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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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NEMO is recruited to the MAVS CARD in a TRAF3/5-dependent manner.A HEK293T cells were transfected with AU1-NEMO and FLAG-MAVS CARD-FPK3 or FLAG-MAVS CARDmt-FPK3 where indicated. MAVS CARDmt contains a substitution of alanine for tryptophan at residue 68. Lysates were prepared and anti-FLAG immunoprecipitates were probed for AU1-NEMO. Lysates were probed for FLAG-MAVS-CARD-FPK3 protein and AU1-NEMO. AP1510 was added to media 12 hrs prior to cell lysis. B HEK293T cells were transfected with AU1-NEMO and FLAG-MAVS CARD-FPK3, HA-TRAF3 or HA-TRAF5 where indicated. Anti-FLAG-immunoprecipitates were probed for AU1-NEMO. Lysates were immunoblotted for FLAG-MAVS proteins and AU1-NEMO. AP1510 was added to media 12 hrs prior to cell lysis. C HEK293T cells first transfected with siRNAs targeting GFP, TRAF3, or TRAF5. 48 hrs later, cells were transfected with FLAG-MAVS CARD-FPK3 and AU1-NEMO. Anti-FLAG-immunoprecipitates were probed for AU1-NEMO. Lysates were immunoblotted for FLAG-MAVS proteins and AU1-NEMO. AP1510 was added to media 12 hrs prior to cell lysis.
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pone-0009172-g006: NEMO is recruited to the MAVS CARD in a TRAF3/5-dependent manner.A HEK293T cells were transfected with AU1-NEMO and FLAG-MAVS CARD-FPK3 or FLAG-MAVS CARDmt-FPK3 where indicated. MAVS CARDmt contains a substitution of alanine for tryptophan at residue 68. Lysates were prepared and anti-FLAG immunoprecipitates were probed for AU1-NEMO. Lysates were probed for FLAG-MAVS-CARD-FPK3 protein and AU1-NEMO. AP1510 was added to media 12 hrs prior to cell lysis. B HEK293T cells were transfected with AU1-NEMO and FLAG-MAVS CARD-FPK3, HA-TRAF3 or HA-TRAF5 where indicated. Anti-FLAG-immunoprecipitates were probed for AU1-NEMO. Lysates were immunoblotted for FLAG-MAVS proteins and AU1-NEMO. AP1510 was added to media 12 hrs prior to cell lysis. C HEK293T cells first transfected with siRNAs targeting GFP, TRAF3, or TRAF5. 48 hrs later, cells were transfected with FLAG-MAVS CARD-FPK3 and AU1-NEMO. Anti-FLAG-immunoprecipitates were probed for AU1-NEMO. Lysates were immunoblotted for FLAG-MAVS proteins and AU1-NEMO. AP1510 was added to media 12 hrs prior to cell lysis.

Mentions: Recently NEMO was identified as an essential adaptor molecule required for IRF3 and IRF7 activation downstream of MAVS in RLR signaling pathways [24]. NEMO was previously suggested to mediate the recruitment of TANK and TBK1 to the MAVS signaling complex although any physical association with NEMO and MAVS has yet to be demonstrated. We first examined whether NEMO could be recruited to the MAVS signaling complex. We tested if the MAVS CARD, when oligomerized and activated through FPK3, could coprecipitate NEMO from cells. We found that NEMO associated with the MAVS CARD-FPK3 fusion when it was oligomerized, but not with the mutant form (Figure 6A). To check if this interaction was influenced by TRAF3 or TRAF5, we examined the effect of cotransfecting an additional expression plasmid for TRAF3 or TRAF5. Additional TRAF3 or TRAF5 expression resulted in an enhancement in the association of NEMO with MAVS CARD-FPK3 (Figure 6B). Conversely, we found that association of NEMO with MAVS CARD-FPK3 was diminished when the expression of TRAF3 or TRAF5 was knocked down by RNAi (Figure 6C). Thus, TRAF3 and TRAF5 both mediate the recruitment of NEMO to the MAVS signaling complex.


