Ube2D3 and Ube2N are essential for RIG-I-mediated MAVS aggregation in antiviral innate immunity

Abstract

Innate immunity plays a pivotal role in virus infection. RIG-I senses viral RNA and initiates an effective innate immune response for type I interferon production. To transduce RIG-I-mediated antiviral signalling, a mitochondrial protein MAVS forms prion-like aggregates to activate downstream kinases and transcription factors. However, the activation mechanism of RIG-I is incompletely understood. Here we identify two ubiquitin enzymes Ube2D3 and Ube2N through chromatographic purification as activators for RIG-I on virus infection. We show that together with ubiquitin ligase Riplet, Ube2D3 promotes covalent conjugation of polyubiquitin chains to RIG-I, while Ube2N preferentially facilitates production of unanchored polyubiquitin chains. In the presence of these polyubiquitin chains, RIG-I induces MAVS aggregation directly on the mitochondria. Our data thus reveal two essential polyubiquitin-mediated mechanisms underlying the activation of RIG-I and MAVS for triggering innate immune signalling in response to viral infection in cells.

Figure 1. Reconstitution of a cell-free assay recapitulating RIG-I and MAVS activation in antiviral signalling.

(a,b) S1 fraction from HEK293T cells (wild type or Rig-i^−/−) was incubated with or without His-RIG-I, His-RIG-I-2CARD, vRNA at 30 °C for 1 h, and NEM (40 μM) or PYR-41 (40 μM) was included as indicated (b). The reaction mixture was then separated with SDD–AGE, SDS-PAGE and Native-PAGE followed by immunoblotting (IB) analysis. The original full blot for SDD-AGE shown in a can be found in Supplementary Fig. 8a. (c) HEK293T cells were treated with PYR-41 at indicated concentrations for two hours before infected with or without Sendai virus, which were collected for subcellular fractionation followed by immunoblotting. (d) HEK293T cells (wild type and knockout lines) were infected with or without Sendai virus, which were collected 12 h post infection and analysed as described in d. See also Supplementary Fig. 1b. The original full blot for SDD-AGE can be found in Supplementary Fig. 8b. (e) HEK293T cells (wild type and knockout lines) were transfected with pcDNA3-flag-MAVS. Thirty six hours after transfection, the cells were collected and IFN induction was measured with quantitative PCR (qPCR). See also Supplementary Fig. 1e. (f) Flag-Riplet (wild type or mutant form) and Flag-TRIM25 were transduced into MEFs (wild type and knockout lines) by retrovirus. Forty eight hours after transduction, the cells were infected with VSV for sixteen hours. The cells were then collected and IFN induction was measured with qPCR. See also Supplementary Fig. 1f. *P<0.05 and ***P<0.001. NS indicates no statistically significant difference. See also Supplementary Fig. 1g.

Figure 2. Purification of multiple E2s required for RIG-I and MAVS activation in the cell-free assay.

(a) The cell-free assay was performed in the presence or absence of Riplet as described in Fig. 1a. (b) S100 fractions were prepared from HEK293T cells (Rig-i^−/− and Riplet^−/−, DKO). The cell-free assay containing S100 and vRNA was performed with or without purified components (E1, Ub, RIG-I and Riplet) as indicated. One hour after incubation, the reaction mixture was further incubated with P5 fraction. P5 was then subjected to SDD-AGE and IRF3 dimerization assay. See also Supplementary Fig. 2b. (c) Fractions (A, B and C) from Q column were used to replace S100 fraction in the cell-free assay and analysed as described in (b, Lane 5). (d) Fractions (from 1 to 9) from Superdex200 column were used to replace S100 fraction in the cell-free assay and analysed as described in b. These fractions were also separated by SDS-PAGE and visualized by silver staining (top panel). The band indicated by the arrow were isolated and subjected to mass spectrometric analysis. The original full blot for silver-staining can be found in Supplementary Fig. 8c.

