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An Internally Translated MAVS Variant Exposes Its Amino-terminal TRAF-Binding Motifs to Deregulate Interferon Induction.

Minassian A, Zhang J, He S, Zhao J, Zandi E, Saito T, Liang C, Feng P - PLoS Pathog. (2015)

Bottom Line: By contrast, MAVS50 inhibits the IRF activation and suppresses IFN induction.Ablation of the TRAF-binding motif of MAVS50 impaired its inhibitory effect on IRF activation and IFN induction.These results collectively identify a new means by which signaling events is differentially regulated via exposing key internally embedded interaction motifs, implying a more ubiquitous regulatory role of truncated proteins arose from internal translation and other related mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology and Immunology, Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, California, United States of America.

ABSTRACT
Activation of pattern recognition receptors and proper regulation of downstream signaling are crucial for host innate immune response. Upon infection, the NF-κB and interferon regulatory factors (IRF) are often simultaneously activated to defeat invading pathogens. Mechanisms concerning differential activation of NF-κB and IRF are not well understood. Here we report that a MAVS variant inhibits interferon (IFN) induction, while enabling NF-κB activation. Employing herpesviral proteins that selectively activate NF-κB signaling, we discovered that a MAVS variant of ~50 kDa, thus designated MAVS50, was produced from internal translation initiation. MAVS50 preferentially interacts with TRAF2 and TRAF6, and activates NF-κB. By contrast, MAVS50 inhibits the IRF activation and suppresses IFN induction. Biochemical analysis showed that MAVS50, exposing a degenerate TRAF-binding motif within its N-terminus, effectively competed with full-length MAVS for recruiting TRAF2 and TRAF6. Ablation of the TRAF-binding motif of MAVS50 impaired its inhibitory effect on IRF activation and IFN induction. These results collectively identify a new means by which signaling events is differentially regulated via exposing key internally embedded interaction motifs, implying a more ubiquitous regulatory role of truncated proteins arose from internal translation and other related mechanisms.

No MeSH data available.


Related in: MedlinePlus

MAVS50 targets TRAF molecules to inhibit MAVS70-mediated IFN induction.(A) Diagram of key signaling molecules of the RIG-I-dependent IFN induction. (B and C) 293T cells were transfected with an IFN-β reporter cocktail, a plasmid containing RIG-I-N (B) or TBK-1 (C), and increasing amount of plasmid containing MAVS50. The IFN-β promoter activity was determined by luciferase assay at 30 hours post-transfection. **p<0.01; ***p<0.005. (D) 293T cells were transfected with plasmids containing MAVS wild-type (WT) or MAVS50 and a plasmid containing TRAF6. Whole cell lysates (WCL) were precipitated with anti-Flag (TRAF6). Precipitated proteins and WCLs were analyzed by immunoblotting with indicated antibodies. (E) 293T cells, depleted with endogenous MAVS and “reconstituted” with MAVS70 or MAVS50, were precipitated with anti-V5 (MAVS70 or MAVS50). Precipitated proteins and WCLs were analyzed by immunoblotting with indicated antibodies. (F) MAVS knockdown cells “reconstituted” with MAVS70-V5 were transfected with increasing amount of MAVS50-Flag. At 30 hours post-transfection, cells were infected with Sendai virus (SeV, 200 HA unit/ml) for two hours. WCLs were prepared and precipitated with anti-V5 (MAVS70) antibody. Precipitated proteins and WCLs were analyzed by immunoblotting with indicated antibodies. V, vector.
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ppat.1005060.g005: MAVS50 targets TRAF molecules to inhibit MAVS70-mediated IFN induction.(A) Diagram of key signaling molecules of the RIG-I-dependent IFN induction. (B and C) 293T cells were transfected with an IFN-β reporter cocktail, a plasmid containing RIG-I-N (B) or TBK-1 (C), and increasing amount of plasmid containing MAVS50. The IFN-β promoter activity was determined by luciferase assay at 30 hours post-transfection. **p<0.01; ***p<0.005. (D) 293T cells were transfected with plasmids containing MAVS wild-type (WT) or MAVS50 and a plasmid containing TRAF6. Whole cell lysates (WCL) were precipitated with anti-Flag (TRAF6). Precipitated proteins and WCLs were analyzed by immunoblotting with indicated antibodies. (E) 293T cells, depleted with endogenous MAVS and “reconstituted” with MAVS70 or MAVS50, were precipitated with anti-V5 (MAVS70 or MAVS50). Precipitated proteins and WCLs were analyzed by immunoblotting with indicated antibodies. (F) MAVS knockdown cells “reconstituted” with MAVS70-V5 were transfected with increasing amount of MAVS50-Flag. At 30 hours post-transfection, cells were infected with Sendai virus (SeV, 200 HA unit/ml) for two hours. WCLs were prepared and precipitated with anti-V5 (MAVS70) antibody. Precipitated proteins and WCLs were analyzed by immunoblotting with indicated antibodies. V, vector.

