Fig 1.
TFG interacts and colocalizes specifically with TRAF3.
(A) HEK293T cells were stably transfected with pcDNA3-FLAG-TRAF3 or pcDNA3-FLAG alone. After G418 selection, cells were lysed and subjected to AP/MS as described here [48]. Data for TFG, which was undetected in control experiments, represent previously unpublished information from two biological replicates. MS; mascot score, TP; average total number of peptides (spectral counts) identified, UP; number of unique peptides observed. (B-C) HEK293T cells were transiently transfected with empty vector (-) or with vectors encoding FLAG-tagged TRAF3 (FLAG-TRAF3) together with Myc-tagged TFG (Myc-TFG) or FLAG-tagged TFG (FLAG-TFG) together with Myc-tagged TRAF3 (Myc-TRAF3). FLAG-tagged proteins were immunoprecipitated (IP) and analysed with anti-FLAG (M2) or anti-Myc (A-14) antibodies. Whole cell extracts (WCE) were also analyzed in parallel. Data represents representative results from at least 3 independent experiments. (D) HeLa cells were transfected with FLAG-TRAF3 encoding plasmids before being fixed, permeabilized and immunostained with anti-FLAG (M2) or anti-TFG antibodies. Nuclei were labeled with DAPI. Cells were then visualized by confocal microscopy. Scaling bars represent identified length. All images for all panels were representative of at least two independent experiments in which cells were examined and displayed similar staining. (E) HEK293T cells were transfected with empty vector (-) or with vectors encoding Myc-tagged TFG (Myc-TFG) together with FLAG-tagged TRAF2, TRAF3 or TRAF6. Myc-TFG was immunoprecipitated and subjected to immunoblot analysis using anti-Myc (A-14) and anti-FLAG (M2) antibodies. WCE were also analyzed in parallel. Data represents representative results from at least three independent experiments.
Fig 2.
TFG accumulates within ER-to-Golgi compartments.
(A) HeLa cells were immunostained for endogenous TFG along with different markers of the ER-to-Golgi associated compartments including Sec31A, ERGIC-53, GM130 and EEA1. Nuclei were labeled with DAPI. Cells were then visualized by confocal microscopy. Scaling bars represent identified length. (B) HeLa cells were transfected with FLAG-TFG encoding plasmids before being fixed, permeabilized, and immunostained with anti-FLAG (M2) or anti-Sec16A antibodies. Nuclei were labeled with DAPI. Cells were then visualized by confocal microscopy. Scaling bars represent identified length. All images for all panels were representative of at least two independent experiments in which cells were examined and displayed similar staining.
Fig 3.
TFG is part of MAVS-TRAF3-TBK1 molecular complex upon activation of intracellular RNA sensors.
(A, B) Whole cell extracts (WCE) were prepared from HeLa cells subjected to SeV infection or transfected with Poly(I:C) for indicated times and were then immunoprecipitated using antibodies directed against endogenous TFG before being immunoblotted for the presence of endogenous TRAF3 (A) and TBK1 (B). WCE were also immunoblotted in parallel. Immunoblots shown are from a single experiment and are representative of at least three independent experiments. Input-normalized TRAF3 and TBK1 densitometric signal is shown below the blot. (C) HeLa cells were either infected by SeV for 4h or transfected with Poly(I:C) for 4h before being fixed, permeabilized and immunostained with anti-MAVS or anti-TFG antibodies. Nuclei were labeled with DAPI. Cells were then visualized by confocal microscopy. Scaling bars represent identified length. White arrows represent sites of close proximity (NS) or colocalization (SeV and Poly(I:C)) between MAVS and TFG. All images for all panels were representative of two (Poly(I:C) or three (SeV) independent experiments in which cells were examined and displayed similar staining.
Fig 4.
TFG is required for the formation of MAVS-TRAF3-TBK1 complex upon cytosolic RNA sensor activation.
(A-B) Co-immunoprecipitation experiments were carried using HEK293T previously transfected with indicated siRNA followed by SeV infection. TRAF3 was immunoprecipitated from the prepared whole cell extracts (WCE) using antibodies directed against endogenous TRAF3 (anti-TRAF3 H-20) or isotype control antibodies (IgG) before being immunoblotted for the presence of endogenous MAVS and TBK1. WCE were also immunoblotted with the indicated antibodies. Immunoblots shown are from a single experiment and are representative of two independent experiments. Input-normalized densitometric signal of MAVS and TBK1 was divided by Input-normalized TRAF3 signal and shown below the blot.