Figure 3. Ub2D3 and Ube2N are required for RIG-I and MAVS activation on virus infection in HEK293T cells.

(a,b) The cell-free assay containing vRNA and purified components (E1, Ub, RIG-I and Riplet) was performed as described in Fig. 2b, except that S100 fraction was substituted with purified E2 recombinant proteins as indicated. Each E2 protein was analysed at three different concentrations. Lane 13 did not contain E2 proteins (−E2). (c) HEK293T cells (wild-type and knockout lines) were transfected with or without si-Ube2D3 RNAs. Seventy two hours after transfection, the cells were infected with VSV. Twelve hours post virus infection, the cells were collected for measuring IFN induction by qPCR. *P<0.05 and ***P<0.001. (d,e) Cells were treated as described in c. P5 fractions were isolated to examine MAVS aggregation (d), and whole cell lysate were used to analyse knockdown or knockout effect in genes as indicated (e). The original full blot for (e) can be found in Supplementary Fig. 8d. (f) si-Ube2D3 RNAs were transfected into HEK293T (Ube2N^−/−) cells. Seventy two hours after siRNA transfection, the cells were further transfected with pcDNA-flag-Ube2D3 or pcDNA-flag-Ube2N as indicated. Twenty four hours after transfection of pcDNA plasmids, the cells were infected with VSV for another twelve hours. The cells were then collected for analysis of MAVS aggregation. See also Supplementary Fig. 3f.

Figure 4. Ube2N is required for RIG-I and MAVS activation in MEFs.

(a–c) Small hairpin RNAs (sh-Ube2D1/2/3/4 & N) as indicated was transduced into MEFs with lentivirus. Eighty four hours after transduction, the cells were infected with VSV. Sixteen hours post infection, the cells were collected and IFN β (a) and IFN α (b) production were measured by qPCR. Immunoblotting were also performed to examine knockdown efficiency (c). *P<0.05 and ***P<0.001. NS indicates no statistically significant difference. (d) MEFs were transduced with or without sh-Ube2N. Eighty four hours after transduction, Flag-MAVS was further transduced into MEFs by retrovirus for forty eight hours. The cells were then infected with VSV. Sixteen hours post infection, the cells were collected to measure cytokine production by qPCR. See also Supplementary Fig. 4d. (e) MEFs were treated as described in a except that the cells were collected to isolate P5 fractions. P5 fractions were subjected to SDD-AGE to examine MAVS aggregation and IRF3 dimerization assay in vitro. The original full blot can be found in Supplementary Fig. 9a. (f) MEFs were treated as described in d except that Flag-Ube2D3 or Flag-Ube2N but not Flag-MAVS was transduced into the cells by retrovirus as indicated. See also Supplementary Fig. 4e.

Figure 5. The association of RIG-I with Riplet and Ube2D3/Ube2N.

(a) pcDNA3-flag-sumo, pcDNA3-flag-RIG-I and pcDNA3-HA-Riplet were transfected into HEK293T cells as indicated. Twenty four hours after transfection, the cells were infected with or without VSV. The cells were collected 12 h post infection and lysed for IP with M2 beads. IP products and whole-cell lysate (WCL) were subjected to immunoblotting. The original full blot can be found in Supplementary Fig. 9b. (b,c) pcDNA3-flag-sumo, pcDNA3-flag-RIG-I (full length and deletions) and pcDNA3-HA-Riplet were transfected into HEK293T cells. Thirty six hours after transfection, IP was performed as described in a. RIG-I-ΔRD covers aa-1-794; RIG-I-ΔCARD covers aa-229-925; RIG-I-Helicase covers aa-229-794; RIG-I-2CARD covers aa-1-200; RIG-I-RD covers aa-795-925. (d–f) pcDNA3-HA-RIG-I, pcDNA3-flag-Riplet, pcDNA3-flag-sumo pcDNA3-HA-Riplet, pcDNA3-flag-Ube2D3 and pcDNA3-flag-Ube2N were transfected into HEK293T cells. Thirty-six hours after transfection, IP was performed. Riplet-SPRY covers aa 261–428.

