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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

The N-terminal TRAF2-binding motif is critical for MAVS50 to inhibit IFN induction.(A) Diagram of the TRAF2-binding motif (T2BM) and TRAF6-binding motif (T6BM) in MAVS50, in relation to MAVS70. (B and C) 293T cells were transfected with plasmids containing Flag-TRAF2 (B) or Flag-TRAF6 (C) and plasmids containing MAVS50 wild-type (WT), mutant of TRAF2-binding (M2) or TRAF6-binding (M6) or both TRAF2- and TRAF6-binding (M2,6). Whole cell lysates (WCLs) were prepared at 30 hours post-transfection and precipitated with anti-Flag agarose or anti-HA agarose (as negative control). Precipitated proteins and WCLs were analyzed by immunoblotting with indicated antibodies. (D) 293T cells were transfected with an IFN-β reporter cocktail, a plasmid containing MAVS70 and increasing amount of a plasmid containing MAVS50 wild-type (WT) or MAVS50 M2,6 mutant. The IFN-β promoter activity was determined by luciferase assay at 30 hours post-transfection. **p<0.01; ***p<0.005. (E) 293T cells were infected with control lentivirus (CTL) or lentivirus containing MAVS50 wild-type or MAVS50 M2,6 mutant. At 48 hours, cells were infected with SeV (100 HA unit/ml) for 8 hours. RNA was extracted and cDNA were prepared for real-time PCR analysis with primers specific for IFNβ and ISG56. (F and G) 293T cells were transfected with an empty plasmid (Vector) or a plasmid containing MAVS50 or MAVS50 M2,6 mutant. At 24 hours post-transfection, cells were infected with VSV-GFP (MOI = 0.01). Cells were photographed at 24 hours post-infection (F) and virus in the supernatant was determined by plaque assay (G).
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ppat.1005060.g006: The N-terminal TRAF2-binding motif is critical for MAVS50 to inhibit IFN induction.(A) Diagram of the TRAF2-binding motif (T2BM) and TRAF6-binding motif (T6BM) in MAVS50, in relation to MAVS70. (B and C) 293T cells were transfected with plasmids containing Flag-TRAF2 (B) or Flag-TRAF6 (C) and plasmids containing MAVS50 wild-type (WT), mutant of TRAF2-binding (M2) or TRAF6-binding (M6) or both TRAF2- and TRAF6-binding (M2,6). Whole cell lysates (WCLs) were prepared at 30 hours post-transfection and precipitated with anti-Flag agarose or anti-HA agarose (as negative control). Precipitated proteins and WCLs were analyzed by immunoblotting with indicated antibodies. (D) 293T cells were transfected with an IFN-β reporter cocktail, a plasmid containing MAVS70 and increasing amount of a plasmid containing MAVS50 wild-type (WT) or MAVS50 M2,6 mutant. The IFN-β promoter activity was determined by luciferase assay at 30 hours post-transfection. **p<0.01; ***p<0.005. (E) 293T cells were infected with control lentivirus (CTL) or lentivirus containing MAVS50 wild-type or MAVS50 M2,6 mutant. At 48 hours, cells were infected with SeV (100 HA unit/ml) for 8 hours. RNA was extracted and cDNA were prepared for real-time PCR analysis with primers specific for IFNβ and ISG56. (F and G) 293T cells were transfected with an empty plasmid (Vector) or a plasmid containing MAVS50 or MAVS50 M2,6 mutant. At 24 hours post-transfection, cells were infected with VSV-GFP (MOI = 0.01). Cells were photographed at 24 hours post-infection (F) and virus in the supernatant was determined by plaque assay (G).

Mentions: A major TRAF2-binding motif (PVQE, [P/S/A/T]x[Q/E]E) locates at the very amino terminus of MAVS50 and a TRAF6-binding motif (PGENSE, PxExx[Ar/Ac]; Ar, aromatic; Ac, acetic)[32,33] immediately follows the TRAF2-binding motif (Fig 6A). To probe the contribution of these N-terminal TRAF-binding motifs, we mutated the critical residues of TRAF2- and TRAF6-binding motifs into alanines, thus named M2 and M6 of MAVS50 (S5A Fig), and examined their interactions with TRAF2 or TRAF6 by co-IP assay. As expected, mutations within the TRAF2-binding motif abolished MAVS50 interaction with TRAF2, while mutations within the TRAF6-binding motif had no effect on MAVS50 interaction with TRAF2 (Fig 6B). MAVS50 mutant ablated both TRAF2- and TRAF6-binding motifs lost the interaction with TRAF2. Surprisingly, mutating the TRAF2-binding motif nearly abolished the MAVS50 interaction with TRAF6 (Fig 6C). Mutations within the immediate downstream TRAF6-binding motif, although reduced MAVS50 interaction with TRAF6, had less effect than mutations within the TRAF2-binding motif (Fig 6C). Simultaneously mutating the TRAF2- and TRAF6-binding motifs reduced MAVS50 association with TRAF6 to residual level. The residual level of TRAF6 interaction of MAVS50 M2,6 mutant is likely due to the transmembrane-proximal TRAF6-binding motif. Additionally, the MAVS50 M2,6 mutant also demonstrated reduced interactions with TRAF3 and TRAF5 in transfected 293T cells (S5B and S5C Fig). Mutations within the TRAF2- and TRAF6-binding motifs had no significant effect on MAVS50 interaction with MAVS70 by co-IP assay (S5D Fig), suggesting that these MAVS50 mutants are functionally competent. These results indicate that the very amino-terminal TRAF2-binding motif is critical for binding to both TRAF2 and TRAF6, and likely other TRAFs, suggesting that the TRAF2-binding motif is a functionally degenerate interaction motif for more than one TRAF molecule.


