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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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TRAF5 is required for the induction of gene expression downstream of RLR signaling.A HEK293T cells were transfected with siRNAs targeting GFP or TRAF5. 48 hrs later, cells were mock infected or infected with SeV for 8 hrs and total RNA was prepared. Ifnb, IP10, and Tnfa mRNA levels were measured by real-time RT-PCR using specific primers as described in the Materials and Methods section. GADPH was used as an internal control. Asterisk, P<0.01 versus GFP siRNA + infection (n = 2), double asterisk, P<0.005 versus GFP siRNA + infection (n = 2). B, HEK293 cells were transfected with siRNAs targeting GFP, MAVS, or TRAF5. 48 hrs later, cells were mock infected or infected with SeV. Tissue culture supernatants were collected 24 hrs following infection and human IFN-β ELISA was performed. The concentration of IFN-β is shown (mean +/- sd). ND, not detectable. Asterisk, P = 0.01 versus GFP siRNA + infection (n = 2), double asterisk, P<0.005 versus GFP siRNA + infection (n = 2). C, HEK293 cells were transfected with siRNAs targeting GFP or TRAF5. 48 hrs later, cells were transfected with poly I:C for 2 or 6 hrs or mock treated for 6 hrs. Nuclear extracts were prepared and analyzed for p65 expression by immunoblotting. TBP was used as a loading control. D, HEK293 cells were transfected with siRNAs targeting GFP, MAVS, TRAF3, or TRAF5. 48 hrs later, cells were transfected with poly dA:dT or mock treated. Tissue culture supernatants were collected for human IFN-β ELISA 24 hrs after poly dA:dT transfection. The concentration of IFN-β is shown (mean +/− sd). ND, not detectable. Asterisk, P<0.025 versus GFP siRNA + poly dA:dT (n = 2), double asterisk, P<0.001 versus GFP siRNA + poly dA:dT (n = 2). E, HEK293 cells were transfected with siRNAs as in (D). 48 hrs later, cells were transfected with poly dA:dT or mock treated for 8 hrs and lysates prepared. Either 1 or 4 ug/ml of poly dA:dT was used as indicated. Lysates were probed for phospho-STAT1 (Y701) or total STAT1. F, Yeast were transformed with the bait or prey constructs indicated as described in the Materials and Methods section and streaked onto minimal SD media lacking leucine and tryptophan (SD-LW) or leucine, tryptophan, adenine, and histidine (SD-LWAH).
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pone-0009172-g005: TRAF5 is required for the induction of gene expression downstream of RLR signaling.A HEK293T cells were transfected with siRNAs targeting GFP or TRAF5. 48 hrs later, cells were mock infected or infected with SeV for 8 hrs and total RNA was prepared. Ifnb, IP10, and Tnfa mRNA levels were measured by real-time RT-PCR using specific primers as described in the Materials and Methods section. GADPH was used as an internal control. Asterisk, P<0.01 versus GFP siRNA + infection (n = 2), double asterisk, P<0.005 versus GFP siRNA + infection (n = 2). B, HEK293 cells were transfected with siRNAs targeting GFP, MAVS, or TRAF5. 48 hrs later, cells were mock infected or infected with SeV. Tissue culture supernatants were collected 24 hrs following infection and human IFN-β ELISA was performed. The concentration of IFN-β is shown (mean +/- sd). ND, not detectable. Asterisk, P = 0.01 versus GFP siRNA + infection (n = 2), double asterisk, P<0.005 versus GFP siRNA + infection (n = 2). C, HEK293 cells were transfected with siRNAs targeting GFP or TRAF5. 48 hrs later, cells were transfected with poly I:C for 2 or 6 hrs or mock treated for 6 hrs. Nuclear extracts were prepared and analyzed for p65 expression by immunoblotting. TBP was used as a loading control. D, HEK293 cells were transfected with siRNAs targeting GFP, MAVS, TRAF3, or TRAF5. 48 hrs later, cells were transfected with poly dA:dT or mock treated. Tissue culture supernatants were collected for human IFN-β ELISA 24 hrs after poly dA:dT transfection. The concentration of IFN-β is shown (mean +/− sd). ND, not detectable. Asterisk, P<0.025 versus GFP siRNA + poly dA:dT (n = 2), double asterisk, P<0.001 versus GFP siRNA + poly dA:dT (n = 2). E, HEK293 cells were transfected with siRNAs as in (D). 48 hrs later, cells were transfected with poly dA:dT or mock treated for 8 hrs and lysates prepared. Either 1 or 4 ug/ml of poly dA:dT was used as indicated. Lysates were probed for phospho-STAT1 (Y701) or total STAT1. F, Yeast were transformed with the bait or prey constructs indicated as described in the Materials and Methods section and streaked onto minimal SD media lacking leucine and tryptophan (SD-LW) or leucine, tryptophan, adenine, and histidine (SD-LWAH).