Fig 5.
TFG is required for the activation of TBK1 and downstream signalling upon cytosolic RNA sensor activation.
(A) HeLa cells previously treated with non-targeting siRNA (-) or siRNA targeting TFG (siTFG #1) were left uninfected or infected with SeV for indicated time. Whole cell extracts (WCE) were used in immunoblot analysis with indicated antibodies. The same WCE were used in native-page under non-denaturing conditions to evaluate IRF3 dimerization. (B) MRC-5 fibroblasts were infected with different lentiviral vectors encoding different TFG-targeting shRNA (shTFG #1, 2 or 3) or a nontargeting (NT) control shRNA (shNT) and then subjected to puromycin selection as described in Materials and Methods. Cells were then left uninfected or infected with SeV for the times indicated. WCE were harvested and used in immunoblot analysis with indicated antibodies. β-actin was used as a loading control. These results are representative of at least three independent experiments with similar results. (C) WCE generated in (A) were used in immunoblot analysis with indicated antibodies. β-actin was used as a loading control. These results are representative of at least three independent experiments with similar results.
Fig 6.
TFG is required for optimal production of ISGs and type I IFN secretion upon viral infection.
(A) MRC-5 fibroblasts were infected with different lentiviral vectors encoding different TFG-targeting shRNA (shTFG #1, 2 or 3) or a nontargeting (NT) control shRNA (shNT) and then subjected to puromycin selection as described in Materials and Methods. Cells were then left uninfected or infected with SeV for the indicated times. RNA was extracted and analyzed by RT-qPCR for indicated gene expression. Mean values and SD of biological triplicates are shown * Significantly below the induction response; * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001. RQ, relative quantification. N.D, Not detected. (B) HeLa cells previously treated with non-targeting siRNA (-) or siRNA targeting TFG (siTFG #1) were left uninfected or infected with SeV for indicated time. Supernatants were collected post-infection and analyzed for IFN-β by ELISA. Mean values and SD of biological triplicates are shown (*** P-value < 0.001).
Fig 7.
TBK1-dependent phosphorylation of mTOR on Ser2159 during viral infection.
(A) Phosphorylation and sequence alignment of different substrates of TBK1 reveal a conserved consensus site. The TBK1 phosphorylation consensus sequence is composed of a central serine that is surrounded by a hydrophobic residue (L/F/M) at the +1 position relative to the phosphorylation site and a polar uncharged side chain (S/T) at the -4 position. TBK1 autophosphorylation at Ser172 closely follows this consensus except for the -4 residue which is represented by a negatively charged side chain. (B) FLAG-GFP and FLAG-mTOR were immunoprecipitated (IP) with FLAG M2 antibody from transfected HEK 293T cells. In vitro kinase assay was conducted by adding indicated amount of recombinant TBK1 and radiolabeled ATP and incubating at 30°C for 30 min followed by detection with autoradiography and immunoblot using indicated antibodies. Data represents representative results from at least 2 independent experiments. (C) HeLa cells were transfected with FLAG- GFP, FLAG-TBK1 and FLAG-TBK1(K38A) mutant. Next day, the media was changed with serum free media for 30h and whole cell extracts (WCE) were subjected to immunoblot analysis with indicated antibodies. Data represents representative results from at least 2 independent experiments. (D, G) HEC-1-B (D), and MRC-5 (G) cells were serum starved for 30h and then left uninfected or infected with SeV for indicated time. WCE were subjected to immunoblot analysis with indicated antibodies. (E, F) HEC-1-B (E), and HeLa (F) cells were serum starved for 30h and then incubated with DMSO and a specific TBK1 inhibitor (MRT67307; 2μM) for 2h before infection with SeV for 16h under the continuous presence of DMSO or inhibitor. WCE were subjected to immunoblot analysis with indicated antibodies. Data represents representative results from at least 2 independent experiments.
Fig 8.
TFG is required for the proper positioning of mTOR with TBK1-TRAF3 complex as well as its phosphorylation on Ser2159, the putative TBK1 phosphoacceptor site.
