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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

Identification of the MAVS50 variant.(A) 293T cells were infected with SeV (100 HA unit/ml) for 2 hours or transfected with a plasmid containing murine γHV68 vGAT for 24 hours. Whole cell lysates (WCL) were prepared in Triton X-100 buffer to separate into soluble and insoluble (pellet) fractions, which were analyzed by immunoblotting with indicated antibodies. (B) Diagram of the mRNA of the full-length MAVS (or MAVS70) and MAVS50. (C) WCLs of indicated amount were analyzed with antibodies against the first 135 amino acids (αMAVS1-135) or an internal sequence encompassing amino acids 150–250 (αMAVS150-250). (D) The expression of MAVS, carrying a N-terminal Flag tag and a C-terminal HA tag, in 293T cells was analyzed by immunoblotting with anti-Flag and anti-HA antibodies, along with endogenous MAVS (left panel). (E) 293T cells were transfected with plasmids containing wild-type MAVS and indicated mutants. WCLs were analyzed by immunoblotting with anti-MAVS antibody (αMAVS150-250). Δ, deletion. (F) WCLs of indicated cells were analyzed by immunoblotting with anti-MAVS (αMAVS150-250) and anti-β-actin. Note, antibody against human MAVS does not react with murine MAVS in NIH 3T3 cells. (G and H) 293T cells were infected with Sendai virus (SeV, 100 HA Unit/ml) (G) and HSV-1 (MOI = 5) (H) and cells were harvested at indicated time points. WCLs were analyzed by immunoblotting with antibody against MAVS.
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ppat.1005060.g001: Identification of the MAVS50 variant.(A) 293T cells were infected with SeV (100 HA unit/ml) for 2 hours or transfected with a plasmid containing murine γHV68 vGAT for 24 hours. Whole cell lysates (WCL) were prepared in Triton X-100 buffer to separate into soluble and insoluble (pellet) fractions, which were analyzed by immunoblotting with indicated antibodies. (B) Diagram of the mRNA of the full-length MAVS (or MAVS70) and MAVS50. (C) WCLs of indicated amount were analyzed with antibodies against the first 135 amino acids (αMAVS1-135) or an internal sequence encompassing amino acids 150–250 (αMAVS150-250). (D) The expression of MAVS, carrying a N-terminal Flag tag and a C-terminal HA tag, in 293T cells was analyzed by immunoblotting with anti-Flag and anti-HA antibodies, along with endogenous MAVS (left panel). (E) 293T cells were transfected with plasmids containing wild-type MAVS and indicated mutants. WCLs were analyzed by immunoblotting with anti-MAVS antibody (αMAVS150-250). Δ, deletion. (F) WCLs of indicated cells were analyzed by immunoblotting with anti-MAVS (αMAVS150-250) and anti-β-actin. Note, antibody against human MAVS does not react with murine MAVS in NIH 3T3 cells. (G and H) 293T cells were infected with Sendai virus (SeV, 100 HA Unit/ml) (G) and HSV-1 (MOI = 5) (H) and cells were harvested at indicated time points. WCLs were analyzed by immunoblotting with antibody against MAVS.

Mentions: We have previously identified gamma herpesviral homologues of glutamine amidotransferase (referred to as vGAT) activate RIG-I via deamidation [27]. We noted a remarkable feature of this innate immune activation is the preferential activation of the NF-κB signaling cascade, but not that of IRF and IFN induction. In an experiment that aims to examine MAVS activation by Sendai virus (SeV) infection or expression of γHV68 vGAT, we observed that a smaller isoform of MAVS, of ~50 kD (designated MAVS50), did not migrate into the Triton X-100-insoluble fraction in cells infected with SeV or expressing γHV68 vGAT (Fig 1A). In contrast, the full-length MAVS, designated as MAVS70, accumulated in the Triton X-insoluble fraction, indicative of its activation [8]. This result suggests that MAVS50 likely possesses function distinct from its kin, MAVS70.


