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TRAF5 is a downstream target of MAVS in antiviral innate immune signaling.

Tang ED, Wang CY - PLoS ONE (2010)

Bottom Line: The recognition of nucleic acids by the innate immune system during viral infection results in the production of type I interferons and the activation of antiviral immune responses.Alternatively, the activation of NF-kappaB leads to proinflammatory cytokine production.However, TRAF3-deficient cells display only a partial reduction in interferon production in response to RNA virus infection and are not defective in NF-kappaB activation.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Signalling, Division of Oral Biology and Medicine, University of California Los Angeles School of Dentistry, Los Angeles, California, United States of America.

ABSTRACT
The recognition of nucleic acids by the innate immune system during viral infection results in the production of type I interferons and the activation of antiviral immune responses. The RNA helicases RIG-I and MDA-5 recognize distinct types of cytosolic RNA species and signal through the mitochondrial protein MAVS to stimulate the phosphorylation and activation of the transcription factors IRF3 and IRF7, thereby inducing type I interferon expression. Alternatively, the activation of NF-kappaB leads to proinflammatory cytokine production. The function of MAVS is dependent on both its C-terminal transmembrane (TM) domain and N-terminal caspase recruitment domain (CARD). The TM domain mediates MAVS dimerization in response to viral RNA, allowing the CARD to bind to and activate the downstream effector TRAF3. Notably, dimerization of the MAVS CARD alone is sufficient to activate IRF3, IRF7, and NF-kappaB. However, TRAF3-deficient cells display only a partial reduction in interferon production in response to RNA virus infection and are not defective in NF-kappaB activation. Here we find that the related ubiquitin ligase TRAF5 is a downstream target of MAVS that mediates both IRF3 and NF-kappaB activation. The TM domain of MAVS allows it to dimerize and thereby associate with TRAF5 and induce its ubiquitination in a CARD-dependent manner. Also, NEMO is recruited to the dimerized MAVS CARD domain in a TRAF3 and TRAF5-dependent manner. Thus, our findings reveal a possible function for TRAF5 in mediating the activation of IRF3 and NF-kappaB downstream of MAVS through the recruitment of NEMO. TRAF5 may be a key molecule in the innate response against viral infection.

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Dimerization of the MAVS CARD restores activation of NF-κB.A, C, D HEK293T cells were transfected with wild-type or mutant MAVS, and an NF-κB reporter, pLuc-PRD(II)2. MAVS CARDmt contains a substitution of alanine for tryptophan at residue 68. Lysates were prepared and luciferase reporter assay was performed 24 hrs later. AP1510 was added 12 hrs prior to cell lysis (C). In the inset figure in (A), a western blot of lysates for FLAG-MAVS proteins was performed (A). B, E HEK293T cells were cotransfected with HA-IkBα and pcDNA3 or MAVS proteins and lysates were prepared. Anti-HA immunoprecipitates were probed for phosphorylated IkBα. Lysates were probed for HA-IkBα and FLAG-tagged MAVS proteins (E).
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pone-0009172-g001: Dimerization of the MAVS CARD restores activation of NF-κB.A, C, D HEK293T cells were transfected with wild-type or mutant MAVS, and an NF-κB reporter, pLuc-PRD(II)2. MAVS CARDmt contains a substitution of alanine for tryptophan at residue 68. Lysates were prepared and luciferase reporter assay was performed 24 hrs later. AP1510 was added 12 hrs prior to cell lysis (C). In the inset figure in (A), a western blot of lysates for FLAG-MAVS proteins was performed (A). B, E HEK293T cells were cotransfected with HA-IkBα and pcDNA3 or MAVS proteins and lysates were prepared. Anti-HA immunoprecipitates were probed for phosphorylated IkBα. Lysates were probed for HA-IkBα and FLAG-tagged MAVS proteins (E).

Mentions: Previously we found that removal of the MAVS TM domain impairs the ability of MAVS to dimerize, to activate IRF3/7 and NF-κB, and to induce IFN-β [25]. Importantly, enforced dimerization of a MAVS deletion mutant lacking its TM domain (MAVSΔTM) can restore its ability to activate IRF3/7 and induce IFN-β. These results suggested that the ability of MAVS to activate IRF3/7 is dependent on its ability to dimerize via the TM domain. To see whether the ability of MAVS to activate NF-κB was also reliant on its ability to dimerize, we examined whether enforced dimerization of MAVSΔTM could restore its ability to activate NF-κB. We tested the ability of a tandem repeat of MAVSΔTM with two copies fused end-to-end (MAVSΔTMx2) to activate NF-κB. In contrast to MAVSΔTM, the MAVSΔTMx2 fusion can phosphorylate IRF3 and activate IRF3/7 [25]. Similarly, we found that the MAVSΔTMx2 fusion, but not MAVSΔTM could elicit NF-κB activation and IkBα phosphorylation (Figure 1A and 1B). In addition, we also examined a fusion of the MAVS CARD to the three tandemly repeated dimerization domains of FPK (MAVS CARD-FPK3). Proteins attached to FPK can be induced to dimerize by the addition of the cell-permeable artificial ligand AP1510 [26]. We previously found that MAVS CARD-FPK3 could activate IRF3/7 activation and IFN-β production only in the presence of the dimerizer AP1510. We also found that this fusion protein could activate NF-κB only in the presence of dimerizer (Figure 1C). Substitution of a conserved tryptophan residue for alanine in the CARD domain (W68A), a mutation that prevents full-length MAVS from inducing IFN-β and activating IRF3 [25], also prevented MAVS CARD-FPK3 from activating NF-κB (Figure 1C).


