Fig 1.
Neddylation facilitates the DNA-triggered signaling pathway.
(A and B) MEFs were pretreated with MLN4924 for 2h, followed by stimulation with HSV-1 (MOI = 1) or ISD for 6h, the transcription of Ifnb, Ifna4 and Cxcl10 were measured by qRT-PCR. (C) THP-1 cells were pretreated with MLN4924 for 2h and then treated with HSV-1 (MOI = 1) for 6h, the transcription of IFNB, CXCL10 and IFIT2 were measured by qRT-PCR. (D) MEFs were pretreated with MLN4924 for 2h and then treated with IFNβ, the induction of Cxcl10 and Ifit1 were measured by qRT-PCR. (E) MEFs were pretreated with MLN4924 for 2h, followed by stimulation with ISD for 6h, then fixed and stained with an antibody specific for IRF3 and imaged by confocal microscopy. Scale bars represent 50 μm. Graphs are presented as means ± SEM, data are representative of three independent experiments, *P <0.05; **P <0.01 (One-way ANOVAs followed by Tukey’s post hoc test).
Fig 2.
Depletion of Ube2m but not Ube2f attenuates dsDNA-induced innate immune response.
(A) Effects of Ube2m deficiency on the expression of Ifnb, Ifna4 and Cxcl10 after ISD stimulation for indicated hours in BMDM. (B) Effects of Ube2m deficiency on the expression of Ifnb, Ifna4, Cxcl10 and Ifit1 after HSV-1 (MOI = 1) stimulation in BMDM. (C) Effect of Ube2f deficiency on the expression of Ifnb, Ifna4 and Cxcl10 after ISD and HT-DNA stimulation in BMDM. (D) Effects of Ube2m deficiency on the phosphorylation of TBK1/IRF3 and the dimerization of IRF3 after ISD stimulation for the indicated time. (E) Effects of Ube2f deficiency on phosphorylation of TBK1/IRF3 and the dimerization of IRF3 after ISD stimulation for the indicated time. (F) Effects of Ube2m deficiency on the protein level of cGAS and STING. Graphs are presented as means ± SEM, data are representative of three independent experiments, *P <0.05; **P <0.01 (One-way ANOVAs followed by Tukey’s post hoc test).
Fig 3.
Rnf111 facilitates dsDNA-triggered signaling.
(A) dsDNA stimulation promoted the expression of Rnf111 in MEFs (left) and BMDM (right). (B) Effect of Rnf111 deficiency on the expression of Ifnb, Ifna4 and Cxcl10 after ISD and HT-DNA stimulation for indicated hours in BMDM. (C) Effect of Rnf111 deficiency on the expression of Ifnb, Ifna4 and Cxcl10 after HSV-1 (MOI = 0.5) infection in BMDM. (D) Effects of Rnf111 deficiency on IRF3 dimerization and phosphorylation of TBK1/IRF3 after ISD (left) or cGAMP (right) stimulation for the indicated time. (E) Effect of Rnf111 deficiency on the expression of Ifnb and Cxcl10 after cGAMP stimulation for 3h in BMDM. (F) Rnf111 deficiency BMDM were transfected with 100 ng of empty vector (EV, pcDNA4.0) or plasmids for the expression of wild-type His-Rnf111 or His-Rnf111 C937A. 48h after transfection, BMDM were stimulated with ISD for 6h, the transcription of Ifnb, Ifna4 and Cxcl10 were measured by qRT-PCR. Graphs are presented as means ± SEM, data are representative of three independent experiments, *P <0.05; **P <0.01 (One-way ANOVAs followed by Tukey’s post hoc test).
Fig 4.
(A) MEFs were stimulated with ISD for 3h, stained with indicated antibodies and imaged by confocal microscopy. Scale bars represent 200 μm. (B) MEFs were stimulated with ISD for 3h, then PLA analysis was applied to detect the interaction between cGAS and Rnf111. Scale bars represent 10 μm. (C) WT or cGAS-/- L929 were stimulated with HT-DNA for 3h, then PLA analysis was applied to detect the interaction between cGAS and Rnf111. Scale bars represent 10 μm. (D) HEK293T cells were transfected with indicated plasmids, 24h after transfection, cell lysis was immunoprecipitated with an anti-Flag antibody and then immunoblotted with indicated antibodies. (E) MEFs were infected with HSV-1 (MOI = 0.5) for the indicated time, cell lysis was immunoprecipitated with an anti-cGAS or IgG antibodies and then immunoblotted with indicated antibodies. (F) Diagrams of Rnf111 truncations used in this paper. (G) HEK293T cells were transfected with indicated plasmids for 24h, then cell lysis was immunoprecipitated with anti-Flag antibody and then immunoblotted with indicated antibodies. (H) HEK293T cells were co-transfected with plasmids of different truncations of cGAS and Rnf111, 24h after transfection, lysates of HEK293T were immunoprecipitated with anti-HA antibody and then immunoblotted with indicated antibodies.
Fig 5.
Molecular characterization of cGAS neddylation.
