Fig 2: The ubiquitin ligase activity of TRIM21 is required for TRIM21-mediated inhibition of PKR activation.(A) Immunoprecipitation analysis the interaction between PKR and TRIM21. HEK293T cells were transfected with p3xFlag-CMV-PKR and pcDNA3.1a-TRIM21 for 48 h. The anti-Flag antibody was used to perform immunoprecipitation and detect Flag-PKR, the anti-V5 antibody was used to detect V5-TRIM21. (B) HEK293T cells were co-transfected with p3xFlag-CMV-TRIM21 and pcDNA3.1a-eIF2α for 48 h. The anti-Flag antibody was used to perform immunoprecipitation and detect Flag-PKR. The anti-V5 antibody was used to detect V5-eIF2α. β-actin was used as an internal control. (C-D) Endogenous PKR and TRIM21 interaction was analyzed by IP in A549 cells with VSV (MOI = 0.2 or 0.5) infection for 6 h (C) or with TG (10 μM) treatment for 12 h (D). The anti-TRIM21 antibody was used to perform immunoprecipitation. The indicated proteins were detected by the corresponding antibodies. GAPDH (C) or β-actin (D) was used as an internal control. (E) Immunoprecipitation analysis of the ubiquitination of PKR. HEK293T cells were co-transfected p3xFlag-CMV-PKR with pcDNA3.1a-vector or pcDNA3.1a-TRIM21 as well as HA-ub for 48 h. The anti-Flag antibody was used to perform immunoprecipitation and detect Flag-PKR. The anti-V5 antibody was used to detect V5-TRIM21. GAPDH was used as an internal control. (F) HLCZ01 cells were infected with lentivirus-Flag-TRIM21-WT or lentivirus-Flag-TRIM21-C16S for 48 h, then infected with VSV (MOI = 0.2) for 6 h. The indicated proteins were detected by western blot. β-actin was used as an internal control. (G) TRIM21-deficient A549 cells (A549-sg-TRIM21) were transfected with p3xFlag-CMV-TRIM21 or p3xFlag-CMV-TRIM21-D-R for 48h, then infected with VSV (MOI = 0.5) for 6 h. The indicated proteins were detected by western blot. GAPDH was used as an internal control. (H) TRIM21-deficient A549 cells (A549-sg-TRIM21) were infected with lentivirus-Flag-TRIM21-WT or lentivirus-Flag-TRIM21-C16S for 48 h, then stimulated with TG (20 μM) for 12 h. The indicated proteins were detected by western blot. β-actin was used as an internal control. The relative interaction ratios of PKR and TRIM21 in (C) were quantified by densiometric analysis, which were normalized to the value in the control group. The relative ratios of p-PKR or p-eIF2α in (F-H) were quantified by densiometric analysis, which were normalized to the value in the control group. Experiments were independently repeated two or three times with similar results, and the data shown are mean ± SD. P values were determined by Student’s t-test. *p<0.05, **P<0.01, NS, no significance difference.

Fig 3: TRIM21 inhibits PKR activation by augmenting PKR-PP1α interaction.(A) A549 cells were infected with lentivirus-sh-vector (sh-vector) or lentivirus-sh- PP1α (sh-PP1α) for 12 h, and infected with lentivirus-sh-vector (sh-vector) or lentivirus-sh-TRIM21 (sh-TRIM21) for 48 h, then infected by VSV (MOI = 0.2 or 0.5) for 6 h. The indicated proteins were detected by western blot by using the corresponding antibodies. GAPDH was used as an internal control. (B) Puromycin incorporation assays demonstrated the cellular protein synthesis in wild-type A549 cells (sh-vector) or PP1α-silenced A549 cells (sh-PP1α). Cells were infected with lentivirus-sh-vector (sh-vector) or lentivirus-sh-TRIM21 (sh-TRIM21) for 48 h, then infected by VSV (MOI = 0.2) for 6 h or stimulated with TG (10 μM) for 12 h. The indicated proteins were detected by western blot by using the corresponding antibodies. GAPDH was used as a control. (C-D) PP1α-silenced A549 cells (sh-PP1α) were infected with lentivirus-Flag-PP1α-WT or lentivirus-Flag-PP1α-K60R for 48 h, then infected with VSV (MOI = 0.5) for 6 h (C) or stimulated with TG (10 μM) for 12 h (D). The indicated proteins were detected by western blot by using the corresponding antibodies. β-actin was used as an internal control. (E) Puromycin incorporation assays demonstrated the cellular protein synthesis in PP1α-silenced A549 cells (sh-PP1α). Cells were infected with lentivirus-sh-vector (sh-vector) or lentivirus-sh-TRIM21 (sh-TRIM21) for 12 h, and infected with lentivirus-Flag-PP1α-WT or Lentivirus-Flag-PP1α-K60R for 48 h, then infected by VSV (MOI = 0.2) for 6 h or stimulated with TG (10 μM) for 12 h. GAPDH was used as a control. (F-G) TRIM21 blocks PKR-PP1α interaction. A549 cells were infected with lentivirus-sh-vector (sh-vector) or lentivirus-sh-TRIM21 (sh-TRIM21) for 48 h, then infected by VSV (MOI = 0.5) for 6 h. The interaction of PP1α and PKR was analyzed by IP and western blot (F). The wild-type A549 cells (sg-vector) or TRIM21-deficient A549 cells (sg-TRIM21) were stimulated with TG (10 μM) for 12 h, The interaction of PP1α and PKR was analyzed by IP and western blot (G). GAPDH (F) or β-actin (G) was used as an internal control. (H) TRIM21-deficient A549 cells (sg-TRIM21) were infected with lentivirus-Flag-TRIM21-WT or lentivirus-Flag-TRIM21-C16S for 48 h, then infected by VSV (MOI = 0.5) for 6 h. The interaction of PP1α and PKR was analyzed by IP and western blot. β-actin was used as an internal control. (I) PP1α-silenced A549 cells (sh-PP1α) were infected with lentivirus-Flag-PP1α-WT or lentivirus-Flag-PP1α-K60R for 48 h. The interaction of PP1α and PKR was analyzed by IP and western blot. β-actin was used as an internal control. (J) Schematic model of TRIM21 regulating the translation initiation. Upon stress stimulation, TRIM21 augments the polyubiquitination of PP1α, enhancing the PKR-PP1α interaction, leading to PKR inactivation and subsequent release of protein synthesis inhibition. The relative ratios of p-PKR or p-eIF2α in (A, C and D), the relative ratios of puro signal in (B, E) and the amounts of PP1α bound PKR in (F-H) were quantified by densiometric analysis, which were normalized to the value in the control group. Experiments were independently repeated two or three times with similar results, and the data shown are mean ± SD. P values were determined by Student’s t-test. *p<0.05, **P<0.01, ***p<0.001, NS, no significance difference.