Figure 6. Covalently anchored and unanchored polyubiquitin chains are two alternative mechanisms for RIG-I and MAVS activation.

(a) The cell-free assay was performed using purified components (E1, Ube2N or Ube2D3, Riplet, ubiquitin, RIG-I) as described in Fig. 3b. The reaction mixtures were treated with or without IsoT or vOTU for one hour before further incubation with P5 fraction to measure MAVS aggregation and activity in stimulating IRF3 dimerization. An aliquot of the reaction mixture was subjected to immunoblotting to examine RIG-I ubiquitination. The original full blot can be found in Supplementary Fig. 9c. (b) The cell-free assay (containing E1, Ube2N, Riplet and ubiquitin) was performed as described in a in the presence (Lanes 1 and 2) or absence of RIG-I (Lanes 3 and 4). Lane 2 also included NEM. After incubation, NEM and RIG-I were added (Lanes 3 and 4). The sample in Lane 3 was further treated with IsoT. The reaction mixtures were then incubated with P5 fraction to examine MAVS aggregation. PU: post ubiquitination, indicating that NEM and RIG-I were added after the incubation of ubiquitin enzymes and ubiquitin (Lanes 3 and 4). (c) The cell-free assay was performed using purified components (E1, Ube2D3, Riplet or TRIM25, ubiquitin, RIG-I) as described in a. The reaction mixtures were treated with or without IsoT or vOTU before further incubation with P5 fraction to measure MAVS aggregation and activity in stimulating IRF3 dimerization. An aliquot of the reaction mixture was subjected to immunoblotting to examine polyubiquitin chain formation. (d) The cell-free assay was performed using purified components (E1, Ube2D3, Riplet, various forms of ubiquitin, RIG-I) as described in a. K48R: lysine48 of ubiquitin was mutated to R; K48: all lysine residues of ubiquitin except lysine48 were mutated to R; K63R: lysine63 of ubiquitin was mutated to R; K63: all lysine residues of ubiquitin except lysine63 were mutated to R; Me-Ub: methylated ubiquitin, as a negative control. (e) The cell-free assay was performed as described in (d) except that Ube2D3 was omitted and Ube2N was used.

Figure 7. Modification sites of RIG-I by polyubiquitin chains.

(a) The ubiquitination reaction was performed using purified components (E1, Ube2D3, Riplet, ubiquitin (WT or K63), His-flag-RIG-I). His-flag-RIG-I was then immunoprecipitated by M2 beads and eluted with flag peptide, followed by SDS-PAGE and silver-staining. (b) Ubiquitination sites of RIG-I identified by mass spectrometric analysis. (c) pcDNA3-flag-RIG-I (wild type and various mutant forms) were transfected into HEK293T (Rig-i^−/−) cells. Thirty six hours after transfection, the cells were infected with or without VSV for twelve hours, which then were collected to measure IFN induction by qPCR. *P<0.05 and ***P<0.001. (d) pcDNA3-HA-ubiquitin and pcDNA3-flag-sumo or pcDNA3-flag-RIG-I (wild type and various mutant forms) were transfected into HEK293T (Rig-i^−/−) cells. Thirty six hours after transfection, the cells were infected with or without VSV. Twelve hours post infection, the cells were collected for IP with M2 beads and IP products were subjected to immunoblotting. The original full blot can be found in Supplementary Fig. 9d. (e) Wild type and various mutant forms of RIG-I were transfected into HEK293T (Rig-i^−/−), HEK293T (Rig-i^−/− & Ube2D3^−/−) or HEK293T (Rig-i^−/− & Ube2N^−/−) cells. Thirty six hours after transfection, the cells were infected with VSV for twelve hours, which then were collected to measure IFN induction by qPCR. *P<0.05 and ***P<0.001.