Mentions: To examine whether TRAF5 in fact plays a role in RLR signaling, we tested the effect of the knockdown of TRAF5 expression on signaling activated by transfected poly I:C and viral infection. Knockdown of TRAF5 or TRAF3 by siRNA transfection diminished Ifnb promoter induction, IRF3 phosphorylation, and STAT1 phosphorylation in response to transfected poly I:C (Figures 4A, 4B, 4C). TRAF5 was also required for the full induction of the Ifnb promoter and activation of IRF3/7 in response to SeV infection (Figures 4D and 4E). Accordingly, TRAF5 knockdown inhibited the upregulation of Ifnb and IP-10 mRNAs in response to SeV infection (Figures 5A). IFN-β production in response to SeV infection was also impaired (Figure 5B). We also examined the effect of TRAF5 knockdown on NF-κB activation by RLR signaling. TRAF5 siRNA transfection inhibited p65/RelA nuclear translocation in response to transfected poly I:C (Figure 4C). Also knockdown of TRAF5 impaired activation of an NF-κB reporter in response to SeV infection (Figure 4E). We also found that the induction of Tnfa mRNA in response to SeV infection was also impaired (Figure 5A). Therefore, like TRAF3, TRAF5 is necessary for full IRF3/7 activation and IFNβ induction in response to RLR signaling, whereas TRAF5 is also required for NF-κB activation.


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

Tang ED, Wang CY - PLoS ONE (2010)

TRAF5 is required for the induction of gene expression downstream of RLR signaling.A HEK293T cells were transfected with siRNAs targeting GFP or TRAF5. 48 hrs later, cells were mock infected or infected with SeV for 8 hrs and total RNA was prepared. Ifnb, IP10, and Tnfa mRNA levels were measured by real-time RT-PCR using specific primers as described in the Materials and Methods section. GADPH was used as an internal control. Asterisk, P<0.01 versus GFP siRNA + infection (n = 2), double asterisk, P<0.005 versus GFP siRNA + infection (n = 2). B, HEK293 cells were transfected with siRNAs targeting GFP, MAVS, or TRAF5. 48 hrs later, cells were mock infected or infected with SeV. Tissue culture supernatants were collected 24 hrs following infection and human IFN-β ELISA was performed. The concentration of IFN-β is shown (mean +/- sd). ND, not detectable. Asterisk, P = 0.01 versus GFP siRNA + infection (n = 2), double asterisk, P<0.005 versus GFP siRNA + infection (n = 2). C, HEK293 cells were transfected with siRNAs targeting GFP or TRAF5. 48 hrs later, cells were transfected with poly I:C for 2 or 6 hrs or mock treated for 6 hrs. Nuclear extracts were prepared and analyzed for p65 expression by immunoblotting. TBP was used as a loading control. D, HEK293 cells were transfected with siRNAs targeting GFP, MAVS, TRAF3, or TRAF5. 48 hrs later, cells were transfected with poly dA:dT or mock treated. Tissue culture supernatants were collected for human IFN-β ELISA 24 hrs after poly dA:dT transfection. The concentration of IFN-β is shown (mean +/− sd). ND, not detectable. Asterisk, P<0.025 versus GFP siRNA + poly dA:dT (n = 2), double asterisk, P<0.001 versus GFP siRNA + poly dA:dT (n = 2). E, HEK293 cells were transfected with siRNAs as in (D). 48 hrs later, cells were transfected with poly dA:dT or mock treated for 8 hrs and lysates prepared. Either 1 or 4 ug/ml of poly dA:dT was used as indicated. Lysates were probed for phospho-STAT1 (Y701) or total STAT1. F, Yeast were transformed with the bait or prey constructs indicated as described in the Materials and Methods section and streaked onto minimal SD media lacking leucine and tryptophan (SD-LW) or leucine, tryptophan, adenine, and histidine (SD-LWAH).
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Related In: Results  -  Collection

