1. Cell Biology
  2. Immunology and Inflammation
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MAVS recruits multiple ubiquitin E3 ligases to activate antiviral signaling cascades

  1. Siqi Liu
  2. Jueqi Chen
  3. Xin Cai
  4. Jiaxi Wu
  5. Xiang Chen
  6. You-Tong Wu
  7. Lijun Sun
  8. Zhijian J Chen  Is a corresponding author
  1. University of Texas Southwestern Medical Center, United States
  2. Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, United States
Research Article
Cite this article as: eLife 2013;2:e00785 doi: 10.7554/eLife.00785
8 figures

Figures

Figure 1 with 1 supplement
TRAF2, TRAF5, and TRAF6 are important for IRF3 and IKK activation in vitro.

(A) Diagram of differential centrifugation of cell homogenates. HEK293T cells infected with Sendai virus (+SeV) or mock treated (−SeV) were homogenized in hypotonic buffer, followed by sequential centrifugation to separate crude mitochondria (P5) from cytosolic supernatant (S5 and S100). (B) IRF3 and NF-κB activation in vitro. Mitochondrial fraction (P5) from Sendai virus-infected HEK293T cells or purified His6-tagged MAVS without transmembrane domain (His6-MAVSΔTM) was incubated with cytosolic extract (S5) from uninfected cells in the presence of ATP and 35S-IRF3. Dimerization of IRF3 was analyzed by native gel electrophoresis, followed by autoradiography. IκBα phosphorylation was analyzed by immunoblotting. (C) NEMO-interacting complex is required for IRF3 activation in vitro. GST-tagged NEMO without its N-terminal IKK-binding region (GST-NEMOΔN) was mixed with cytosolic extract from Nemo−/− MEF cells to collect GST-NEMOΔN pull down (NEMOΔN PD). This material, or GST-NEMO, was incubated with cytosolic extract (S100) from HeLa cells depleted of NEMO with a NEMO antibody. Activation of IRF3 was analyzed as described in (B). (D) Reconstitution of IRF3 dimerization in vitro. NEMOΔN PD, His8-E1, Ubc5c, His6-TRAF6, ubiquitin, His6-MAVSΔTM, His8-IRF3, and 35S-Flag-IRF3 were incubated together with ATP as indicated, followed by analysis of IRF3 dimerization. (E and F) TRAF6 is important for IRF3 and NF-κB activation by MAVS in vitro. Cytosolic extracts from wild-type or Traf6−/− MEF cells were incubated with His6-MAVSΔTM together with WT or mutant TRAF6 protein as indicated, followed by analysis of IRF3 dimerization (E) or IκBα phosphorylation (F). T6RZC: TRAF6 containing the RING, zinc, and coiled-coil domains, with the TRAF-C domain replaced by a fragment of bacterial gyrase B. (G) TRAF2 and 5 are important for IRF3 activation by MAVS in vitro. Cytosolic extracts from WT or different TRAF deficient MEF cells were incubated with His6-MAVSΔTM, followed by IRF3 dimerization assay. (H and I) Either TRAF2 or TRAF5 rescues IRF3 and IKK activation by MAVS in the Traf2/5 DKO extract. Traf2/5 DKO extracts were supplemented with TRAF2 or TRAF5 proteins as indicated, together with His6-MAVSΔTM and ATP, followed by measurement of IRF3 dimerization and IκBα phosphorylation.

https://doi.org/10.7554/eLife.00785.003
Figure 1—figure supplement 1
TRAF6 and TRAF2/5 are IRF3 activators.