(A) HEK293T cells were transfected with indicated plasmids. 48h after transfection, cells were denatured with 1% SDS, then cells were lysed and immunoprecipitation with anti-Flag antibody and then analyzed by immunoblotting with indicated antibodies. (B) HEK293T cells were transfected with indicated plasmids. 45h after transfection, one group of cells was treated with 5mM MLN4924 for 3h, then cells were subjected to denatured immunoprecipitation with anti-Flag antibody and then analyzed by immunoblotting with indicated antibodies. (C) HEK293T cells were transfected with HA-Nedd8, Flag-cGAS, myc-Rnf111 (WT), myc-Rnf111(C937A). 48h after transfection, cells were subjected to denatured immunoprecipitation with anti-Flag antibody and then analyzed by immunoblotting with indicated antibodies. (D) Recombinant cGAS was incubated with NEDD8, E1, UBE2M and RNF111 in the presence of ATP, after the reaction, proteins were subjected to SDS-PAGE, and immunoblotting with indicated antibodies. (E) MEFs were infected with HSV-1 (MOI = 0.3) for indicated times, then cell lysis was subjected to denatured immunoprecipitation with anti-NEDD8 antibody and then analyzed by immunoblotting with indicated antibodies.
Fig 6.
Neddylation promotes the activation of cGAS.
(A) HEK293T cells were transfected with indicated plasmids. 24h after transfection, cells were stimulated with HT-DNA for 4h before cells were harvested for cGAMP detection. (B) Rnf111 deficiency or control BMDM were transfected with ISD for 4h, cell lysates were heat denatured, and cGAMP was measured by ELISA. (C) Ube2m deficiency BMDMs were infected with HSV-1 (MOI = 0.5) for 3h, after adding human cDNA as an external reference, cell lysates were immunoprecipitated with anti-cGAS antibody, then cGAS-bound DNA was extracted and quantified by qRT-PCR by normalized to human GAPDH. (D) Rnf111 deficiency BMDMs were infected with HSV-1 (MOI = 0.5) for 3h, after adding human cDNA as an external reference, cell lysates were immunoprecipitated with anti-cGAS antibody, then cGAS-bound DNA was extracted and quantified by qRT-PCR by normalized to human GAPDH. (E) Rnf111 deficiency BMDMs were transfected with an empty plasmid for 3h, after adding human cDNA as an external reference, cell lysates were immunoprecipitated with anti-cGAS antibody, then cGAS-bound DNA was extracted and quantified by qRT-PCR by normalized to human GAPDH. (F) HEK293T cells were transfected with indicated plasmids for 45h and then infected with HSV-1 (MOI = 0.3) for 4h, the cell lysates were immunoprecipitated with an anti-Flag antibody and then immunoblotted with indicated antibodies. (G) HA-NEDD8 and Flag-cGAS were coexpressed in HEK293T cells, 48h later cells were collected and cell lysates were incubated with recombinant His-cGAS, the interaction was analyzed by immunoprecipitated with anti-Flag beads and immunoblotted with indicated antibodies. (H) Poly-NEDD8 chains were generated in vitro, followed by incubating with His-cGAS. After immunoprecipitated with anti-His antibody, and then immunoblotted with indicated antibodies. (I) Poly-NEDD8 chains were generated in vitro, followed by incubating with His-cGAS. After immunoprecipitated with anti-NEDD8 antibody, and then immunoblotted with indicated antibodies. Graphs are presented as means ± SEM, data are representative of three independent experiments, *P <0.05; **P <0.01 (One-way ANOVAs followed by Tukey’s post hoc test).
Fig 7.
UBE2M-RNF111 mediated neddylation pathway is indispensable for the innate defense against HSV-1 infection.
(A) Mice were injected intravenously with HSV-1 (1.5×107 pfu per mouse) for 12h, tissues from Ube2m cKO or the control group were harvest and the relative expression of Ifnb and Cxcl10 in livers, spleens and lungs were measured by qRT-PCR, respectively (n = 6–10). (B) Mice were injected intravenously with HSV-1 (1.5×107 pfu per mouse) for 12h, the serum was harvested and the concentration IFNβ was analyzed with ELISA (n = 6–8). (C) Mice were injected intravenously with HSV-1 (1.5×107 pfu per mouse) for 72h, lungs were harvested and inflammatory cells infiltration were analyzed by H&E staining (left), scale bars represent 100 μm. The presence of inflammation was scored and counted (right, n = 5). (D) Mice of the Ube2m cKO or the control groups were injected intravenously with HSV-1 (6×107 pfu per mouse), and the survival rates were monitored for 7 days (n = 10). (E) Mice were injected intravenously with HSV-1 (1.5×107 pfu per mouse) for 12h, tissues from Rnf111 cKO or the control group was harvest, and the relative expression of Ifnb and Cxcl10 in livers, spleens and lungs were measured by qRT-PCR (n = 6–10). (F) Mice were injected intravenously with HSV-1 (1.5×107 pfu per mouse) for 12h, the serum was harvested and the concentration IFNβ was measured with ELISA (n = 6–10). (G) Mice were injected intravenously with HSV-1 (1.5×107 pfu per mouse) for 72h, lungs were harvested and inflammatory cells infiltration were analyzed by H&E staining (left), scale bars represent 100 μm. The presence of inflammation was scored and counted (right, n = 5). (H) Mice of the Rnf111 cKO or the control groups were injected intravenously with HSV-1 (6×107 pfu per mouse), and the survival rates were monitored for 7 days (n = 10). Graphs are presented as means ± SEM, data are representative of three independent experiments, *P <0.05; **P <0.01 (Mann-Whitney test for C and G, Mantel-Cox test for D and H, One-way ANOVAs followed by Tukey’s post hoc test for the rest).
Fig 8.
Models of RNF111-facilitated neddylation potentiated the activation of cGAS.
In the presence of cytosolic DNA, the Ube2m-Rnf111 neddylation axis facilitated the neddylation of cGAS, which in turn promoted the dimerization and enhanced the DNA-binding ability of it. In the end, the Ube2m-Rnf111 neddylation axis potentiated the cGAS-STING antiviral signaling.