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Regulation of MDA5-MAVS Antiviral Signaling Axis by TRIM25 through TRAF6-Mediated NF-κB Activation.

Lee NR, Kim HI, Choi MS, Yi CM, Inn KS - Mol. Cells (2015)

Bottom Line: Tripartite motif protein 25 (TRIM25), mediates K63-linked polyubiquitination of Retinoic acid inducible gene I (RIG-I) that is crucial for downstream antiviral interferon signaling.Mechanical studies showed that TRIM25 is involved in TRAF6-mediated NF-κB activation.These collectively indicate that TRIM25 plays an additional role in RIG-I/MDA5 signaling other than RIG-I ubiquitination via activation of NF-κB.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Science, College of Pharmacy, Kyung Hee University, Seoul 130-701, Korea.

ABSTRACT
Tripartite motif protein 25 (TRIM25), mediates K63-linked polyubiquitination of Retinoic acid inducible gene I (RIG-I) that is crucial for downstream antiviral interferon signaling. Here, we demonstrate that TRIM25 is required for melanoma differentiation-associated gene 5 (MDA5) and MAVS mediated activation of NF-κB and interferon production. TRIM25 is required for the full activation of NF-κB at the downstream of MAVS, while it is not involved in IRF3 nuclear translocation. Mechanical studies showed that TRIM25 is involved in TRAF6-mediated NF-κB activation. These collectively indicate that TRIM25 plays an additional role in RIG-I/MDA5 signaling other than RIG-I ubiquitination via activation of NF-κB.

No MeSH data available.


Related in: MedlinePlus

TRIM25 is involved in MDA5-mediated antiviral signaling. (A) Effect of TRIM25 depletion on polyI:C induced interferon-β synthesis. HEK293T cells were transfected with control shRNA or shRNAs against TRIM25. Cells were transfected with polyI:C 24 h later. Cells were lysed at 6 h after transfection and analyzed for interferon (IFN)-β mRNA levels by RT-qPCR, as described in “Materials and Methods”. KD efficiency (%): Knock-down efficiency compared to control (Ctrl). (B) Enhanced RIG-I and MDA5-mediated IFN-β promoter activity by ectopic expression of TRIM25. HEK293T cells were transfected with a RIG-IN, MDA5N expression plasmids or control vector together with vector or TRIM25 expression plasmids as indicated. Cells were co-transfected with IFN-β promoter-reporter and TK-Renilla reporter plasmids. Promoter activities were determined by Dual-Luciferase assay 16 h after transfection. (C) Enhanced RIG-I and MDA5-mediated NF-κB promoter activity by ectopic expression of TRIM25. HEK293T cells were transfected with expression plasmids and NF-κB reporter plasmids as indicated. Promoter activities were determined using procedures similar to those in (B). (D) Requirement of TRIM25 for efficient activation IFN-β promoter by MDA5. Wild-type (WT) and TRIM25 knock-out (TRIM25 KO) MEFs were transfected with vector or MDA5N plasmids together with reporter plasmids. IFN-β promoter activity was determined by luciferase assay as describe above. (E) Inhibition of MDA5 signaling by influenza A NS1. HEK293T cells were transfected with vector or MDA5N plasmids together with wild-type influenza A NS1 (WT) or mutant NS1 (E96A/E97A) as indicated. IFN-β promoter activity was determined by luciferase assay as describe above. Results of experiments, performed in triplicate, are presented as means ± standard deviation. *p < 0.05, **p < 0.01
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f1-molce-38-9-759: TRIM25 is involved in MDA5-mediated antiviral signaling. (A) Effect of TRIM25 depletion on polyI:C induced interferon-β synthesis. HEK293T cells were transfected with control shRNA or shRNAs against TRIM25. Cells were transfected with polyI:C 24 h later. Cells were lysed at 6 h after transfection and analyzed for interferon (IFN)-β mRNA levels by RT-qPCR, as described in “Materials and Methods”. KD efficiency (%): Knock-down efficiency compared to control (Ctrl). (B) Enhanced RIG-I and MDA5-mediated IFN-β promoter activity by ectopic expression of TRIM25. HEK293T cells were transfected with a RIG-IN, MDA5N expression plasmids or control vector together with vector or TRIM25 expression plasmids as indicated. Cells were co-transfected with IFN-β promoter-reporter and TK-Renilla reporter plasmids. Promoter activities were determined by Dual-Luciferase assay 16 h after transfection. (C) Enhanced RIG-I and MDA5-mediated NF-κB promoter activity by ectopic expression of TRIM25. HEK293T cells were transfected with expression plasmids and NF-κB reporter plasmids as indicated. Promoter activities were determined using procedures similar to those in (B). (D) Requirement of TRIM25 for efficient activation IFN-β promoter by MDA5. Wild-type (WT) and TRIM25 knock-out (TRIM25 KO) MEFs were transfected with vector or MDA5N plasmids together with reporter plasmids. IFN-β promoter activity was determined by luciferase assay as describe above. (E) Inhibition of MDA5 signaling by influenza A NS1. HEK293T cells were transfected with vector or MDA5N plasmids together with wild-type influenza A NS1 (WT) or mutant NS1 (E96A/E97A) as indicated. IFN-β promoter activity was determined by luciferase assay as describe above. Results of experiments, performed in triplicate, are presented as means ± standard deviation. *p < 0.05, **p < 0.01

