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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

Characterize the roles of MAVS50 in RIG-I-dependent signaling.(A and B) 293T cells were transfected with an NF-κB (A) or IFN-β (B) reporter cocktail and increasing amount of MAVS wild-type (WT), MAVS70 or MAVS50. Reporter activation was determined by luciferase assay at 30 hours post-transfection. (C) 293T cells were transfected with plasmids containing indicated genes. At 48 hours post-transfection, IKKβ kinase was precipitated and analyzed by in vitro kinase assay and immunoblotting with anti-IKKβ. Whole cell lysates were analyzed with anti-Flag (MAVS) and anti-GST (RIG-I-N). (D) 293T cells were transfected with plasmids containing Flag-MAVS70 and Flag-MAVS50. MAVS70 and MAVS50 were purified by affinity chromatography, eluted and analyzed by gel filtration chromatography with Superdex 200. Fractions (30 μl) were analyzed by immunoblotting with anti-Flag antibody. V0, void volume; numbers at the top indicate molecular weight in kDa. (E and F) MAVS in 293T cells was depleted with shRNA and analyzed by immunoblotting (E) and MAVS expression was “reconstituted” with lentivirus containing MAVS wild-type (WT), MAVS70 or MAVS50. Whole cell lysates were analyzed with anti-V5 antibody (F). (G) MAVS knockdown 293T cells “reconstituted” with control lentivirus (Vec) or lentivirus containing MAVS wild-type (WT), MAVS70 (70) or MAVS50 (50) as shown in (F), were mock- or infected with Sendai virus (SeV, 100 HAU/ml) for 8 hours, WCLs were prepared and analyzed by immunoblotting with indicated antibodies. (H) Infection of “reconstituted” 293T cells as described in (G). Mitochondrion-enriched fraction was obtained and analyzed by immunoblotting with indicated antibodies. (I and J) MAVS knockdown 293T cells, “reconstituted” with MAVS expression as described in (F), were infected with SeV (100 HA unit/ml) for 8 hours, RNA was extracted, cDNA was prepared and real-time PCR with primers specific for hIFNb and CCL5 were performed (I). Supernatants were collected and hIFNβ and hCCL5 were determined by ELISA (J).
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ppat.1005060.g002: Characterize the roles of MAVS50 in RIG-I-dependent signaling.(A and B) 293T cells were transfected with an NF-κB (A) or IFN-β (B) reporter cocktail and increasing amount of MAVS wild-type (WT), MAVS70 or MAVS50. Reporter activation was determined by luciferase assay at 30 hours post-transfection. (C) 293T cells were transfected with plasmids containing indicated genes. At 48 hours post-transfection, IKKβ kinase was precipitated and analyzed by in vitro kinase assay and immunoblotting with anti-IKKβ. Whole cell lysates were analyzed with anti-Flag (MAVS) and anti-GST (RIG-I-N). (D) 293T cells were transfected with plasmids containing Flag-MAVS70 and Flag-MAVS50. MAVS70 and MAVS50 were purified by affinity chromatography, eluted and analyzed by gel filtration chromatography with Superdex 200. Fractions (30 μl) were analyzed by immunoblotting with anti-Flag antibody. V0, void volume; numbers at the top indicate molecular weight in kDa. (E and F) MAVS in 293T cells was depleted with shRNA and analyzed by immunoblotting (E) and MAVS expression was “reconstituted” with lentivirus containing MAVS wild-type (WT), MAVS70 or MAVS50. Whole cell lysates were analyzed with anti-V5 antibody (F). (G) MAVS knockdown 293T cells “reconstituted” with control lentivirus (Vec) or lentivirus containing MAVS wild-type (WT), MAVS70 (70) or MAVS50 (50) as shown in (F), were mock- or infected with Sendai virus (SeV, 100 HAU/ml) for 8 hours, WCLs were prepared and analyzed by immunoblotting with indicated antibodies. (H) Infection of “reconstituted” 293T cells as described in (G). Mitochondrion-enriched fraction was obtained and analyzed by immunoblotting with indicated antibodies. (I and J) MAVS knockdown 293T cells, “reconstituted” with MAVS expression as described in (F), were infected with SeV (100 HA unit/ml) for 8 hours, RNA was extracted, cDNA was prepared and real-time PCR with primers specific for hIFNb and CCL5 were performed (I). Supernatants were collected and hIFNβ and hCCL5 were determined by ELISA (J).

