TRIM68 negatively regulates IFN-β production by degrading TRK fused gene, a novel driver of IFN-β downstream of anti-viral detection systems

Abstract

In recent years members of the tripartite motif-containing (TRIM) family of E3 ubiquitin ligases have been shown to both positively and negatively regulate viral defence and as such are emerging as compelling targets for modulating the anti-viral immune response. In this study we identify TRIM68, a close homologue of TRIM21, as a novel regulator of Toll-like receptor (TLR)- and RIG-I-like receptor (RLR)-driven type I IFN production. Proteomic analysis of TRIM68-containing complexes identified TRK-fused gene (TFG) as a potential TRIM68 target. Overexpression of TRIM68 and TFG confirmed their ability to associate, with TLR3 stimulation appearing to enhance the interaction. TFG is a known activator of NF-κB via its ability to interact with inhibitor of NF-κB kinase subunit gamma (IKK-γ) and TRAF family member-associated NF-κB activator (TANK). Our data identifies a novel role for TFG as a positive regulator of type I IFN production and suggests that TRIM68 targets TFG for lysosomal degradation, thus turning off TFG-mediated IFN-β production. Knockdown of TRIM68 in primary human monocytes resulted in enhanced levels of type I IFN and TFG following poly(I:C) treatment. Thus TRIM68 targets TFG, a novel regulator of IFN production, and in doing so turns off and limits type I IFN production in response to anti-viral detection systems.

Figure 1. TRIM68 is structurally similar to TRIM21 and down-regulates type I IFN production downstream of TBK1.

(A) Human TRIM68 and TRIM21 amino acid sequence alignment using T-Coffee (http://tcoffee.crg.cat/apps/tcoffee/do:regular). An * (asterisk) indicates positions which have a single, fully conserved residue. A: (colon) indicates conservation between groups of strongly similar properties - scoring >0.5 in the Gonnet PAM 250 matrix. A. (period) indicates conservation between groups of weakly similar properties - scoring <0.5 in the Gonnet PAM 250 matrix. (B–C) HeLa cells were transfected with 40 ng full-length IFN-β promoter, empty vector (EV) control and increasing amounts of TRIM68 as indicated for 18 hr and were then stimulated with 20 µg/ml poly(I:C) for 24 hr. In all other cases HEK293T cells were transfected with 40 ng full-length IFN-β promoter and were co-transfected with 50 ng of the relevant promoter drivers TRIF, TBK1, IRF3 (B), or dsRNA, RIG-I, MAVS (C) as well as with empty vector (EV) control and increasing amounts of TRIM68 as indicated. Cells were assayed for relative fold increase of reporter gene activity 18–24 hr post-transfection. (D) HeLa cells were transiently transfected with 1 µg of TRIM68 together with 1 µg/ml 5′ppp-dsRNA for 6 and 24 hr or 18 hr later with 20 µg/ml poly(I:C) for 6 and 24 hr before supernatants were collected for RANTES ELISA. In all cases presented data is graphed from the average of three separate experiments ± S.E.M. *p<0.05 was considered significant.

Figure 2. TRIM68 interaction with TRK-fused gene protein (TFG) increases post TLR3 stimulation.

(A) TRIM68 bound proteins were immunoprecipitated from lysates with anti-FLAG coated agarose beads (Sigma). Immunoprecipitation using normal mouse IgG functioned as a negative control. Selected bands were excised, trypsin digested and analysed by mass spectrometry. A western blot showing corresponding lysates from the silver stained gel was immunoblotted for FLAG to verify successful transfection and immunoprecipitation of FLAG-TRIM68. Immunoblots shown are from a single experiment and are representative of two independent experiments. (B) HEK293T cells were transfected with 2 µg of EV and FLAG-TRIM68. TRIM68 containing complexes were immunoprecipitated using anti-FLAG coated beads and blotted for TFG and FLAG. Lysates were immunoblotted for TFG and FLAG. Immunoblots shown are from a single experiment and are representative of three independent experiments. (C) HEK293T cells were transfected with 2 µg of EV and FLAG-TRIM68. TFG containing complexes were immunoprecipitated using anti-TFG coated beads and immunoblotted for FLAG and TFG. Lysates were immunoblotted for TFG and FLAG. Immunoblots shown are from a single experiment and are representative of three independent experiments. (D) HeLa cells were transfected with 2 µg of GFP-TRIM68 (green) expression plasmid and stimulated with 20 µg/ml of poly(I:C) for 6 and 24 hr. Cells were fixed and then stained with anti-TFG before mounting in DAPI in order to visualise nuclei (blue). Images were taken under oil immersion at 63X magnification. Panels on the right contain zoomed images of white box in previous panel. Images shown are from a single experiment and are representative of three independent experiments. The raw integrated density value (sum of pixel values) was calculated using ImageJ software (1.48 4-bit Java) for TFG only images for each poly(I:C) time point. Presented data is graphed from the average of sixteen separate images ± S.E.M. *p<0.05 was considered significant.