(A) Wild-type and IKKα/IKKβ double knockout MEF cells were infected with VSV for the indicated time. IRF3 dimerization was analyzed by immunoblotting. (B) Flag-tagged NEMO WT and ΔN were tested for their ability to rescue IRF3 dimerization in Ikbkg−/−(Nemo−/−) MEF extracts in the presence or absence of mitochondria (P5) isolated from Sendai virus-infected cells. (C) Ikbkg−/−(Nemo−/−) MEF cells stably expressing Flag-NEMOΔN were used to isolate endogenous NEMO–TBK1 complex, which was analyzed by immunoblotting. (D) Partially purified fraction from HeLa S100 that contains IRF3 stimulatory activity was further fractionated on Heparin-Sepharose, then each fraction was assayed for IRF3 dimerization in the presence or absence of the NEMO pull down (PD) as shown in (C). The reactions also contained His8-E1, Ubc5c, ubiquitin, ATP, and virus-activated mitochondria (P5). (E) Scheme for purification of an IRF3 activator. (F) Fractions from the last step (monoQ) shown in (E) were tested for their ability to stimulate IRF3 dimerization in the presence of NEMO-PD and virus-activated mitochondria (top). Aliquots of the fractions were immunoblotted with a TRAF6 antibody (bottom). (G) Recombinant TRAF6 activates IRF3. Indicated amount of His-tagged TRAF6 purified from Sf9 cells was added into the IRF3 dimerization assay that contains E1, UbcH5, ubiquitin, 35S-IRF3, NEMO-PD (containing TBK1 complex), and mitochondria from Sendai virus-infected cells (P5). (H) Wild-type and Traf6−/− primary MEF cells were infected with Sendai virus for the indicated time, and phosphorylation of IRF3 and IκBα was analyzed by immunoblotting (top). As a control, the same cells were treated with IL-1β, and the cell extract was analyzed by immunoblotting with an IκBα antibody (bottom).

https://doi.org/10.7554/eLife.00785.004
Figure 2 with 2 supplements
TRAF6 functions redundantly with TRAF2 and TRAF5 to activate IRF3 in cells.

(A) Depletion of TRAF6 in Traf2/5 DKO cells abolishes both IRF3 and NF-κB activation by virus. Traf2/5 DKO MEF cells stably expressing GFP (as a control) or an shRNA against TRAF6 were infected with Sendai virus for the indicated time followed by immunoblotting of the cell extracts with the indicated antibodies (top). As a control, IFNγ induced STAT-1 phosphorylation was also analyzed by immunoblotting (bottom). (B) Depletion of TRAF6 in Traf2/5 DKO cells abolishes IFNβ mRNA induction by virus. The cells described in (A) were treated with Sendai virus for the indicated time before total RNA was isolated. IFNβ mRNA level was analyzed by q-RT-PCR. (CE) The catalytic activity of TRAF6 is required for antiviral immune responses. Traf2/5 DKO MEF cells stably expressing shRNA against TRAF6 (DKO+shT6) and those in which endogenous TRAF6 was replaced with WT or RING mutant (C70A) Flag-TRAF6 were stimulated with Sendai Virus or VSV for the indicated time. Phosphorylation of IRF3 and IκBα was analyzed by immunoblotting. Total RNA was also isolated for the measurement of IFNβ and IL6 RNA by q-RT-PCR. T6-K0: a TRAF6 mutant in which all lysine residues were substituted with arginine. (FH) The RING domain of TRAF2 is dispensable for its signaling functions. Traf2/5 DKO cells stably expressing WT or RING mutant (C34A) Flag-TRAF2 were stimulated with Sendai virus or VSV for the indicated time. Activation of IRF3 and phosphorylation of IκBα was analyzed by immunoblotting. Cytokine RNA levels were measured by q-RT-PCR. Unless indicated otherwise, error bars in this and other figures of this paper represent standard deviations of triplicate experiments.

https://doi.org/10.7554/eLife.00785.005
Figure 2—figure supplement 1
TRAF2, 5, and 6 function redundantly to activate IRF3 in cells.

(A and B) Wild-type, Traf6−/−, and Traf2−/− primary MEF cells were infected with Sendai virus for the indicated time. The RNA levels of IFNβ and IL-6 were measured by q-RT-PCR. (C and D) Traf2/5 DKO MEF cells stably expressing an shRNA against TRAF6 were reconstituted with WT or mutant TRAF6 as indicated. The cells were infected with VSV for the indicated time. Cytokine RNA levels were measured by q-RT-PCR. (EG) Traf2/5 DKO MEF cells stably expressing an shRNA against TRAF3 were infected with VSV as indicated, and then RNA levels of IFNβ, IL6, and TRAF3 were analyzed by q-RT-PCR. (H) Traf3−/− MEF cells stably expressing TRAF6 shRNA were infected with Sendai virus, followed by immunoblotting with phospho-IRF3 and TRAF6 antibodies.

https://doi.org/10.7554/eLife.00785.006
Figure 2—figure supplement 2
Multiple E3 ligases function redundantly to activate IRF3 in cells.