Mentions: MAVS serves as an adaptor to relay signaling from RIG-I and MDA5 receptor to downstream kinases that bifurcate to activate NF-κB and IRF transcription factors [7,8]. To examine the roles of MAVS50 in these signaling cascades, we over-expressed MAVS50 and examined signaling events leading to NF-κB and IRF activation. Using reporter assays, we found that MAVS50 expression activated NF-κB (Fig 2A) in a dose-dependent manner. By contrast, MAVS50 expression did not up-regulate the promoter of IFN-β, but MAVS wild-type and MAVS70 did (Fig 2B). Consistent with the NF-κB activation, over-expressed MAVS50 also up-regulated the kinase activity of IKKβ by an in vitro kinase assay, in comparison to RIG-I-N and MAVS70 (Fig 2C). TRAF molecules are important adaptors downstream MAVS and are implicated in specific activation of NF-κB and IRF transcription factors. Thus, we examined MAVS50 interactions with a panel of six TRAF molecules by co-IP assays in transfected 293T cells. While MAVS70 interacted with all TRAF molecules except TRAF4, MAVS50 demonstrated preferential interaction with TRAF2 and TRAF6 (S1A and S1B Fig). This result suggests that MAVS50, in comparison to MAVS70, is distinct in interacting with downstream TRAF adaptors. When signaling events of TBK-1 and IRF activation were examined, we found that MAVS50 expression had no detectable effect on the kinase activity of TBK-1, nor the dimerization of IRF3 (S1C and S1D Fig), supporting the conclusion that MAVS50 does not activate the IRF signaling cascades. As controls, RIG-I-N (2CARDs) and MAVS70 potently activated TBK-1 by kinase assay and induced IRF3 dimerization by native gel electrophoresis. These results collectively show that MAVS50 preferentially activates the IKKβ-NF-κB signaling cascade.


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)

Characterize the roles of MAVS50 in RIG-I-dependent signaling.(A and B) 293T cells were transfected with an NF-κB (A) or IFN-β (B) reporter cocktail and increasing amount of MAVS wild-type (WT), MAVS70 or MAVS50. Reporter activation was determined by luciferase assay at 30 hours post-transfection. (C) 293T cells were transfected with plasmids containing indicated genes. At 48 hours post-transfection, IKKβ kinase was precipitated and analyzed by in vitro kinase assay and immunoblotting with anti-IKKβ. Whole cell lysates were analyzed with anti-Flag (MAVS) and anti-GST (RIG-I-N). (D) 293T cells were transfected with plasmids containing Flag-MAVS70 and Flag-MAVS50. MAVS70 and MAVS50 were purified by affinity chromatography, eluted and analyzed by gel filtration chromatography with Superdex 200. Fractions (30 μl) were analyzed by immunoblotting with anti-Flag antibody. V0, void volume; numbers at the top indicate molecular weight in kDa. (E and F) MAVS in 293T cells was depleted with shRNA and analyzed by immunoblotting (E) and MAVS expression was “reconstituted” with lentivirus containing MAVS wild-type (WT), MAVS70 or MAVS50. Whole cell lysates were analyzed with anti-V5 antibody (F). (G) MAVS knockdown 293T cells “reconstituted” with control lentivirus (Vec) or lentivirus containing MAVS wild-type (WT), MAVS70 (70) or MAVS50 (50) as shown in (F), were mock- or infected with Sendai virus (SeV, 100 HAU/ml) for 8 hours, WCLs were prepared and analyzed by immunoblotting with indicated antibodies. (H) Infection of “reconstituted” 293T cells as described in (G). Mitochondrion-enriched fraction was obtained and analyzed by immunoblotting with indicated antibodies. (I and J) MAVS knockdown 293T cells, “reconstituted” with MAVS expression as described in (F), were infected with SeV (100 HA unit/ml) for 8 hours, RNA was extracted, cDNA was prepared and real-time PCR with primers specific for hIFNb and CCL5 were performed (I). Supernatants were collected and hIFNβ and hCCL5 were determined by ELISA (J).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4519330&req=5