(A) Co-immunoprecipitation (IP) experiments from whole cell extracts (WCE) prepared from HEK293T cells infected with SeV using antibodies directed against endogenous TFG and immunoblotted for the presence of endogenous mTOR and TBK1. WCE were also immunoblotted with the indicated antibodies. Immunoblots shown are from a single experiment and are representative of three independent experiments. (B) Co-IP experiments from WCE prepared from HEK293T cells following SeV infection using antibodies directed against endogenous TRAF3 and immunoblotted for the presence of endogenous mTOR, MAVS and TBK1. WCE were also immunoblotted with the indicated antibodies. Immunoblots shown are from a single experiment and are representative of three independent experiments. (C) Co-IP experiments were carried out from HEK293T WCE previously treated with indicated siRNA followed by SeV infection using TRAF3 antibody. The same WCE were also used in immunoblot analysis with indicated antibodies. Immunoblots shown are from a single experiment and are representative of two independent experiments. (D) Following the depletion of TFG, HeLa cells were infected with SeV for the indicated time. WCE were subjected to immunoblot analysis using the indicated antibodies. N.B: Data from Fig 8C was obtained simultaneously with that for Fig 4B. It was separated for clarity of presentation. Hence, the data are the same, except for the presentation of the immunoblot for mTOR.
Fig 9.
Implication of mTOR in the induction of selected sets of Interferon-Stimulated Genes (ISGs) during RLR signaling.
Serum starved HeLa cells (A) or primary MRC5 fibroblasts (B) were pretreated with 0.5 μM Ku-0063794, a highly selective mTOR inhibitor, or vehicle for 30 minutes and then left uninfected or infected with SeV (200 HAU/ml) for the indicated times in the continuous presence of the drug. Whole cell extracts (WCE) were subjected to immunoblot analysis using the indicated antibodies. (C) Serum starved primary MRC5 fibroblasts were treated as described above. Crude nuclear and cytoplasmic fractions were prepared and subjected to immunoblot analysis using the indicated antibodies. Immunoblots shown are from a single experiment and are representative of three independent experiments.
Fig 10.
Knockdown of TFG increases viral replication and dissemination.
(A) HeLa cells were treated with siNT or with four different siTFG constructs. Cells were then infected with VSV-GFP for 16h at an MOI of 0.1 and monolayers were analysed with an inverted fluorescence microscope. Observation from one experiment out of three experiments is shown. Quantification of fluorescence from 3 independent biological replicates is shown below the images. Mean values and SD of pooled data are shown (* P-value < 0.05; *** P-value < 0.001). (B) HeLa cells were treated with siNT or with siTFG #1 before being infected with VSV-GFP for 16h at indicated MOI. Monolayers were analyzed by fluorescence microscopy before whole cell extracts (WCE) were prepared. WCE were then immunoblotted with indicated antibodies. (C) MRC-5 fibroblasts were infected with different lentiviral vectors encoding different TFG-targeting shRNA (shTFG #1, 2 or 3) or a nontargeting (NT) control shRNA (shNT) and then subjected to puromycin selection. Cells were then infected with VSV-GFP for 16h at an MOI of 0.01. and monolayers were analysed with an inverted microscope. Then, the extent of VSV-GFP infection was further analysed by quantifying GFP-positive cells by flow cytometry. Data were pooled from three independent experiments and are expressed relative to their cognate shNT control from each experiment to account for day-to-day variation. Mean values and SD of pooled data are shown (* P-value < 0.05; *** P-value < 0.001).
Fig 11.
Proposed unified model representing the implication of TFG in the organization of RLR-dependent antiviral innate immunity.
The ER-to-Golgi vesicular transport system serves as an organizing membrane-rich platform allowing the organization of RLR-dependent antiviral innate immunity. TFG is involved in optimizing COPII assembly at the ERES and disassembly at the ER/ERGIC interface (pink vesicles located between the RER and ERGIC). TFG thereby enables an efficient export of proteins from the ER to other organelles. Possibly through its ability to self-associate and to generate larger polymers, TFG also allows the proper positioning of essential effectors (TRAF3, TBK1) with MAVS onto an interface between mitochondria and ER-related membranes where they can functionally interact upon viral infection. These signaling events result in the phosphorylation of IRF3, its dimerization and nuclear translocation where it rapidly induces the transcription of type I IFN genes and a subset of ISGs. TFG also allows the positioning of mTOR with TRAF3-TBK1 complexes resulting in mTORC1 signaling pathway activation. The translation of a subset of ISGs mRNA is also under the control of mTORC1 pathway, which is regulated, at least in part, by TBK1. SER: smooth endoplasmic reticulum, RER: rough endoplasmic reticulum. The model was created using Servier Medical Art templates (www.servier.com) licensed under a CC BY 3.0 license (https://creativecommons.org/licenses/by/3.0/).