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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 associates with the dimerized MAVS CARD.A, B, C HEK293T cells were cotransfected with FLAG-tagged MAVS constructs together with HA-tagged TRAF5 and whole cell lysates were prepared. Anti-FLAG immunoprecipitates were probed for HA-TRAF5 or FLAG-MAVS. Lysates were immunoblotted for HA-TRAF5. D 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). E HEK293T cells were transfected with FLAG-tagged RIG-I or MAVS constructs, HA-TRAF5, and AU1-Ub. Whole cell lysates were prepared and anti-HA immunoprecipitates were probed for Ub-conjugated TRAF3 and TRAF5 proteins using AU1 antibody from or total protein using HA antibody. F HEK293T cells were transfected with FLAG-MAVS CARD-FPK3, HA-TRAF3 or HA-TRAF5, and AU1-Ub. Anti-HA immunoprecipitates were probed for AU1-Ub conjugates or HA-TRAF3/5. Lysates were immunoblotted for FLAG-MAVS CARD-FPK3. AP1510 was added 12 hrs prior to cell lysis. G, H HEK293T cells transfected initially with siRNAs targeting GFP or TRAF5. 48 hrs later, cells were transfected with FLAG-RIG-IΔRD, FLAG-MAVS, or FLAG-p65, and pLuc-PRD(III-I)3 (IRF3/7) (G) or pLuc-PRD(II)2 (NF-κB) (H). Luciferase activity was measured 24 hrs following plasmid transfection. Discontinuity in graph indicates a broken Y-axis (H).
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pone-0009172-g003: TRAF5 associates with the dimerized MAVS CARD.A, B, C HEK293T cells were cotransfected with FLAG-tagged MAVS constructs together with HA-tagged TRAF5 and whole cell lysates were prepared. Anti-FLAG immunoprecipitates were probed for HA-TRAF5 or FLAG-MAVS. Lysates were immunoblotted for HA-TRAF5. D 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). E HEK293T cells were transfected with FLAG-tagged RIG-I or MAVS constructs, HA-TRAF5, and AU1-Ub. Whole cell lysates were prepared and anti-HA immunoprecipitates were probed for Ub-conjugated TRAF3 and TRAF5 proteins using AU1 antibody from or total protein using HA antibody. F HEK293T cells were transfected with FLAG-MAVS CARD-FPK3, HA-TRAF3 or HA-TRAF5, and AU1-Ub. Anti-HA immunoprecipitates were probed for AU1-Ub conjugates or HA-TRAF3/5. Lysates were immunoblotted for FLAG-MAVS CARD-FPK3. AP1510 was added 12 hrs prior to cell lysis. G, H HEK293T cells transfected initially with siRNAs targeting GFP or TRAF5. 48 hrs later, cells were transfected with FLAG-RIG-IΔRD, FLAG-MAVS, or FLAG-p65, and pLuc-PRD(III-I)3 (IRF3/7) (G) or pLuc-PRD(II)2 (NF-κB) (H). Luciferase activity was measured 24 hrs following plasmid transfection. Discontinuity in graph indicates a broken Y-axis (H).

Mentions: We next examined whether TRAF5 may physically and functionally interact with MAVS. In coimmunoprecipitation experiments, TRAF5 bound to MAVS (Figure 3A). However, TRAF5 failed to bind to an inactive deletion mutant of MAVS lacking its TM domain (MAVSΔTM), demonstrating that the TM was important for MAVS to bind to TRAF5, as was previously shown for TRAF3 [25]. TRAF5 binding was restored when the TM domain of MAVS was replaced with the analogous domain from Bcl-xL (MAVS-TM) (Figure 3B), a mutant protein that regains function and binding to TRAF3 also [25], [31]. Mutation of the conserved tryptophan residue in the MAVS CARD (W68A) that is essential for function, also impaired TRAF5 binding (Figure 3B). Thus, the TM domain and CARD are both necessary for MAVS to bind to TRAF5. In addition, as previously shown for TRAF3 [25], we found that TRAF5 associated with MAVS in yeast suggesting a likely direct interaction (Figure 3D).