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

Tang ED, Wang CY - PLoS ONE (2010)

NEMO is recruited to the MAVS CARD in a TRAF3/5-dependent manner.A HEK293T cells were transfected with AU1-NEMO and FLAG-MAVS CARD-FPK3 or FLAG-MAVS CARDmt-FPK3 where indicated. MAVS CARDmt contains a substitution of alanine for tryptophan at residue 68. Lysates were prepared and anti-FLAG immunoprecipitates were probed for AU1-NEMO. Lysates were probed for FLAG-MAVS-CARD-FPK3 protein and AU1-NEMO. AP1510 was added to media 12 hrs prior to cell lysis. B HEK293T cells were transfected with AU1-NEMO and FLAG-MAVS CARD-FPK3, HA-TRAF3 or HA-TRAF5 where indicated. Anti-FLAG-immunoprecipitates were probed for AU1-NEMO. Lysates were immunoblotted for FLAG-MAVS proteins and AU1-NEMO. AP1510 was added to media 12 hrs prior to cell lysis. C HEK293T cells first transfected with siRNAs targeting GFP, TRAF3, or TRAF5. 48 hrs later, cells were transfected with FLAG-MAVS CARD-FPK3 and AU1-NEMO. Anti-FLAG-immunoprecipitates were probed for AU1-NEMO. Lysates were immunoblotted for FLAG-MAVS proteins and AU1-NEMO. AP1510 was added to media 12 hrs prior to cell lysis.
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pone-0009172-g006: NEMO is recruited to the MAVS CARD in a TRAF3/5-dependent manner.A HEK293T cells were transfected with AU1-NEMO and FLAG-MAVS CARD-FPK3 or FLAG-MAVS CARDmt-FPK3 where indicated. MAVS CARDmt contains a substitution of alanine for tryptophan at residue 68. Lysates were prepared and anti-FLAG immunoprecipitates were probed for AU1-NEMO. Lysates were probed for FLAG-MAVS-CARD-FPK3 protein and AU1-NEMO. AP1510 was added to media 12 hrs prior to cell lysis. B HEK293T cells were transfected with AU1-NEMO and FLAG-MAVS CARD-FPK3, HA-TRAF3 or HA-TRAF5 where indicated. Anti-FLAG-immunoprecipitates were probed for AU1-NEMO. Lysates were immunoblotted for FLAG-MAVS proteins and AU1-NEMO. AP1510 was added to media 12 hrs prior to cell lysis. C HEK293T cells first transfected with siRNAs targeting GFP, TRAF3, or TRAF5. 48 hrs later, cells were transfected with FLAG-MAVS CARD-FPK3 and AU1-NEMO. Anti-FLAG-immunoprecipitates were probed for AU1-NEMO. Lysates were immunoblotted for FLAG-MAVS proteins and AU1-NEMO. AP1510 was added to media 12 hrs prior to cell lysis.
Mentions: Recently NEMO was identified as an essential adaptor molecule required for IRF3 and IRF7 activation downstream of MAVS in RLR signaling pathways [24]. NEMO was previously suggested to mediate the recruitment of TANK and TBK1 to the MAVS signaling complex although any physical association with NEMO and MAVS has yet to be demonstrated. We first examined whether NEMO could be recruited to the MAVS signaling complex. We tested if the MAVS CARD, when oligomerized and activated through FPK3, could coprecipitate NEMO from cells. We found that NEMO associated with the MAVS CARD-FPK3 fusion when it was oligomerized, but not with the mutant form (Figure 6A). To check if this interaction was influenced by TRAF3 or TRAF5, we examined the effect of cotransfecting an additional expression plasmid for TRAF3 or TRAF5. Additional TRAF3 or TRAF5 expression resulted in an enhancement in the association of NEMO with MAVS CARD-FPK3 (Figure 6B). Conversely, we found that association of NEMO with MAVS CARD-FPK3 was diminished when the expression of TRAF3 or TRAF5 was knocked down by RNAi (Figure 6C). Thus, TRAF3 and TRAF5 both mediate the recruitment of NEMO to the MAVS signaling complex.

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