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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 inhibits MAVS70-dependent IFN induction.(A and B) 293T cells were transfected with an IFN-β (A) or NF-κB reporter cocktail (B), a plasmid containing MAVS70 and increasing amount of a plasmid containing MAVS50. The promoter activity of IFN-β and NF-κB was determined by luciferase assay at 30 hours post-transfection. **p<0.01; ***p<0.005. (C and D) 293T cells were infected with control (CTL) lentivirus or lentivirus containing MAVS50. Whole cell lysates were analyzed with indicated antibodies (C). Stable 293T cells were infected with SeV (100 HA unit/ml) for 8 hours and RNA was extracted. cDNA was prepared and analyzed by real-time PCR with primers specific for IFNβ and ISG56 (D). (E and F) 293T cells were transfected with vector or plasmids containing MAVS wild-type (WT), MAVS70 or MAVS50. At 24 hours post-transfection, cells were infected with VSV-GFP (MOI = 0.01). Cells were photographed at 24 hours post-infection (E) and VSV in the supernatant was determined by plaque assay (F).
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ppat.1005060.g004: MAVS50 inhibits MAVS70-dependent IFN induction.(A and B) 293T cells were transfected with an IFN-β (A) or NF-κB reporter cocktail (B), a plasmid containing MAVS70 and increasing amount of a plasmid containing MAVS50. The promoter activity of IFN-β and NF-κB was determined by luciferase assay at 30 hours post-transfection. **p<0.01; ***p<0.005. (C and D) 293T cells were infected with control (CTL) lentivirus or lentivirus containing MAVS50. Whole cell lysates were analyzed with indicated antibodies (C). Stable 293T cells were infected with SeV (100 HA unit/ml) for 8 hours and RNA was extracted. cDNA was prepared and analyzed by real-time PCR with primers specific for IFNβ and ISG56 (D). (E and F) 293T cells were transfected with vector or plasmids containing MAVS wild-type (WT), MAVS70 or MAVS50. At 24 hours post-transfection, cells were infected with VSV-GFP (MOI = 0.01). Cells were photographed at 24 hours post-infection (E) and VSV in the supernatant was determined by plaque assay (F).

Mentions: To determine the effect of MAVS50 on MAVS70-mediated signaling, we assessed activation of NF-κB and IFN-β promoter by reporter assays. We found that MAVS50 inhibited MAVS70-induced transcription of the IFN-β promoter in a dose-dependent manner (Fig 4A). By contrast, MAVS50 did not significantly impact the NF-κB activation by MAVS70 (Fig 4B). We noted that MAVS50 was capable of activating NF-κB. We then expressed exogenous MAVS50 either by lentivirus transduction or transient transfection, and examined host cytokine gene expression and viral replication. When MAVS50 was expressed in 293T cells by lentivirus transduction (Fig 4C), while ISG56 expression was not significantly impacted, the expression of IFNβ was reduced by 50% in response to SeV infection (Fig 4D). Conversely, exogenously expressed MAVS50 enhanced the replication of vesicular stomatitis virus (VSV), a prototype RNA virus, by fluorescence microscopy (Fig 4E). Plaque assay further showed that MAVS50 expression increased VSV replication by more than 5-fold (Fig 4F). Taken together, MAVS50 inhibits IFN-β induction in response to viral infection.


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 inhibits MAVS70-dependent IFN induction.(A and B) 293T cells were transfected with an IFN-β (A) or NF-κB reporter cocktail (B), a plasmid containing MAVS70 and increasing amount of a plasmid containing MAVS50. The promoter activity of IFN-β and NF-κB was determined by luciferase assay at 30 hours post-transfection. **p<0.01; ***p<0.005. (C and D) 293T cells were infected with control (CTL) lentivirus or lentivirus containing MAVS50. Whole cell lysates were analyzed with indicated antibodies (C). Stable 293T cells were infected with SeV (100 HA unit/ml) for 8 hours and RNA was extracted. cDNA was prepared and analyzed by real-time PCR with primers specific for IFNβ and ISG56 (D). (E and F) 293T cells were transfected with vector or plasmids containing MAVS wild-type (WT), MAVS70 or MAVS50. At 24 hours post-transfection, cells were infected with VSV-GFP (MOI = 0.01). Cells were photographed at 24 hours post-infection (E) and VSV in the supernatant was determined by plaque assay (F).
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ppat.1005060.g004: MAVS50 inhibits MAVS70-dependent IFN induction.(A and B) 293T cells were transfected with an IFN-β (A) or NF-κB reporter cocktail (B), a plasmid containing MAVS70 and increasing amount of a plasmid containing MAVS50. The promoter activity of IFN-β and NF-κB was determined by luciferase assay at 30 hours post-transfection. **p<0.01; ***p<0.005. (C and D) 293T cells were infected with control (CTL) lentivirus or lentivirus containing MAVS50. Whole cell lysates were analyzed with indicated antibodies (C). Stable 293T cells were infected with SeV (100 HA unit/ml) for 8 hours and RNA was extracted. cDNA was prepared and analyzed by real-time PCR with primers specific for IFNβ and ISG56 (D). (E and F) 293T cells were transfected with vector or plasmids containing MAVS wild-type (WT), MAVS70 or MAVS50. At 24 hours post-transfection, cells were infected with VSV-GFP (MOI = 0.01). Cells were photographed at 24 hours post-infection (E) and VSV in the supernatant was determined by plaque assay (F).
Mentions: To determine the effect of MAVS50 on MAVS70-mediated signaling, we assessed activation of NF-κB and IFN-β promoter by reporter assays. We found that MAVS50 inhibited MAVS70-induced transcription of the IFN-β promoter in a dose-dependent manner (Fig 4A). By contrast, MAVS50 did not significantly impact the NF-κB activation by MAVS70 (Fig 4B). We noted that MAVS50 was capable of activating NF-κB. We then expressed exogenous MAVS50 either by lentivirus transduction or transient transfection, and examined host cytokine gene expression and viral replication. When MAVS50 was expressed in 293T cells by lentivirus transduction (Fig 4C), while ISG56 expression was not significantly impacted, the expression of IFNβ was reduced by 50% in response to SeV infection (Fig 4D). Conversely, exogenously expressed MAVS50 enhanced the replication of vesicular stomatitis virus (VSV), a prototype RNA virus, by fluorescence microscopy (Fig 4E). Plaque assay further showed that MAVS50 expression increased VSV replication by more than 5-fold (Fig 4F). Taken together, MAVS50 inhibits IFN-β induction in response to viral infection.

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