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

Tang ED, Wang CY - PLoS ONE (2010)

TRAF5 associates with the dimerized MAVS CARD.A, B, C HEK293T cells were cotransfected with FLAG-tagged MAVS constructs together with HA-tagged TRAF5 and whole cell lysates were prepared. Anti-FLAG immunoprecipitates were probed for HA-TRAF5 or FLAG-MAVS. Lysates were immunoblotted for HA-TRAF5. D 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). E HEK293T cells were transfected with FLAG-tagged RIG-I or MAVS constructs, HA-TRAF5, and AU1-Ub. Whole cell lysates were prepared and anti-HA immunoprecipitates were probed for Ub-conjugated TRAF3 and TRAF5 proteins using AU1 antibody from or total protein using HA antibody. F HEK293T cells were transfected with FLAG-MAVS CARD-FPK3, HA-TRAF3 or HA-TRAF5, and AU1-Ub. Anti-HA immunoprecipitates were probed for AU1-Ub conjugates or HA-TRAF3/5. Lysates were immunoblotted for FLAG-MAVS CARD-FPK3. AP1510 was added 12 hrs prior to cell lysis. G, H HEK293T cells transfected initially with siRNAs targeting GFP or TRAF5. 48 hrs later, cells were transfected with FLAG-RIG-IΔRD, FLAG-MAVS, or FLAG-p65, and pLuc-PRD(III-I)3 (IRF3/7) (G) or pLuc-PRD(II)2 (NF-κB) (H). Luciferase activity was measured 24 hrs following plasmid transfection. Discontinuity in graph indicates a broken Y-axis (H).
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pone-0009172-g003: TRAF5 associates with the dimerized MAVS CARD.A, B, C HEK293T cells were cotransfected with FLAG-tagged MAVS constructs together with HA-tagged TRAF5 and whole cell lysates were prepared. Anti-FLAG immunoprecipitates were probed for HA-TRAF5 or FLAG-MAVS. Lysates were immunoblotted for HA-TRAF5. D 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). E HEK293T cells were transfected with FLAG-tagged RIG-I or MAVS constructs, HA-TRAF5, and AU1-Ub. Whole cell lysates were prepared and anti-HA immunoprecipitates were probed for Ub-conjugated TRAF3 and TRAF5 proteins using AU1 antibody from or total protein using HA antibody. F HEK293T cells were transfected with FLAG-MAVS CARD-FPK3, HA-TRAF3 or HA-TRAF5, and AU1-Ub. Anti-HA immunoprecipitates were probed for AU1-Ub conjugates or HA-TRAF3/5. Lysates were immunoblotted for FLAG-MAVS CARD-FPK3. AP1510 was added 12 hrs prior to cell lysis. G, H HEK293T cells transfected initially with siRNAs targeting GFP or TRAF5. 48 hrs later, cells were transfected with FLAG-RIG-IΔRD, FLAG-MAVS, or FLAG-p65, and pLuc-PRD(III-I)3 (IRF3/7) (G) or pLuc-PRD(II)2 (NF-κB) (H). Luciferase activity was measured 24 hrs following plasmid transfection. Discontinuity in graph indicates a broken Y-axis (H).
Mentions: We next examined whether TRAF5 may physically and functionally interact with MAVS. In coimmunoprecipitation experiments, TRAF5 bound to MAVS (Figure 3A). However, TRAF5 failed to bind to an inactive deletion mutant of MAVS lacking its TM domain (MAVSΔTM), demonstrating that the TM was important for MAVS to bind to TRAF5, as was previously shown for TRAF3 [25]. TRAF5 binding was restored when the TM domain of MAVS was replaced with the analogous domain from Bcl-xL (MAVS-TM) (Figure 3B), a mutant protein that regains function and binding to TRAF3 also [25], [31]. Mutation of the conserved tryptophan residue in the MAVS CARD (W68A) that is essential for function, also impaired TRAF5 binding (Figure 3B). Thus, the TM domain and CARD are both necessary for MAVS to bind to TRAF5. In addition, as previously shown for TRAF3 [25], we found that TRAF5 associated with MAVS in yeast suggesting a likely direct interaction (Figure 3D).

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