TRAF5 is a downstream target of MAVS in antiviral innate immune signaling.

Tang ED, Wang CY - PLoS ONE (2010)

Dimerization of the MAVS CARD restores activation of NF-κB.A, C, D HEK293T cells were transfected with wild-type or mutant MAVS, and an NF-κB reporter, pLuc-PRD(II)2. MAVS CARDmt contains a substitution of alanine for tryptophan at residue 68. Lysates were prepared and luciferase reporter assay was performed 24 hrs later. AP1510 was added 12 hrs prior to cell lysis (C). In the inset figure in (A), a western blot of lysates for FLAG-MAVS proteins was performed (A). B, E HEK293T cells were cotransfected with HA-IkBα and pcDNA3 or MAVS proteins and lysates were prepared. Anti-HA immunoprecipitates were probed for phosphorylated IkBα. Lysates were probed for HA-IkBα and FLAG-tagged MAVS proteins (E).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2820086&req=5

pone-0009172-g001: Dimerization of the MAVS CARD restores activation of NF-κB.A, C, D HEK293T cells were transfected with wild-type or mutant MAVS, and an NF-κB reporter, pLuc-PRD(II)2. MAVS CARDmt contains a substitution of alanine for tryptophan at residue 68. Lysates were prepared and luciferase reporter assay was performed 24 hrs later. AP1510 was added 12 hrs prior to cell lysis (C). In the inset figure in (A), a western blot of lysates for FLAG-MAVS proteins was performed (A). B, E HEK293T cells were cotransfected with HA-IkBα and pcDNA3 or MAVS proteins and lysates were prepared. Anti-HA immunoprecipitates were probed for phosphorylated IkBα. Lysates were probed for HA-IkBα and FLAG-tagged MAVS proteins (E).
Mentions: Previously we found that removal of the MAVS TM domain impairs the ability of MAVS to dimerize, to activate IRF3/7 and NF-κB, and to induce IFN-β [25]. Importantly, enforced dimerization of a MAVS deletion mutant lacking its TM domain (MAVSΔTM) can restore its ability to activate IRF3/7 and induce IFN-β. These results suggested that the ability of MAVS to activate IRF3/7 is dependent on its ability to dimerize via the TM domain. To see whether the ability of MAVS to activate NF-κB was also reliant on its ability to dimerize, we examined whether enforced dimerization of MAVSΔTM could restore its ability to activate NF-κB. We tested the ability of a tandem repeat of MAVSΔTM with two copies fused end-to-end (MAVSΔTMx2) to activate NF-κB. In contrast to MAVSΔTM, the MAVSΔTMx2 fusion can phosphorylate IRF3 and activate IRF3/7 [25]. Similarly, we found that the MAVSΔTMx2 fusion, but not MAVSΔTM could elicit NF-κB activation and IkBα phosphorylation (Figure 1A and 1B). In addition, we also examined a fusion of the MAVS CARD to the three tandemly repeated dimerization domains of FPK (MAVS CARD-FPK3). Proteins attached to FPK can be induced to dimerize by the addition of the cell-permeable artificial ligand AP1510 [26]. We previously found that MAVS CARD-FPK3 could activate IRF3/7 activation and IFN-β production only in the presence of the dimerizer AP1510. We also found that this fusion protein could activate NF-κB only in the presence of dimerizer (Figure 1C). Substitution of a conserved tryptophan residue for alanine in the CARD domain (W68A), a mutation that prevents full-length MAVS from inducing IFN-β and activating IRF3 [25], also prevented MAVS CARD-FPK3 from activating NF-κB (Figure 1C).

Bottom Line: The recognition of nucleic acids by the innate immune system during viral infection results in the production of type I interferons and the activation of antiviral immune responses.Alternatively, the activation of NF-kappaB leads to proinflammatory cytokine production.However, TRAF3-deficient cells display only a partial reduction in interferon production in response to RNA virus infection and are not defective in NF-kappaB activation.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Signalling, Division of Oral Biology and Medicine, University of California Los Angeles School of Dentistry, Los Angeles, California, United States of America.

ABSTRACT
The recognition of nucleic acids by the innate immune system during viral infection results in the production of type I interferons and the activation of antiviral immune responses. The RNA helicases RIG-I and MDA-5 recognize distinct types of cytosolic RNA species and signal through the mitochondrial protein MAVS to stimulate the phosphorylation and activation of the transcription factors IRF3 and IRF7, thereby inducing type I interferon expression. Alternatively, the activation of NF-kappaB leads to proinflammatory cytokine production. The function of MAVS is dependent on both its C-terminal transmembrane (TM) domain and N-terminal caspase recruitment domain (CARD). The TM domain mediates MAVS dimerization in response to viral RNA, allowing the CARD to bind to and activate the downstream effector TRAF3. Notably, dimerization of the MAVS CARD alone is sufficient to activate IRF3, IRF7, and NF-kappaB. However, TRAF3-deficient cells display only a partial reduction in interferon production in response to RNA virus infection and are not defective in NF-kappaB activation. Here we find that the related ubiquitin ligase TRAF5 is a downstream target of MAVS that mediates both IRF3 and NF-kappaB activation. The TM domain of MAVS allows it to dimerize and thereby associate with TRAF5 and induce its ubiquitination in a CARD-dependent manner. Also, NEMO is recruited to the dimerized MAVS CARD domain in a TRAF3 and TRAF5-dependent manner. Thus, our findings reveal a possible function for TRAF5 in mediating the activation of IRF3 and NF-kappaB downstream of MAVS through the recruitment of NEMO. TRAF5 may be a key molecule in the innate response against viral infection.

Show MeSH
Related in: MedlinePlus