Mentions: It has been shown that commercial polyI:C is mainly recognized by MDA5 rather than RIG-I (Kato et al., 2006; 2008). Thus, we initially tested the effect of depletion of TRIM25 on polyI:C-induced interferon-β production. Knock-down of TRIM25 using shRNAs resulted in a decrease of interferon-β mRNA synthesis in polyI:C transfected cells (Fig. 1A). To confirm the activation of MDA5 signaling activity by TRIM25, TRIM25 was co-expressed with MDA5-2Card (MDA5N), a constitutively active form of MDA5. As shown in Figs. 1B and 1C, TRIM25 enhanced both RIG-IN- and MDA5N-mediated interferon-β promoter activity and NF-κB transcriptional activity to a similar extent. In addition, MDA5N-mediated interferon-β promoter activity in TRIM25 deficient cells (TRIM25−/− MEF) was much lower compared to wild-type MEF, suggesting that TRIM25 is required for efficient activation of the MDA5 signaling pathway (Fig. 1D).


Regulation of MDA5-MAVS Antiviral Signaling Axis by TRIM25 through TRAF6-Mediated NF-κB Activation.

Lee NR, Kim HI, Choi MS, Yi CM, Inn KS - Mol. Cells (2015)

TRIM25 is involved in MDA5-mediated antiviral signaling. (A) Effect of TRIM25 depletion on polyI:C induced interferon-β synthesis. HEK293T cells were transfected with control shRNA or shRNAs against TRIM25. Cells were transfected with polyI:C 24 h later. Cells were lysed at 6 h after transfection and analyzed for interferon (IFN)-β mRNA levels by RT-qPCR, as described in “Materials and Methods”. KD efficiency (%): Knock-down efficiency compared to control (Ctrl). (B) Enhanced RIG-I and MDA5-mediated IFN-β promoter activity by ectopic expression of TRIM25. HEK293T cells were transfected with a RIG-IN, MDA5N expression plasmids or control vector together with vector or TRIM25 expression plasmids as indicated. Cells were co-transfected with IFN-β promoter-reporter and TK-Renilla reporter plasmids. Promoter activities were determined by Dual-Luciferase assay 16 h after transfection. (C) Enhanced RIG-I and MDA5-mediated NF-κB promoter activity by ectopic expression of TRIM25. HEK293T cells were transfected with expression plasmids and NF-κB reporter plasmids as indicated. Promoter activities were determined using procedures similar to those in (B). (D) Requirement of TRIM25 for efficient activation IFN-β promoter by MDA5. Wild-type (WT) and TRIM25 knock-out (TRIM25 KO) MEFs were transfected with vector or MDA5N plasmids together with reporter plasmids. IFN-β promoter activity was determined by luciferase assay as describe above. (E) Inhibition of MDA5 signaling by influenza A NS1. HEK293T cells were transfected with vector or MDA5N plasmids together with wild-type influenza A NS1 (WT) or mutant NS1 (E96A/E97A) as indicated. IFN-β promoter activity was determined by luciferase assay as describe above. Results of experiments, performed in triplicate, are presented as means ± standard deviation. *p < 0.05, **p < 0.01
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4588718&req=5