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)

The N-terminal TRAF2-binding motif is critical for MAVS50 to inhibit IFN induction.(A) Diagram of the TRAF2-binding motif (T2BM) and TRAF6-binding motif (T6BM) in MAVS50, in relation to MAVS70. (B and C) 293T cells were transfected with plasmids containing Flag-TRAF2 (B) or Flag-TRAF6 (C) and plasmids containing MAVS50 wild-type (WT), mutant of TRAF2-binding (M2) or TRAF6-binding (M6) or both TRAF2- and TRAF6-binding (M2,6). Whole cell lysates (WCLs) were prepared at 30 hours post-transfection and precipitated with anti-Flag agarose or anti-HA agarose (as negative control). Precipitated proteins and WCLs were analyzed by immunoblotting with indicated antibodies. (D) 293T cells were transfected with an IFN-β reporter cocktail, a plasmid containing MAVS70 and increasing amount of a plasmid containing MAVS50 wild-type (WT) or MAVS50 M2,6 mutant. The IFN-β promoter activity was determined by luciferase assay at 30 hours post-transfection. **p<0.01; ***p<0.005. (E) 293T cells were infected with control lentivirus (CTL) or lentivirus containing MAVS50 wild-type or MAVS50 M2,6 mutant. At 48 hours, cells were infected with SeV (100 HA unit/ml) for 8 hours. RNA was extracted and cDNA were prepared for real-time PCR analysis with primers specific for IFNβ and ISG56. (F and G) 293T cells were transfected with an empty plasmid (Vector) or a plasmid containing MAVS50 or MAVS50 M2,6 mutant. At 24 hours post-transfection, cells were infected with VSV-GFP (MOI = 0.01). Cells were photographed at 24 hours post-infection (F) and virus in the supernatant was determined by plaque assay (G).
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ppat.1005060.g006: The N-terminal TRAF2-binding motif is critical for MAVS50 to inhibit IFN induction.(A) Diagram of the TRAF2-binding motif (T2BM) and TRAF6-binding motif (T6BM) in MAVS50, in relation to MAVS70. (B and C) 293T cells were transfected with plasmids containing Flag-TRAF2 (B) or Flag-TRAF6 (C) and plasmids containing MAVS50 wild-type (WT), mutant of TRAF2-binding (M2) or TRAF6-binding (M6) or both TRAF2- and TRAF6-binding (M2,6). Whole cell lysates (WCLs) were prepared at 30 hours post-transfection and precipitated with anti-Flag agarose or anti-HA agarose (as negative control). Precipitated proteins and WCLs were analyzed by immunoblotting with indicated antibodies. (D) 293T cells were transfected with an IFN-β reporter cocktail, a plasmid containing MAVS70 and increasing amount of a plasmid containing MAVS50 wild-type (WT) or MAVS50 M2,6 mutant. The IFN-β promoter activity was determined by luciferase assay at 30 hours post-transfection. **p<0.01; ***p<0.005. (E) 293T cells were infected with control lentivirus (CTL) or lentivirus containing MAVS50 wild-type or MAVS50 M2,6 mutant. At 48 hours, cells were infected with SeV (100 HA unit/ml) for 8 hours. RNA was extracted and cDNA were prepared for real-time PCR analysis with primers specific for IFNβ and ISG56. (F and G) 293T cells were transfected with an empty plasmid (Vector) or a plasmid containing MAVS50 or MAVS50 M2,6 mutant. At 24 hours post-transfection, cells were infected with VSV-GFP (MOI = 0.01). Cells were photographed at 24 hours post-infection (F) and virus in the supernatant was determined by plaque assay (G).
Mentions: A major TRAF2-binding motif (PVQE, [P/S/A/T]x[Q/E]E) locates at the very amino terminus of MAVS50 and a TRAF6-binding motif (PGENSE, PxExx[Ar/Ac]; Ar, aromatic; Ac, acetic)[32,33] immediately follows the TRAF2-binding motif (Fig 6A). To probe the contribution of these N-terminal TRAF-binding motifs, we mutated the critical residues of TRAF2- and TRAF6-binding motifs into alanines, thus named M2 and M6 of MAVS50 (S5A Fig), and examined their interactions with TRAF2 or TRAF6 by co-IP assay. As expected, mutations within the TRAF2-binding motif abolished MAVS50 interaction with TRAF2, while mutations within the TRAF6-binding motif had no effect on MAVS50 interaction with TRAF2 (Fig 6B). MAVS50 mutant ablated both TRAF2- and TRAF6-binding motifs lost the interaction with TRAF2. Surprisingly, mutating the TRAF2-binding motif nearly abolished the MAVS50 interaction with TRAF6 (Fig 6C). Mutations within the immediate downstream TRAF6-binding motif, although reduced MAVS50 interaction with TRAF6, had less effect than mutations within the TRAF2-binding motif (Fig 6C). Simultaneously mutating the TRAF2- and TRAF6-binding motifs reduced MAVS50 association with TRAF6 to residual level. The residual level of TRAF6 interaction of MAVS50 M2,6 mutant is likely due to the transmembrane-proximal TRAF6-binding motif. Additionally, the MAVS50 M2,6 mutant also demonstrated reduced interactions with TRAF3 and TRAF5 in transfected 293T cells (S5B and S5C Fig). Mutations within the TRAF2- and TRAF6-binding motifs had no significant effect on MAVS50 interaction with MAVS70 by co-IP assay (S5D Fig), suggesting that these MAVS50 mutants are functionally competent. These results indicate that the very amino-terminal TRAF2-binding motif is critical for binding to both TRAF2 and TRAF6, and likely other TRAFs, suggesting that the TRAF2-binding motif is a functionally degenerate interaction motif for more than one TRAF molecule.

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