Mentions: The cytosolic sensor-mediated IFN induction pathway constitutes of key signaling molecules, including RIG-I/MDA5, MAVS, TBK-1/IKKε, and IRF3 (Fig 5A). Over-expression of these components is sufficient to activate downstream signaling events, cumulating in the up-regulation of IFN expression. To identify the point of inhibition by MAVS50, we employed reporter assay that takes advantage of the IFN-β promoter as a surrogate and the over-expressed key components outlined in Fig 5A. While MAVS50 inhibited transcription of the IFN-β promoter induced by RIG-I-N and MAVS70 (Figs 5B and 3D), MAVS50 had no inhibitory effect on the IFN-β promoter induced by TBK-1, IKKε and the constitutively active IRF3-5D mutant (Fig 5C and S4A and S4B Fig). These results suggest that MAVS50 targets a step between MAVS70 and TBK-1, a link between the common adaptor and bifurcated downstream signaling events that trigger IRF activation and IFN induction.


An Internally Translated MAVS Variant Exposes Its Amino-terminal TRAF-Binding Motifs to Deregulate Interferon Induction.

Minassian A, Zhang J, He S, Zhao J, Zandi E, Saito T, Liang C, Feng P - PLoS Pathog. (2015)

MAVS50 targets TRAF molecules to inhibit MAVS70-mediated IFN induction.(A) Diagram of key signaling molecules of the RIG-I-dependent IFN induction. (B and C) 293T cells were transfected with an IFN-β reporter cocktail, a plasmid containing RIG-I-N (B) or TBK-1 (C), and increasing amount of plasmid containing MAVS50. The IFN-β promoter activity was determined by luciferase assay at 30 hours post-transfection. **p<0.01; ***p<0.005. (D) 293T cells were transfected with plasmids containing MAVS wild-type (WT) or MAVS50 and a plasmid containing TRAF6. Whole cell lysates (WCL) were precipitated with anti-Flag (TRAF6). Precipitated proteins and WCLs were analyzed by immunoblotting with indicated antibodies. (E) 293T cells, depleted with endogenous MAVS and “reconstituted” with MAVS70 or MAVS50, were precipitated with anti-V5 (MAVS70 or MAVS50). Precipitated proteins and WCLs were analyzed by immunoblotting with indicated antibodies. (F) MAVS knockdown cells “reconstituted” with MAVS70-V5 were transfected with increasing amount of MAVS50-Flag. At 30 hours post-transfection, cells were infected with Sendai virus (SeV, 200 HA unit/ml) for two hours. WCLs were prepared and precipitated with anti-V5 (MAVS70) antibody. Precipitated proteins and WCLs were analyzed by immunoblotting with indicated antibodies. V, vector.
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Related In: Results  -  Collection