ppat.1005060.g002: Characterize the roles of MAVS50 in RIG-I-dependent signaling.(A and B) 293T cells were transfected with an NF-κB (A) or IFN-β (B) reporter cocktail and increasing amount of MAVS wild-type (WT), MAVS70 or MAVS50. Reporter activation was determined by luciferase assay at 30 hours post-transfection. (C) 293T cells were transfected with plasmids containing indicated genes. At 48 hours post-transfection, IKKβ kinase was precipitated and analyzed by in vitro kinase assay and immunoblotting with anti-IKKβ. Whole cell lysates were analyzed with anti-Flag (MAVS) and anti-GST (RIG-I-N). (D) 293T cells were transfected with plasmids containing Flag-MAVS70 and Flag-MAVS50. MAVS70 and MAVS50 were purified by affinity chromatography, eluted and analyzed by gel filtration chromatography with Superdex 200. Fractions (30 μl) were analyzed by immunoblotting with anti-Flag antibody. V0, void volume; numbers at the top indicate molecular weight in kDa. (E and F) MAVS in 293T cells was depleted with shRNA and analyzed by immunoblotting (E) and MAVS expression was “reconstituted” with lentivirus containing MAVS wild-type (WT), MAVS70 or MAVS50. Whole cell lysates were analyzed with anti-V5 antibody (F). (G) MAVS knockdown 293T cells “reconstituted” with control lentivirus (Vec) or lentivirus containing MAVS wild-type (WT), MAVS70 (70) or MAVS50 (50) as shown in (F), were mock- or infected with Sendai virus (SeV, 100 HAU/ml) for 8 hours, WCLs were prepared and analyzed by immunoblotting with indicated antibodies. (H) Infection of “reconstituted” 293T cells as described in (G). Mitochondrion-enriched fraction was obtained and analyzed by immunoblotting with indicated antibodies. (I and J) MAVS knockdown 293T cells, “reconstituted” with MAVS expression as described in (F), were infected with SeV (100 HA unit/ml) for 8 hours, RNA was extracted, cDNA was prepared and real-time PCR with primers specific for hIFNb and CCL5 were performed (I). Supernatants were collected and hIFNβ and hCCL5 were determined by ELISA (J).
Mentions: MAVS serves as an adaptor to relay signaling from RIG-I and MDA5 receptor to downstream kinases that bifurcate to activate NF-κB and IRF transcription factors [7,8]. To examine the roles of MAVS50 in these signaling cascades, we over-expressed MAVS50 and examined signaling events leading to NF-κB and IRF activation. Using reporter assays, we found that MAVS50 expression activated NF-κB (Fig 2A) in a dose-dependent manner. By contrast, MAVS50 expression did not up-regulate the promoter of IFN-β, but MAVS wild-type and MAVS70 did (Fig 2B). Consistent with the NF-κB activation, over-expressed MAVS50 also up-regulated the kinase activity of IKKβ by an in vitro kinase assay, in comparison to RIG-I-N and MAVS70 (Fig 2C). TRAF molecules are important adaptors downstream MAVS and are implicated in specific activation of NF-κB and IRF transcription factors. Thus, we examined MAVS50 interactions with a panel of six TRAF molecules by co-IP assays in transfected 293T cells. While MAVS70 interacted with all TRAF molecules except TRAF4, MAVS50 demonstrated preferential interaction with TRAF2 and TRAF6 (S1A and S1B Fig). This result suggests that MAVS50, in comparison to MAVS70, is distinct in interacting with downstream TRAF adaptors. When signaling events of TBK-1 and IRF activation were examined, we found that MAVS50 expression had no detectable effect on the kinase activity of TBK-1, nor the dimerization of IRF3 (S1C and S1D Fig), supporting the conclusion that MAVS50 does not activate the IRF signaling cascades. As controls, RIG-I-N (2CARDs) and MAVS70 potently activated TBK-1 by kinase assay and induced IRF3 dimerization by native gel electrophoresis. These results collectively show that MAVS50 preferentially activates the IKKβ-NF-κB signaling cascade.

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