f1-molce-38-9-759: TRIM25 is involved in MDA5-mediated antiviral signaling. (A) Effect of TRIM25 depletion on polyI:C induced interferon-β synthesis. HEK293T cells were transfected with control shRNA or shRNAs against TRIM25. Cells were transfected with polyI:C 24 h later. Cells were lysed at 6 h after transfection and analyzed for interferon (IFN)-β mRNA levels by RT-qPCR, as described in “Materials and Methods”. KD efficiency (%): Knock-down efficiency compared to control (Ctrl). (B) Enhanced RIG-I and MDA5-mediated IFN-β promoter activity by ectopic expression of TRIM25. HEK293T cells were transfected with a RIG-IN, MDA5N expression plasmids or control vector together with vector or TRIM25 expression plasmids as indicated. Cells were co-transfected with IFN-β promoter-reporter and TK-Renilla reporter plasmids. Promoter activities were determined by Dual-Luciferase assay 16 h after transfection. (C) Enhanced RIG-I and MDA5-mediated NF-κB promoter activity by ectopic expression of TRIM25. HEK293T cells were transfected with expression plasmids and NF-κB reporter plasmids as indicated. Promoter activities were determined using procedures similar to those in (B). (D) Requirement of TRIM25 for efficient activation IFN-β promoter by MDA5. Wild-type (WT) and TRIM25 knock-out (TRIM25 KO) MEFs were transfected with vector or MDA5N plasmids together with reporter plasmids. IFN-β promoter activity was determined by luciferase assay as describe above. (E) Inhibition of MDA5 signaling by influenza A NS1. HEK293T cells were transfected with vector or MDA5N plasmids together with wild-type influenza A NS1 (WT) or mutant NS1 (E96A/E97A) as indicated. IFN-β promoter activity was determined by luciferase assay as describe above. Results of experiments, performed in triplicate, are presented as means ± standard deviation. *p < 0.05, **p < 0.01
Mentions: It has been shown that commercial polyI:C is mainly recognized by MDA5 rather than RIG-I (Kato et al., 2006; 2008). Thus, we initially tested the effect of depletion of TRIM25 on polyI:C-induced interferon-β production. Knock-down of TRIM25 using shRNAs resulted in a decrease of interferon-β mRNA synthesis in polyI:C transfected cells (Fig. 1A). To confirm the activation of MDA5 signaling activity by TRIM25, TRIM25 was co-expressed with MDA5-2Card (MDA5N), a constitutively active form of MDA5. As shown in Figs. 1B and 1C, TRIM25 enhanced both RIG-IN- and MDA5N-mediated interferon-β promoter activity and NF-κB transcriptional activity to a similar extent. In addition, MDA5N-mediated interferon-β promoter activity in TRIM25 deficient cells (TRIM25−/− MEF) was much lower compared to wild-type MEF, suggesting that TRIM25 is required for efficient activation of the MDA5 signaling pathway (Fig. 1D).

Bottom Line: Tripartite motif protein 25 (TRIM25), mediates K63-linked polyubiquitination of Retinoic acid inducible gene I (RIG-I) that is crucial for downstream antiviral interferon signaling.Mechanical studies showed that TRIM25 is involved in TRAF6-mediated NF-κB activation.These collectively indicate that TRIM25 plays an additional role in RIG-I/MDA5 signaling other than RIG-I ubiquitination via activation of NF-κB.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Science, College of Pharmacy, Kyung Hee University, Seoul 130-701, Korea.

ABSTRACT
Tripartite motif protein 25 (TRIM25), mediates K63-linked polyubiquitination of Retinoic acid inducible gene I (RIG-I) that is crucial for downstream antiviral interferon signaling. Here, we demonstrate that TRIM25 is required for melanoma differentiation-associated gene 5 (MDA5) and MAVS mediated activation of NF-κB and interferon production. TRIM25 is required for the full activation of NF-κB at the downstream of MAVS, while it is not involved in IRF3 nuclear translocation. Mechanical studies showed that TRIM25 is involved in TRAF6-mediated NF-κB activation. These collectively indicate that TRIM25 plays an additional role in RIG-I/MDA5 signaling other than RIG-I ubiquitination via activation of NF-κB.

No MeSH data available.


Related in: MedlinePlus