(AD) Wild-type, Traf6−/−, and Traf2/5 DKO MEF cells were treated with or without a SMAC mimetic compound (SM) for 1 hr before VSV infection. Cell extracts were analyzed by immunoblotting with the indicated antibodies (A and B). The RNA levels of IFNβ and IL6 were measured by q-RT-PCR (C and D). (E and F) Similar to Figure 2—figure supplement 1C,D, except that the cells were reconstituted with WT or C34A TRAF2. (G) Cells described in (E and F) were stimulated with either TNF-α or IL-1β, followed by immunoblotting with a phospho-IκBα antibody.

https://doi.org/10.7554/eLife.00785.007
Figure 3 with 2 supplements
LUBAC functions redundantly with TRAF2 to support MAVS signaling.

(A and B) WT MEF cells stably expressing GFP or shRNA against HOIL-1, Sharpin, HOIP, MAVS, or TRAF3 were infected with VSV for the indicated time. IRF3 dimerization and IκBα phosphorylation were analyzed by immunoblotting (A). The levels of IFNβ RNA were measured by q-RT-PCR (B). The efficiency of RNAi is shown in Figure 3—figure supplement 1A,B. (C) Primary MEF cells from heterozygous or Sharpincpdm mice were infected with VSV for the indicated time, and then IFNβ RNA levels were measured by q-RT-PCR. (D) Traf2/5 DKO MEF cells stably expressing an shRNA against TRAF6 were reconstituted with TRAF2 WT or ΔRING (ΔR) mutant (lower panels). These cells, as well as WT MEF (upper panel), were further depleted of HOIP (lane 4–6) by lentiviral shRNA and then rescued with WT or the active site mutant (CS) of HOIP (lanes 7–12). In lanes 1–3, a lentiviral vector expressing GFP was used as a control. The cells were infected with VSV for the indicated time, followed by measurement of IRF3 dimerization. (E and F) The cells described in (D) were analyzed for the expression of HOIP by q-RT-PCR (E) or immunoblotting with an HA antibody (F).

https://doi.org/10.7554/eLife.00785.008
Figure 3—figure supplement 1
LUBAC is largely dispensable for MAVS signaling when it is depleted from wild-type cells.

(A and B) Wild-type MEFs stably expressing shRNA against the indicated proteins were used for the experiments described in Figure 3A,B. The knockdown efficiency of each protein was analyzed by immunoblotting with the indicated antibody (A). For mouse HOIP, for which no antibody was available, q-RT-PCR was used to measure its RNA levels (B). (C) Primary MEFs from heterozygous or homozygous Sharpincpdm(cpdm) mice were infected with VSV or Sendai virus as indicated for 0–48 hr as indicated. IFNβ RNA levels were measured by q-RT-PCR. (D) Immortalized Sharpincpdm(cpdm) MEF cells stably expressing GFP or HA-Sharpin were infected with Sendai virus. IRF3 dimerization, IκBα phosphorylation (left), and Sharpin expression (right) were analyzed by immunoblotting.

https://doi.org/10.7554/eLife.00785.009
Figure 3—figure supplement 2
LUBAC is required for MAVS signaling only when the functions of TRAF proteins are compromised.