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ppat.1005060.g005: MAVS50 targets TRAF molecules to inhibit MAVS70-mediated IFN induction.(A) Diagram of key signaling molecules of the RIG-I-dependent IFN induction. (B and C) 293T cells were transfected with an IFN-β reporter cocktail, a plasmid containing RIG-I-N (B) or TBK-1 (C), and increasing amount of plasmid containing MAVS50. The IFN-β promoter activity was determined by luciferase assay at 30 hours post-transfection. **p<0.01; ***p<0.005. (D) 293T cells were transfected with plasmids containing MAVS wild-type (WT) or MAVS50 and a plasmid containing TRAF6. Whole cell lysates (WCL) were precipitated with anti-Flag (TRAF6). Precipitated proteins and WCLs were analyzed by immunoblotting with indicated antibodies. (E) 293T cells, depleted with endogenous MAVS and “reconstituted” with MAVS70 or MAVS50, were precipitated with anti-V5 (MAVS70 or MAVS50). Precipitated proteins and WCLs were analyzed by immunoblotting with indicated antibodies. (F) MAVS knockdown cells “reconstituted” with MAVS70-V5 were transfected with increasing amount of MAVS50-Flag. At 30 hours post-transfection, cells were infected with Sendai virus (SeV, 200 HA unit/ml) for two hours. WCLs were prepared and precipitated with anti-V5 (MAVS70) antibody. Precipitated proteins and WCLs were analyzed by immunoblotting with indicated antibodies. V, vector.
Mentions: The cytosolic sensor-mediated IFN induction pathway constitutes of key signaling molecules, including RIG-I/MDA5, MAVS, TBK-1/IKKε, and IRF3 (Fig 5A). Over-expression of these components is sufficient to activate downstream signaling events, cumulating in the up-regulation of IFN expression. To identify the point of inhibition by MAVS50, we employed reporter assay that takes advantage of the IFN-β promoter as a surrogate and the over-expressed key components outlined in Fig 5A. While MAVS50 inhibited transcription of the IFN-β promoter induced by RIG-I-N and MAVS70 (Figs 5B and 3D), MAVS50 had no inhibitory effect on the IFN-β promoter induced by TBK-1, IKKε and the constitutively active IRF3-5D mutant (Fig 5C and S4A and S4B Fig). These results suggest that MAVS50 targets a step between MAVS70 and TBK-1, a link between the common adaptor and bifurcated downstream signaling events that trigger IRF activation and IFN induction.

Bottom Line: By contrast, MAVS50 inhibits the IRF activation and suppresses IFN induction.Ablation of the TRAF-binding motif of MAVS50 impaired its inhibitory effect on IRF activation and IFN induction.These results collectively identify a new means by which signaling events is differentially regulated via exposing key internally embedded interaction motifs, implying a more ubiquitous regulatory role of truncated proteins arose from internal translation and other related mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology and Immunology, Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, California, United States of America.

ABSTRACT
Activation of pattern recognition receptors and proper regulation of downstream signaling are crucial for host innate immune response. Upon infection, the NF-κB and interferon regulatory factors (IRF) are often simultaneously activated to defeat invading pathogens. Mechanisms concerning differential activation of NF-κB and IRF are not well understood. Here we report that a MAVS variant inhibits interferon (IFN) induction, while enabling NF-κB activation. Employing herpesviral proteins that selectively activate NF-κB signaling, we discovered that a MAVS variant of ~50 kDa, thus designated MAVS50, was produced from internal translation initiation. MAVS50 preferentially interacts with TRAF2 and TRAF6, and activates NF-κB. By contrast, MAVS50 inhibits the IRF activation and suppresses IFN induction. Biochemical analysis showed that MAVS50, exposing a degenerate TRAF-binding motif within its N-terminus, effectively competed with full-length MAVS for recruiting TRAF2 and TRAF6. Ablation of the TRAF-binding motif of MAVS50 impaired its inhibitory effect on IRF activation and IFN induction. These results collectively identify a new means by which signaling events is differentially regulated via exposing key internally embedded interaction motifs, implying a more ubiquitous regulatory role of truncated proteins arose from internal translation and other related mechanisms.

No MeSH data available.


Related in: MedlinePlus