Figure 3. TRIM68 negatively regulates type I IFN production via polyubiquitination and lysosomal degradation of TFG.

(A) HEK293T cells were co-transfected with MYC-TFG, HA-Ubiquitin, FLAG-TRIM68 and EV. Lysates were immunoblotted for TFG, alpha actinin, HA and FLAG. Immunoblots shown are from a single experiment and are representative of three independent experiments. (B) HEK293T cells were transfected with 1 µg of EV, MYC-TFG, HA-Ubiquitin and FLAG-TRIM68. Ubiquitin containing complexes were immunoprecipitated using anti-HA coated beads and immunoblotted for MYC, FLAG and HA. Lysates were immunoblotted for FLAG and HA. Immunoblots shown are from a single experiment and are representative of three independent experiments. (C) Cells were treated with proteasomal inhibitor MG132 (10 µM) and DMSO vehicle control for 2 hr or lysosomal inhibitor NH₄Cl (20 mM) or PBS vehicle control for 18 hr before cells were lysed and immunblotted for MYC, alpha actinin, FLAG and HA. In all cases the immunoblots shown are from a single experiment and are representative of three independent experiments. Densitometry data is graphed from the average of three separate experiments ± S.E.M. of TFG normalised to alpha actinin.

Figure 4. TRIM68 knockdown results in an increase in IFN-β and TFG levels post TLR3 stimulation.

(A) HEK293T cells were transfected with 40 ng full-length IFN-β promoter and increasing amounts of TFG as indicated. Cells were assayed for relative fold increase of reporter gene activity 18–24 hr post-transfection. (B) HEK293T cells were transfected with 400 ng full-length IFN-β promoter and were co-transfected with 250 ng of Scrambled (Scr) or TFG shRNA as indicated and 24 hr later 50 ng of TRIF. Cells were assayed for relative fold increase of reporter gene activity 18–24 hr post-transfection. Presented data is graphed from one experiment. (C) HeLa cells were transiently transfected with 1 µg of TFG and 18 hr later were stimulated with 20 µg/ml poly(I:C) for 24 hr before supernatants were collected for RANTES ELISA. (D) HEK293T cells were transfected with 40 ng full-length IFN-β promoter and were co-transfected with 50 ng of TFG, TBK1 or dsRNA as well as with empty vector (EV) control and 50 ng of full length TRIM68 or TRIM68 mutant lacking the RING and B-box domain (TRIM PRY/SPRY) as indicated. Cells were assayed for relative fold increase of reporter gene activity 18–24 hr post-transfection and % inhibition on promoter activity was calculated. Presented data is graphed from the average of three separate experiments ± S.E.M. *p<0.05 was considered significant. (E) A schematic of the IFN-β promoter which consists of three principle transcription factor binding sites, the PRD IV region which binds the transcription factor ATF-2/c-Jun, the PRD III–I region which binds the transcription factors IRF3 and IRF7 and the PRD II region which binds NF-κB. (F) HEK293T cells were transfected with 40 ng full-length IFN-β promoter or the PRD segments (PRD II, PRD III-I, PRD IV) and 50 ng of TFG as well as with EV control and increasing amounts of TRIM68 as indicated. Cells were assayed for relative fold increase of reporter gene activity 18–24 hr post-transfection. (G–I) Human monocytes were treated with PLGA microparticles containing either Scrambled (Scr) or TRIM68 shRNA for 48 hr. Real-time PCR was used to measure TRIM68 mRNA levels post treatment (G). After treatment with PLGA microparticles, monocytes were stimulated with poly(I:C) for 24 hr before IFN-β mRNA was measured (H). Human monocytes which have been treated with shRNA containing microparticles as above were immunoblotted for TFG and alpha actinin. Immunoblots shown are from a single experiment and are representative of three independent experiments (I). In all other cases, presented data is graphed from the average of three separate experiments ± S.E.M. *p<0.05 was considered significant.