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pone-0009172-g005: TRAF5 is required for the induction of gene expression downstream of RLR signaling.A HEK293T cells were transfected with siRNAs targeting GFP or TRAF5. 48 hrs later, cells were mock infected or infected with SeV for 8 hrs and total RNA was prepared. Ifnb, IP10, and Tnfa mRNA levels were measured by real-time RT-PCR using specific primers as described in the Materials and Methods section. GADPH was used as an internal control. Asterisk, P<0.01 versus GFP siRNA + infection (n = 2), double asterisk, P<0.005 versus GFP siRNA + infection (n = 2). B, HEK293 cells were transfected with siRNAs targeting GFP, MAVS, or TRAF5. 48 hrs later, cells were mock infected or infected with SeV. Tissue culture supernatants were collected 24 hrs following infection and human IFN-β ELISA was performed. The concentration of IFN-β is shown (mean +/- sd). ND, not detectable. Asterisk, P = 0.01 versus GFP siRNA + infection (n = 2), double asterisk, P<0.005 versus GFP siRNA + infection (n = 2). C, HEK293 cells were transfected with siRNAs targeting GFP or TRAF5. 48 hrs later, cells were transfected with poly I:C for 2 or 6 hrs or mock treated for 6 hrs. Nuclear extracts were prepared and analyzed for p65 expression by immunoblotting. TBP was used as a loading control. D, HEK293 cells were transfected with siRNAs targeting GFP, MAVS, TRAF3, or TRAF5. 48 hrs later, cells were transfected with poly dA:dT or mock treated. Tissue culture supernatants were collected for human IFN-β ELISA 24 hrs after poly dA:dT transfection. The concentration of IFN-β is shown (mean +/− sd). ND, not detectable. Asterisk, P<0.025 versus GFP siRNA + poly dA:dT (n = 2), double asterisk, P<0.001 versus GFP siRNA + poly dA:dT (n = 2). E, HEK293 cells were transfected with siRNAs as in (D). 48 hrs later, cells were transfected with poly dA:dT or mock treated for 8 hrs and lysates prepared. Either 1 or 4 ug/ml of poly dA:dT was used as indicated. Lysates were probed for phospho-STAT1 (Y701) or total STAT1. F, Yeast were transformed with the bait or prey constructs indicated as described in the Materials and Methods section and streaked onto minimal SD media lacking leucine and tryptophan (SD-LW) or leucine, tryptophan, adenine, and histidine (SD-LWAH).
Mentions: To examine whether TRAF5 in fact plays a role in RLR signaling, we tested the effect of the knockdown of TRAF5 expression on signaling activated by transfected poly I:C and viral infection. Knockdown of TRAF5 or TRAF3 by siRNA transfection diminished Ifnb promoter induction, IRF3 phosphorylation, and STAT1 phosphorylation in response to transfected poly I:C (Figures 4A, 4B, 4C). TRAF5 was also required for the full induction of the Ifnb promoter and activation of IRF3/7 in response to SeV infection (Figures 4D and 4E). Accordingly, TRAF5 knockdown inhibited the upregulation of Ifnb and IP-10 mRNAs in response to SeV infection (Figures 5A). IFN-β production in response to SeV infection was also impaired (Figure 5B). We also examined the effect of TRAF5 knockdown on NF-κB activation by RLR signaling. TRAF5 siRNA transfection inhibited p65/RelA nuclear translocation in response to transfected poly I:C (Figure 4C). Also knockdown of TRAF5 impaired activation of an NF-κB reporter in response to SeV infection (Figure 4E). We also found that the induction of Tnfa mRNA in response to SeV infection was also impaired (Figure 5A). Therefore, like TRAF3, TRAF5 is necessary for full IRF3/7 activation and IFNβ induction in response to RLR signaling, whereas TRAF5 is also required for NF-κB activation.

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