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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 interacts with MAVS70.(A) 293T cells were transfected with plasmids containing MAVS wild-type, MAVS70 or MAVS50. Whole cell lysates (WCLs) were precipitated with anti-Flag (MAVS50). Precipitated proteins and WCLs were analyzed by immunoblotting with indicated antibodies. (B) 293T-Rex/MAVS50-Flag cell line was induced with doxycycline (100 ng/ml) for 24 hours. WCLs were precipitated with anti-Flag agarose (MAVS50). Precipitated proteins and WCLs were analyzed by immunoblotting with indicated antibodies. (C) 293T cells were transfected with a plasmid containing MAVS50-Flag, with or without a plasmid containing MAVS70-V5. MAVS50 was purified by affinity chromatography, eluted and analyzed by gel filtration chromatography. Fractions (30 μl) were analyzed by immunoblotting with anti-Flag and anti-V5 antibodies. (D) Whole cell lysates of 293T, HeLa and THP-1 macrophage were analyzed by gel filtration chromatography. Fractions (50 μl) were analyzed by immunoblotting with anti-MAVS antibody. For C and D, V0, void volume; numbers indicate molecule weight in kDa.
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ppat.1005060.g003: MAVS50 interacts with MAVS70.(A) 293T cells were transfected with plasmids containing MAVS wild-type, MAVS70 or MAVS50. Whole cell lysates (WCLs) were precipitated with anti-Flag (MAVS50). Precipitated proteins and WCLs were analyzed by immunoblotting with indicated antibodies. (B) 293T-Rex/MAVS50-Flag cell line was induced with doxycycline (100 ng/ml) for 24 hours. WCLs were precipitated with anti-Flag agarose (MAVS50). Precipitated proteins and WCLs were analyzed by immunoblotting with indicated antibodies. (C) 293T cells were transfected with a plasmid containing MAVS50-Flag, with or without a plasmid containing MAVS70-V5. MAVS50 was purified by affinity chromatography, eluted and analyzed by gel filtration chromatography. Fractions (30 μl) were analyzed by immunoblotting with anti-Flag and anti-V5 antibodies. (D) Whole cell lysates of 293T, HeLa and THP-1 macrophage were analyzed by gel filtration chromatography. Fractions (50 μl) were analyzed by immunoblotting with anti-MAVS antibody. For C and D, V0, void volume; numbers indicate molecule weight in kDa.

Mentions: MAVS50 lacks the CARD domain and fails to relay signal transduction downstream of RIG-I. Considering that MAVS50 possesses most of the sequence of MAVS70, we reasoned that MAVS50 likely regulates MAVS70-mediated signaling. To test this hypothesis, we first examined whether MAVS50 can interact with MAVS70 and itself. In transfected 293T cells, V5-tagged MAVS70 and MAVS50 were readily detected in protein complexes precipitated with anti-Flag antibody against Flag-MAVS50 (Fig 3A), indicating that MAVS50 can interact with MAVS70 and MAVS50. This result is consistent with our observation that MAVS50 eluted as ~120 kDa in gel filtration, which implies self-oligomerization of MAVS50 (Fig 2D). Due to the largely overlapping sequence between MAVS70 and MAVS50, it is technically challenging to probe the interaction between endogenous MAVS70 and MAVS50. Thus, we established a stable 293 cell line that expresses Flag-tagged MAVS50 under the control of doxycycline in a dose-dependent manner (S2D Fig). Precipitation of MAVS50 effectively pulled down MAVS70, indicating that MAVS50 physically associates with endogenous MAVS70 (Fig 3B).


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 interacts with MAVS70.(A) 293T cells were transfected with plasmids containing MAVS wild-type, MAVS70 or MAVS50. Whole cell lysates (WCLs) were precipitated with anti-Flag (MAVS50). Precipitated proteins and WCLs were analyzed by immunoblotting with indicated antibodies. (B) 293T-Rex/MAVS50-Flag cell line was induced with doxycycline (100 ng/ml) for 24 hours. WCLs were precipitated with anti-Flag agarose (MAVS50). Precipitated proteins and WCLs were analyzed by immunoblotting with indicated antibodies. (C) 293T cells were transfected with a plasmid containing MAVS50-Flag, with or without a plasmid containing MAVS70-V5. MAVS50 was purified by affinity chromatography, eluted and analyzed by gel filtration chromatography. Fractions (30 μl) were analyzed by immunoblotting with anti-Flag and anti-V5 antibodies. (D) Whole cell lysates of 293T, HeLa and THP-1 macrophage were analyzed by gel filtration chromatography. Fractions (50 μl) were analyzed by immunoblotting with anti-MAVS antibody. For C and D, V0, void volume; numbers indicate molecule weight in kDa.
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ppat.1005060.g003: MAVS50 interacts with MAVS70.(A) 293T cells were transfected with plasmids containing MAVS wild-type, MAVS70 or MAVS50. Whole cell lysates (WCLs) were precipitated with anti-Flag (MAVS50). Precipitated proteins and WCLs were analyzed by immunoblotting with indicated antibodies. (B) 293T-Rex/MAVS50-Flag cell line was induced with doxycycline (100 ng/ml) for 24 hours. WCLs were precipitated with anti-Flag agarose (MAVS50). Precipitated proteins and WCLs were analyzed by immunoblotting with indicated antibodies. (C) 293T cells were transfected with a plasmid containing MAVS50-Flag, with or without a plasmid containing MAVS70-V5. MAVS50 was purified by affinity chromatography, eluted and analyzed by gel filtration chromatography. Fractions (30 μl) were analyzed by immunoblotting with anti-Flag and anti-V5 antibodies. (D) Whole cell lysates of 293T, HeLa and THP-1 macrophage were analyzed by gel filtration chromatography. Fractions (50 μl) were analyzed by immunoblotting with anti-MAVS antibody. For C and D, V0, void volume; numbers indicate molecule weight in kDa.
Mentions: MAVS50 lacks the CARD domain and fails to relay signal transduction downstream of RIG-I. Considering that MAVS50 possesses most of the sequence of MAVS70, we reasoned that MAVS50 likely regulates MAVS70-mediated signaling. To test this hypothesis, we first examined whether MAVS50 can interact with MAVS70 and itself. In transfected 293T cells, V5-tagged MAVS70 and MAVS50 were readily detected in protein complexes precipitated with anti-Flag antibody against Flag-MAVS50 (Fig 3A), indicating that MAVS50 can interact with MAVS70 and MAVS50. This result is consistent with our observation that MAVS50 eluted as ~120 kDa in gel filtration, which implies self-oligomerization of MAVS50 (Fig 2D). Due to the largely overlapping sequence between MAVS70 and MAVS50, it is technically challenging to probe the interaction between endogenous MAVS70 and MAVS50. Thus, we established a stable 293 cell line that expresses Flag-tagged MAVS50 under the control of doxycycline in a dose-dependent manner (S2D Fig). Precipitation of MAVS50 effectively pulled down MAVS70, indicating that MAVS50 physically associates with endogenous MAVS70 (Fig 3B).

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