mRNA levels were determined by RT-QPCR as described in (A), in HeLa cells transfected with non-target siRNAs or with Optn-specific siRNAs and left unsynchronized (AS), blocked in G2/M phases by RO-336 treatment (RO) or blocked in G1/S transition by double thymidine block and release for the time indicated (in hours). Mean ± SD values of expression levels, significance and average % of cells in G2/M are calculated as in (A). (F) Western blot analyses of extracts from asynchronous and RO-treated cells before and after different time (in hours) of SeV-infection using Hemagglutinin-Neuraminidase Protein of the Sendai virus (SeV-HN) and anti-actin-α antibodies. (G) Quantification of the signals obtained in (F) is presented as a ratio of SeV-HN protein levels versus actin levels. Paired t-test was used to determine the significance of the difference between the SeV-HN protein levels observed in asynchronous versus G2/M-treated cells after infection. * p values < 0.05, ** p values < 0.01, *** p values < 0.001.
Mentions: Enhancement of TBK1 activity in G2/M synchronized cells should lead to increased IFN-B gene expression at this cell cycle phase independently of viral infection. Indeed, the basal expression level of IFN-B, that was barely detectable by RT-QPCR in asynchronous (AS) cells, was 7-fold increased when more than 50% of cells were blocked at G2/M phase following RO treatment (Figs 7A and S7A and S7B). Similarly, the enhancement of IFN-B expression observed following cell synchronization by thymidine block and release was correlated with the percentage of cells in G2/M phase. Accordingly, ELISA experiments indicated a 3- to 4-fold increase in the RO-induced IFN-β protein level, although this amount remains 10-fold lower than the IFN-β production following poly(I:C) stimulation (Fig 7B). This enhancement was linked to activation of the TBK1/IRF3 pathway as depletion of TBK1 resulted in strong inhibition of the RO-induced IFN-B gene expression, as for poly(I:C) stimulation (S7C and S7D Fig). The dsRNA-induced levels of IFN-B transcription was also increased in G2/M-arrested cells, since an almost 2-fold higher level of IFN-B gene expression was observed in RO-treated cells compared to unsynchronized cells following dsRNA stimulation (Fig 7C). Such a cell cycle-dependent regulation of the IFN-B gene expression was not observed in Optn-depleted cells (after siRNAs transfection), in which IFN-B transcription levels were high and unregulated regardless of the cell cycle stage (Fig 7A). Similar results were obtained in HeLa cells stably depleted for Optn and synchronized at the G2/M transition following RO treatment (Fig 7D).

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