(AC) U2OS cells stably expressing a tetracycline-inducible shRNA against HOIP were treated with tetracycline (Tet; 1 μg/ml) for 7 days, then infected with VSV for the indicated time, followed by immunoblotting of cell extracts with the indicated antibodies (A). IFNβ RNA levels were measured by q-RT-PCR (B and C). (D) Traf2/5 DKO MEF cells stably expressing shRNA against TRAF6 and HOIP (or GFP) were reconstituted with WT TRAF2, TRAF6, or ΔRING TRAF2. The cells were infected with VSV for the indicated time, followed by immunoblotting with a phospho-IRF3 specific antibody. (E) Traf2/5 DKO MEF cells stably expressing the TRAF6 shRNA were reconstituted with TRAF2 WT or ΔRING mutant. These cells were then further depleted of HOIL-1, Sharpin, or HOIP by lentiviral shRNA. After VSV infection, cell extracts were prepared for immunoblotting with the indicated antibodies.

https://doi.org/10.7554/eLife.00785.010
Figure 4 with 2 supplements
MAVS recruits multiple TRAF proteins to activate IRF3 upon virus infection.

(A) The conserved binding motifs of TRAF2, TRAF5, and TRAF6 in human MAVS. Residues in red indicate mutation sites. (BE) The TRAF-binding motifs on MAVS are essential for IRF3 and NF-κB activation by virus. Mavs−/− MEF cells reconstituted with WT or TRAF-binding mutant MAVS were infected with Sendai virus for the indicated time. IRF3 dimerization and the expression of different MAVS mutants were analyzed by immunoblotting. Cytokine RNA levels were measured by q-RT-PCR. 2ED: E155D/E457D; QN2ED: Q145N/E155D/E457D. (F) The TRAF-binding motifs on MAVS are important for restricting viral replication. MEF cells described in (B) were infected with VSV-ΔM51-GFP for the indicated time, and the GFP-positive cells were visualized under fluorescence microscope. (G) MAVS recruits TRAF6 after virus infection through TRAF6-binding motifs. Traf6−/− MEF cells were depleted of endogenous MAVS by shRNA, then reconstituted with Flag-TRAF6 and WT or mutant MAVS as indicated. The cells were infected with Sendai virus for the indicated time followed by immunoprecipitation of TRAF6 with a Flag antibody. The immunoprecipitates and whole cell lysates (WCL) were analyzed by immunoblotting with the indicated antibodies. (H) MAVS recruits TRAF2 upon virus infection through TRAF2 binding motif. Similar to (G), except that Traf2/5 DKO MEF cells were reconstituted with Flag-TRAF2 and the binding between TRAF2 and different MAVS mutants was analyzed.

https://doi.org/10.7554/eLife.00785.011
Figure 4—figure supplement 1
MAVS recruits multiple TRAF proteins upon virus infection.

(A) Mavs−/− MEF cells reconstituted with MAVS WT or QN2ED were infected with Sendai virus for the indicated time. Crude mitochondria (P5) were isolated, solubilized in 1% DDM, and then separated by sucrose gradient ultracentrifugation. Fractions were analyzed by immunoblotting with a MAVS antibody. (B) MAVS (Q145N) only functions through TRAF6. Traf6−/− MEF cells in which endogenous MAVS was knocked down by shRNA and replaced by wild-type or TRAF-binding mutant human MAVS were infected with Sendai virus as indicated. IFNβ RNA level was analyzed by q-RT-PCR. (C) Traf6−/− MEF cells stably expressing an shRNA against MAVS were reconstituted with MAVS Q145N and TRAF6 WT or C70A mutant. The cells were infected with Sendai virus to induce IFNβ RNA, which was measured by q-RT-PCR. (D) MAVS (2ED) only functions through TRAF2 and TRAF5. Traf2/5 DKO MEF cells with endogenous MAVS replaced by wild-type or TRAF-binding mutant human MAVS were infected with Sendai virus for the indicated time. IFNβ RNA levels were measured by q-RT-PCR. (E) Traf2/5 DKO MEF cells with endogenous MAVS replaced by MAVS (2ED) were reconstituted with WT or RING mutant TRAF2. The cells were infected with Sendai virus, and then IFNβ RNA induction was analyzed by q-RT-PCR. (F and G) Similar to (E), except that Traf2−/− or Traf5−/− MEFs were used.

https://doi.org/10.7554/eLife.00785.012
Figure 4—figure supplement 2
MAVS recruits multiple TRAF proteins upon virus infection.

