TRIM28 interacts with IRF7. (A) Identification of TRIM28 as an interferon-regulatory factor 7 (IRF7) binding partner. HEK293T cells were transfected with plasmids expressing double-tagged Flag-IRF7-HA and Flag-luciferase-HA as control. The whole-cell lysates were immunoprecipitated with anti-Flag and anti-hemagglutinin (anti-HA) resins sequentially. The eluted IP complexes were analyzed by SDS-PAGE and visualized by silver staining (left panel). The 100-kDa protein band (marked by an asterisk) was identified as tripartite motif–containing (TRIM) protein 28 (TRIM28) by liquid chromatography–tandem mass spectrometry, and its identity was confirmed by western blot. (B) Interaction of endogenous TRIM28 with IRF7. A549 cells were left uninfected or infected with Sendai virus for 24 h. The cell lysates were immunoprecipitated with anti-IRF7 or control serum. The immunoprecipitation (IP) complexes and cell lysates were analyzed by western blot with the antibodies indicated. (C) Schematic representation of domains of human IRF7 and TRIM28. DBD, DNA bind domain; ID, internal inhibitory domain; RING, RING-finger domain; B1 and B2, B-box 1 and B-box 2; CC, coiled-coil domain; Middle, middle region; PHD, plant homeodomain; BR, Bromodomain. (D) Association of TRIM28 with IRF7 ID domain. HEK293T cells were transfected with plasmids expressing GFP-tagged TRIM28 and full length IRF7 or truncation mutants. The cell lysates were immunoprecipitated with anti-Flag affinity resins. The IP complexes and lysates were analyzed by western blot with the indicated antibodies. (E) Association of IRF7 with the RING-finger domain, two B-box domains, and coiled-coil domain of TRIM28. HEK293T cells were transfected with plasmids expressing Flag-IRF7 with full length TRIM28 or truncation mutants. Immunoprecipitation experiments were performed similarly as in (D). The asterisk marks a nonspecific band. (F) Direct association of IRF7 with TRIM28 in vitro. GST-TRIM28 full length or its truncated fusion proteins bound on the glutathione beads were incubated with ³⁵S-labeled full-length IRF7 for 30 min at 4°C. After extensive washes, the bound proteins were resolved by SDS-PAGE and detected by autoradiography. The input GST proteins were revealed by Coomassie staining. The mapping experiments have been repeated at least three times, and representative results are shown.

TRIM28 increases the SUMOylation of IRF7 in cells. (A) IRF7 was SUMOylated in cells. HEK293T cells were transfected with plasmids expressing Flag-IRF7 plus HA-SUMOs or conjugation-deficient (G/A) mutants. Forty-eight h after transfection, cells were lysed in buffer with high concentration of SDS, diluted, and then immunoprecipitated with anti-Flag affinity resins. The IP complexes were analyzed by western blot with anti-HA antibody, which detected SUMOylated protein, about 20 kDa larger than the unmodified one. We also probed the IP complexes and cell lysates with the indicated antibodies to reveal protein input and overall SUMOylation of cellular proteins. (B) TRIM28 promoted SUMOylation of IRF7. HEK293T cells were transfected with Flag-IRF7, HA-SUMO1, or HA-SUMO2 in the presence or absence of GFP-TRIM28–expression plasmids. The cell lysates were analyzed as described above for detection of the SUMOylated IRF7. (C) TRIM28 specifically increased SUMOylation of IRF7 but not that of IRF3. HEK293T cells were transfected with HA-SUMO2, Flag-IRF7, or Flag-IRF3 in the presence or absence of GFP-TRIM28–expression plasmids. SUMOylated IRF7 or IRF3 were detected as described above. (D) The TRIM28-mediated SUMOylation sites were located mainly in the ID domain of IRF7. HEK293T cells were transfected with HA-SUMO2, full-length Flag-IRF7, or its truncated mutants in the presence or absence of GFP-TRIM28–expression plasmids. SUMOylation assays were performed as described above. (E) TRIM28 induced SUMOylation of IRF7 as efficiently as did PIAS1. HEK293T cells were transfected with Flag-IRF7 ID (aa283–466), HA-SUMO2 in the presence of GFP-TRIM28, GFP-PIAS1, or empty vector–control plasmids. The experiments were performed as above for detection of the SUMOylated IRF7. (F) Identification of the major SUMOylation sites of the IRF7 ID domain. HEK293T cells were transfected with plasmids expressing HA-SUMO2, GFP-TRIM28, and Flag-IRF7 ID or K-to-R substitution mutants. The assays were performed as described above for detection of SUMOylation of IRF7. All the experiments have been repeated at least three times and representative results are shown.

TRIM28 is a SUMO E3 ligase of IRF7. (A) The RING-finger domain of TRIM28 mediated the SUMOylation of IRF7. HEK293T cells were transfected with Flag-IRF7 ID, HA-SUMO2 in the presence of GFP-TRIM28, or its truncated-mutant expression plasmids. The experiments were performed as described in Fig. 2A for detection of SUMOylated IRF7. (B) TRIM28 interacted with Ubc9 mainly through the RING domain. HEK293T cells were transfected with Flag-Ubc9 and GFP-tagged TRIM28 full length and truncation mutants. The cell lysates were immunoprecipitated with anti-Flag affinity resins. The IP complexes and the input cell lysates were analyzed by western blot with antibodies as indicated. (C) TRIM28 catalyzed SUMOylation of IRF7 in vitro. Purified proteins SUMO2 (2 μg), E1 (200 ng), Ubc9 (100 ng), Flag-IRF7 ID (1 μg), and HA-TRIM28 (2 μg) were mixed with the indicated combinations and incubated at 30°C for 3 h. The mixtures were immunoprecipitated with anti-Flag resins, and the IP complexes were then immunoblotted with anti-SUMO2 and anti-Flag. The experiments were repeated at least twice, and representative results are shown.