Fig 4: Trim21 exposes AdV genomes to cGAS and STING to trigger NLRP3‐dependent inflammasome activation AIM2 mRNA levels from PBMCs, CD19+ve B cells, CD14+ve monocytes or HMDM derived from CD14+ve monocytes were assessed by qPCR, and copy number was determined relative to actin copy number (n = 5 mean ± s.e.m).HMDM were stimulated with AdV (50,000 pp/cell) and 20 μg/ml h9C12 for 1 h, washed 2× with SFM and then whole cell lysates harvested after a further 5 h. Viral hexon and transgene (GFP) expression in the cytosol was assessed by Western blot. Data are representative of two independent experiments.THP‐1s expressing either a control guide RNA or targeting cGAS and STING were generated and stimulated with 1,000 U/ml IFN‐α for 4 h and protein levels assessed by Western blot.THP‐1s deficient in cGAS and STING were stimulated with AdV (50,000 pp/cell) and 20 μg/ml h9C12, 200 ng/well HT‐DNA, 10 μM Nigericin or 10 ng/ml LPS for 16 h. Data show combined data (mean ± s.e.m) of three experiments with absolute protein values (upper panel) or as % cytokine output of KO cells relative to Ctrl‐treated cells (lower panel), *P ≤ 0.05, ***P ≤ 0.001 unpaired, two‐tailed t‐test).WT THP‐1s were treated with 5 μM H151 for 30 min before stimulation as in (D). Data in upper panel are representative of three independent experiments. Data in lower panel show combined data of these three experiments (mean ± s.e.m) showing H151‐treated cells relative to media treated cells (*P ≤ 0.05, ***P ≤ 0.001 unpaired, two‐tailed t‐test).ASC‐GFP THP‐1s were treated with 5 μM H151 for 30 min before stimulation with AdV‐mCherry (50,000 pp/cell) + 20 μg/ml h9C12 or 20 mg/ml IVIg or 200 ng/ well HT‐DNA for 8 h. A representative image (scale bar 50 μm) and quantification of number of cells with ASC specks from one representative experiment of three are shown. Source data are available online for this figure.

Fig 5: TRIM21 destabilizes Chaperonin‐containing TCP1 subunit 6A (CCT6A) through K48‐linked polyubiquitination. (A) Immunoprecipitation assay followed by silver staining of the CCT6A‐binding proteins. (B) Immunoprecipitation and western blot analysis of the interaction of Flag‐CCT6A with HA‐TRIM21 in HEK‐293T cells (n = 3). (C) Co‐immunoprecipitation of endogenous TRIM21 with an anti‐CCT6A antibody in MDA‐MB‐231 cells. (D) Co‐immunoprecipitation of endogenous CCT6A with an anti‐TRIM21 antibody in BT‐549 cells (n = 3). (E) Immunofluorescence assay was performed on MDA‐MB‐231 cells with an anti‐CCT6A antibody (green), an anti‐TRIM21 antibody (red) and DAPI (blue). The above data were derived from more than three independent experiments. (F) Different domains of TRIM21 were cotransfected with CCT6A into HEK‐293T cells. The cell lysates were subjected to immunoprecipitation with an anti‐Flag antibody as described, and the immunoprecipitates were then analyzed by western blotting. (G) A panel of CCT6A expression constructs was cotransfected with TRIM21 into HEK‐293T cells, followed by IP and western blotting as described. (H) Immunoblot analysis of Flag‐CCT6A and HA‐TRIM21 in the lysates of HEK‐293T cells cotransfected with Flag‐CCT6A and various amounts of HA‐TRIM21. (I) Immunoblot analysis was performed to evaluate the half‐life of Flag‐CCT6A in HEK‐293T cells transfected with Flag‐CCT6A and HA‐TRIM21. (J) HEK‐293T cells were cotransfected with HA‐TRIM21, Flag‐CCT6A and His‐ubiquitin and were then treated with 20 µM MG132 for 4 h. A ubiquitin pulldown assay for Flag‐tagged CCT6A was carried out as described in the Materials and Methods section. (K) K48‐only and K63‐only ubiquitin plasmids were transfected alone or with the HA‐TRIM21 and Flag‐CCT6A plasmids into 293T cells for 48 h, after which the cells were harvested and subjected to IP with an anti‐His antibody.