(A) Traf6−/− or Traf3−/− MEF cells stably expressing an shRNA against MAVS were reconstituted with WT or TRAF-binding mutant human MAVS. These cells were infected with Sendai virus followed by the analysis of endogenous IRF3 dimerization. (B) A summary of results from the mutagenesis and complementation experiments that reveal the essential role of the distinct TRAF binding motifs in recruiting specific TRAF proteins.

https://doi.org/10.7554/eLife.00785.013
Figure 5 with 1 supplement
Prion-like polymerization of MAVS is required for IRF3 activation and TRAF6 recruitment.

(A) Sequence alignment of the CARD domain of MAVS from different species. Conserved residues mutated in this study are colored and shaded. (B) MAVS WT or CARD mutants were transfected into HEK293-IFNβ-luciferase reporter cells. Cells were lysed 24 hr later, followed by luciferase reporter assay. (C) Mavs−/− MEF cells reconstituted with Flag-MAVS WT or CARD mutants were infected with Sendai virus or mock treated for 12 hr, then mitochondrial extracts were separated by SDD-AGE (top) or SDS-PAGE (middle) followed by immunoblotting with a Flag antibody. Aliquots of the cytosolic extracts were separated by native gel electrophoresis (bottom), followed by immunoblotting with an IRF3 antibody. (D) HEK293 cells in which endogenous MAVS was knocked down by shRNA and replaced by RNAi resistant MAVS WT or CARD mutants were infected with Sendai virus for the indicated time. Crude mitochondria were solubilized in a buffer containing 1% DDM and then subjected to sucrose gradient ultracentrifugation. Aliquots of the fractions were immunoblotted with a MAVS antibody. (E) MAVS CARD mutants defective in polymerization failed to recruit TRAF6 upon virus infection. Traf6−/− MEF cells were depleted of MAVS by shRNA and reconstituted with Flag-TRAF6 and WT or mutant MAVS as indicated. The cells were infected with Sendai virus for the indicated time, and TRAF6 was then immunoprecipitated with a Flag antibody. Co-immunoprecipitated MAVS was analyzed by immunoblotting.

https://doi.org/10.7554/eLife.00785.014
Figure 5—figure supplement 1
Mutations that disrupt MAVS polymerization abolish viral activation of IRF3.

(A) HEK293T cells stably expressing an shRNA against human MAVS were reconstituted with WT or mutant MAVS as indicated (see also Figure 5D). The cells were infected with Sendai virus followed by analysis of endogenous IRF3 dimerization. Aliquots of the cell lysates were immunoblotted with a MAVS antibody. (B and C) Mavs−/− MEFs stably expressing Flag-TRAF2 (B) or Flag-TRAF5 (C) were infected with Sendai virus, then the Flag-tagged protein complexes were immunoprecipitated and analyzed by immunoblotting with the indicated antibodies.

https://doi.org/10.7554/eLife.00785.015
Figure 6 with 1 supplement
Ubiquitination-dependent assembly of a MAVS signaling complex.

(A) Lys63-linked polyubiquitination is important for MAVS-mediated IRF3 and IKK activation. U2OS cells stably integrated with tetracycline-inducible shRNA against ubiquitin genes were grown in the presence of tetracycline for 48 hr to deplete endogenous ubiquitin. The cytosolic extracts were then supplemented with WT or mutant ubiquitin (1 μg) and 35S-IRF3 in the presence or absence of MAVSΔTM, followed by analyses of IRF3 dimerization and IκBα phosphorylation. K63-only: containing only one lysine at residue 63 of ubiquitin. Ubiquitin WT and mutants were analyzed by immunoblotting with an ubiquitin antibody. (B) Purified Flag-NEMO was incubated with HeLa S100 and His6-MAVSΔTM at the indicated temperatures in the presence or absence of vOTU, a viral deubiquitination enzyme. After the reaction, NEMO was immunoprecipitated with Flag antibody, and the co-precipitated proteins were detected with specific antibodies. (C) Similar to (B), except that Flag-NEMO WT and UBD mutant were tested for their ability to interact with TRAF2 and MAVS. (D) A schematic diagram of TRAF2 protein. The ubiquitination sites identified by mass spectrometry are highlighted. (EG) Traf2/5 DKO MEF cells stably expressing shRNA against TRAF6 were reconstituted with WT TRAF2 or a TRAF2 mutant containing arginine at each of the five ubiquitination sites (K31/148/195/313/481R, 5KR). In some experiments, HOIP was further knocked down by shRNA as indicated. These cells were infected with VSV for the indicated time and cytokine RNA levels were analyzed by q-RT-PCR.

https://doi.org/10.7554/eLife.00785.016
Figure 6—figure supplement 1
SILAC experiments to identify ubiquitin-dependent NEMO-signaling complex both in vitro and in cells.