TRIM28-mediated IRF7 SUMOylation is increased during viral infections. (A) TRIM28 specifically increased virus-induced SUMOylation of IRF7 but not that of IRF3. HEK293T cells were transfected with indicated plasmids (each 7 μg) and infected with Sendai virus for different lengths of time before cell harvest. Forty-eight h after transfection, in vivo SUMOylation assays were performed, similar to those described in Fig. 2. (B) Sendai-virus infection increased the SUMO E3 activity of TRIM28. HEK293T cells were transfected with 10 μg of HA-TRIM28 plasmid. After 24 h, the transfected cells were infected with Sendai virus or mock-infected for 24 h before cell harvest. HA-TRIM28 proteins were purified from HEK293T cells and used in in vitro SUMOylation assays (2 μg SUMO2, 100 ng E1, 200 ng Ubc9, 1 μg Flag-IRF7 ID, and 2 μg HA-TRIM28). After 3 h incubation at 30°C, the mixtures were immunoprecipitated with anti-Flag affinity resins. The IP complexes were analyzed by immunoblotting with anti-SUMO2 and anti-Flag. All the experiments were repeated at least three times, and representative results are shown.

TRIM28 negatively regulated IRF7 activity. (A), (B), and (C) TRIM28 inhibited the transactivation activity of IRF7. HEK293T cells seeded in 24-well plates were transfected with the IFNα1-luc (A), IFNβ-luc (B), or ISRE-luc (C) luciferase promoter reporter (100 ng per well), pRL-TK (10 ng, as an internal control), pCR3.1-IRF7 (20 ng), and increasing amounts of TRIM28-expression plasmids (50 ng, 100 ng, and 250 ng). Eight h after transfection, cells were infected with Sendai virus or left untreated. Dual luciferase assays were performed 16 h after viral infection. (D) TRIM28 RING-finger domain was required for inhibition on IRF7 transactivation activity. HEK293T cells were transfected with IFNα1 promoter reporter (100 ng), pRL-TK (10 ng), pCR3.1-IRF7 (20 ng), and TRIM28 or its truncated-mutants expression plasmids (100 ng). Experiments were performed as in (A). (E) Mutation of the main SUMOylation sites (K444 and K446) potentiated IRF7 transactivation activity. HEK293T cells were transfected with IFNα1 promoter reporter (100 ng), pRL-TK (10 ng), and Flag-IRF7 or its K to R mutants expression plasmids (20 ng). Luciferase assays were performed as described above. Data from luciferase experiments represent the average of at least three independent experiments, and error bars represent standard deviation.

Knockdown of TRIM28 potentiated IFN expression and antiviral responses. (A) Knockdown of TRIM28 expression by siRNAs impaired SUMOylation of IRF7. HEK293 cells were transduced with retroviral vectors expressing siTRIM28 or siControl siRNAs. After selection with puromycin (2 μg/ml) for 2 weeks, the stably transduced cells were then transfected with plasmids expressing HA-SUMO2 and Flag-IRF7 for in vivo SUMOylation assays as described in Figure 2. (B) and (C) Knockdown of TRIM28 potentiated transactivation activity of IRF7. HEK293 siControl or HEK293 siTRIM28 cells were transfected with the IFNα1 (B) or IFNβ (C) promoter reporters (100 ng), pRL-TK (10 ng) and IRF7 (20 ng). Eight h after transfection, cells were infected with Sendai virus or left untreated. Dual luciferase assays were performed 16 h after infection. (D) TRIM28 contributed to the inhibition of IRF7 by SUMOylation on K444 and K446. HEK293 siControl cells or siTRIM28 cells were transfected with IFNα1 promoter reporter (100 ng), pRL-TK (10 ng), and Flag-IRF7 or its K to R mutants expression plasmids (20 ng). Luciferase assays were performed as described above. (E) Knockdown of IRF7 had more profound effect on induction of type I IFN than knockdown of IRF3. A549 cells stably transduced with siControl, siIRF7, or siIRF3 siRNAs were infected with Sendai virus for 24 h. The culture medium was then collected and used for measurement of IFNα by ELISA. (F) Knockdown of TRIM28 potentiated IFNα production. A549 siControl or A549 siTRIM28 cells were treated with Sendai virus for 24 h. The culture medium was collected after treatment and used for measurement of IFNα by ELISA. (G) and (H) Knockdown of TRIM28 inhibited VSV replication. A549 siControl and A549 siTRIM28 cells were infected with increasing amounts of VSV. Twenty-four h after infection, cells lysates were tested with anti-VSV-G (E), and cell medium was used for plaque assays (F). Data represent the average of at least three independent experiments, and error bars represent standard deviation.