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)

Identification of the MAVS50 variant.(A) 293T cells were infected with SeV (100 HA unit/ml) for 2 hours or transfected with a plasmid containing murine γHV68 vGAT for 24 hours. Whole cell lysates (WCL) were prepared in Triton X-100 buffer to separate into soluble and insoluble (pellet) fractions, which were analyzed by immunoblotting with indicated antibodies. (B) Diagram of the mRNA of the full-length MAVS (or MAVS70) and MAVS50. (C) WCLs of indicated amount were analyzed with antibodies against the first 135 amino acids (αMAVS1-135) or an internal sequence encompassing amino acids 150–250 (αMAVS150-250). (D) The expression of MAVS, carrying a N-terminal Flag tag and a C-terminal HA tag, in 293T cells was analyzed by immunoblotting with anti-Flag and anti-HA antibodies, along with endogenous MAVS (left panel). (E) 293T cells were transfected with plasmids containing wild-type MAVS and indicated mutants. WCLs were analyzed by immunoblotting with anti-MAVS antibody (αMAVS150-250). Δ, deletion. (F) WCLs of indicated cells were analyzed by immunoblotting with anti-MAVS (αMAVS150-250) and anti-β-actin. Note, antibody against human MAVS does not react with murine MAVS in NIH 3T3 cells. (G and H) 293T cells were infected with Sendai virus (SeV, 100 HA Unit/ml) (G) and HSV-1 (MOI = 5) (H) and cells were harvested at indicated time points. WCLs were analyzed by immunoblotting with antibody against MAVS.
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ppat.1005060.g001: Identification of the MAVS50 variant.(A) 293T cells were infected with SeV (100 HA unit/ml) for 2 hours or transfected with a plasmid containing murine γHV68 vGAT for 24 hours. Whole cell lysates (WCL) were prepared in Triton X-100 buffer to separate into soluble and insoluble (pellet) fractions, which were analyzed by immunoblotting with indicated antibodies. (B) Diagram of the mRNA of the full-length MAVS (or MAVS70) and MAVS50. (C) WCLs of indicated amount were analyzed with antibodies against the first 135 amino acids (αMAVS1-135) or an internal sequence encompassing amino acids 150–250 (αMAVS150-250). (D) The expression of MAVS, carrying a N-terminal Flag tag and a C-terminal HA tag, in 293T cells was analyzed by immunoblotting with anti-Flag and anti-HA antibodies, along with endogenous MAVS (left panel). (E) 293T cells were transfected with plasmids containing wild-type MAVS and indicated mutants. WCLs were analyzed by immunoblotting with anti-MAVS antibody (αMAVS150-250). Δ, deletion. (F) WCLs of indicated cells were analyzed by immunoblotting with anti-MAVS (αMAVS150-250) and anti-β-actin. Note, antibody against human MAVS does not react with murine MAVS in NIH 3T3 cells. (G and H) 293T cells were infected with Sendai virus (SeV, 100 HA Unit/ml) (G) and HSV-1 (MOI = 5) (H) and cells were harvested at indicated time points. WCLs were analyzed by immunoblotting with antibody against MAVS.
Mentions: We have previously identified gamma herpesviral homologues of glutamine amidotransferase (referred to as vGAT) activate RIG-I via deamidation [27]. We noted a remarkable feature of this innate immune activation is the preferential activation of the NF-κB signaling cascade, but not that of IRF and IFN induction. In an experiment that aims to examine MAVS activation by Sendai virus (SeV) infection or expression of γHV68 vGAT, we observed that a smaller isoform of MAVS, of ~50 kD (designated MAVS50), did not migrate into the Triton X-100-insoluble fraction in cells infected with SeV or expressing γHV68 vGAT (Fig 1A). In contrast, the full-length MAVS, designated as MAVS70, accumulated in the Triton X-insoluble fraction, indicative of its activation [8]. This result suggests that MAVS50 likely possesses function distinct from its kin, MAVS70.

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