(A) Design of the in vitro SILAC experiments. Cytosolic extracts from MEF cells cultured with heavy (Lys8, Arg10) or light (Lys0, Arg0) isotopes were incubated with Flag-NEMO protein and ATP in the presence (A1) or absence (A2) of His6-MAVSΔTM. In another set of experiments, the ‘heavy’ extracts were incubated with WT Flag-NEMO (B1), whereas the ‘light’ extracts were incubated with Flag-NEMO UBD mutant (UBDm, B2). Both samples were incubated with His6-MAVSΔTM. After incubation at 30°C for 1 hr, NEMO and its associated proteins were immunoprecipitated with a Flag antibody, mixed in pairs (A1 and A2; B1 and B2), and resolved by SDS-PAGE. The proteins were identified and analyzed by quantitative mass spectrometry. (B) Of the 187 proteins identified in both in vitro SILAC experiments as described in (A), the ratios of ‘heavy’ to ‘light’ labeled proteins are plotted as indicated. Proteins known to function in the innate immunity pathways are highlighted in red. NEMO (Cyan) and Krt76 (keratin, Green) have low H/L ratios because these proteins were not labeled with the heavy isotopes. (C) Similar to (A) except that Ikbkg−/−(Nemo−/−) MEF cells reconstituted with Flag-NEMOΔN or Flag-NEMOΔN-UBDm were labeled with heavy or light isotopes and infected with VSV as indicated. (D) Of the 762 proteins identified in both cell-based SILAC experiments, the H/L ratios are plotted as indicated. Proteins that associate with WT NEMO in response to viral infection are shown in the upper right quadrant. (E) Venn diagram showing proteins with H/L ratios above 1.2 in both in vitro and cell-based SILAC experiments.

https://doi.org/10.7554/eLife.00785.017
Mutation of NEMO ubiquitination sites does not impair viral induction of interferons.

(AC) Nemo−/− MEF cells stably expressing GFP, Flag-NEMO WT or mutants were infected with VSV for the indicated time. Cytokine RNA levels were measured by q-RT-PCR (A and B). Expression of the NEMO proteins was analyzed by immunoblotting (C). (D) A schematic diagram of human NEMO and the ubiquitination sites identified by mass spectrometry. (E and F) Nemo−/− MEF cells stably expressing GFP, Flag-NEMO WT, or 6KR (K111/285/309/325/342/344R) were infected with VSV for the indicated time. Cytokine RNA levels were analyzed by q-RT-PCR. (G and H) Nemo−/− MEF cell extracts (S5) were supplemented with Flag-NEMO WT or 13KR (K111/139/143/165/283/285/321/325/326/342/344/399R) protein and incubated with 35S-IRF3 and His6-MAVSΔTM. IRF3 dimerization was detected by native gel electrophoresis followed by autoradiography (G). Aliquots of the NEMO proteins were analyzed by immunoblotting (H).

https://doi.org/10.7554/eLife.00785.018
A Model for MAVS-mediated IRF3 and NF-κB activation.

Upon virus infection, MAVS undergoes prion-like polymerization to recruit and activate E3 ligases TRAF2, TRAF5, and TRAF6 (possibly also LUBAC). These E3 ligases in turn synthesize polyubiquitin chains on TRAF2 and other proteins, resulting in the recruitment of NEMO through its ubiquitin-binding domains. NEMO then recruits IKK and TBK1 complexes to the MAVS polymer, where the kinases phosphorylate IκBα and IRF3, respectively, leading to the induction of type-I interferons and other cytokines.

https://doi.org/10.7554/eLife.00785.019

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