Effects of TRIM38 knockdown on TLR3-induced IFN-β activation.

<p>(A) TRIM38 protein level in 293/TLR3 cell line stably expressing TRIM38-specific or non-targeting shRNA. 293/TLR3 cells were transfected with TRIM38-specific shRNA or non-targeting shRNA (NT) followed by puromycin selection and cloning. Screened cell populations were analyzed by immunoblot with an antibody specific for TRIM38. (B) Effects of TRIM38 knockdown on poly(I:C)-induced IFN-β activation. TRIM38 knockdown or control 293/TLR3 cells were transfected with IFN-β-luc plasmid. Twenty-four hours after transfection, cells were left untreated or stimulated with 100 µg/ml of poly(I:C) for 4 h before luciferase analysis was performed. (C) Effects of TRIM38 knockdown on poly(I:C)-induced IRF3 phosphorylation. TRIM38 knockdown or control 293/TLR3 cells were incubated with 100 µg/ml of poly(I:C) for 4 h before the immunoblot analysis was performed. (D, E) Effects of TRIM38 knockdown on poly(I:C)-induced transcription of IFN-β and ISG56 genes. Indicated cells were stimulated with poly(I:C) for 4 h, then total RNA was extracted for real-time PCR analysis. Similar results were obtained from three independent experiments.</p

TRIM38 induces TRIF degradation through proteasomal pathway.

<p>(A) Overexpression of TRIM38 does not affect endogenous TRIF mRNA expression. HeLa cells were transfected with increasing amounts of a TRIM38-Flag plasmid (0, 0.5, and 2 µg). After 48 h, total RNA was prepared and used for RT-PCR analysis of the indicated genes. GAPDH was used as an internal control. (B) Overexpression of TRIM38 reduces TRIF protein. 293T cells were transfected with plasmids expressing Flag-TRIF or TRAF3-Flag, together with increasing amounts of TRIM38-Flag plasmid (0, 50, 100, and 200 ng). After 24 h, immunoblot analysis was performed with indicated antibodies. (C) Overexpression of TRIM38 reduces endogenous TRIF. HeLa cells were transfected with increasing amounts of TRIM38-Flag plasmid (0, 0.5, and 2 µg). Fourty-eight hours after transfection, immunoblot analysis was performed using indicated antibodies. (D) Caspase inhibitor does not inhibit TRIM38-mediated TRIF degradation. 293T cells were transfected with Flag-TRIF plasmid and increasing amounts of TRIM38-Flag plasmid (0, 10, 50, 100, 150, and 200 ng) for 6 h. Then cells were treated with DMSO (negative control) or 5 µM Z-VAD-FMK for 16 h before immunoblot analysis was performed. (E) Overexpression of TRIM38 promotes degradation of caspase-resistant TRIF. 293T cells were transfected with plasmids expressing Flag-TRIF or TRIF mutant carrying D284E and D289E substitutions, together with increasing amounts of TRIM38-Flag plasmid (0, 50, 100, and 200 ng). After 24 h, immunoblot analysis was performed. (F) TRIM38 promotes proteasomal degradation of TRIF. 293T cells were transfected with a Flag-TRIF plasmid and increasing amounts of TRIM38-Flag plasmid (0, 10, 50, 100, 150, and 200 ng) for 6 h. Then cells were treated with DMSO, 0.1 µM MG132, or 10 mM NH₄Cl for 16 h before immunoblot analysis was performed.</p

TRIM38 targets TRIF.

<p>(A–C) Effects of TRIM38 on TRIF, TBK1, or IKKi-induced IFN-β activation. 293T cells were transfected with an IFN-β-luc plasmid, together with a plasmid expressing TRIF (A), TBK1 (B), or IKKi (C), and a TRIM38 plasmid (0, 50, 100, and 200 ng), respectively. Luciferase assays were performed after 24 h post-transfection. (D) Effect of TRIM38 on TRIF-mediated IRF3 phosphorylation. 293T cells were transfected with a TRIF plasmid and a TRIM38 plasmid (0, 0.5, and 2 µg). Twenty-four hours post transfection, cell lysates were analyzed by immunoblot with indicated antibodies.</p

RING/B-box of TRIM38 is critical for TRIF degradation.

<p>(A) TRIM38 catalyzes K48-linked ubiquitination of TRIF. 293T cells were transfected with plasmids expressing Myc-tagged full-length or RING/B-box domain deleted (ΔRING/B-box) TRIM38, Flag-TRIF, and HA-ubiquitin plasmids. At 24 h post-transfection, cell lysates were denatured and immunoprecipitated using anti-Flag agrose beads. Immunoblot analysis was performed using an antibody specific against K48-linkage polyubiquitin. (B) Effect of TRIM38ΔRING/B-box mutant on TRIF degradation. 293T cells were transfected with Flag-TRIF plasmid and Flag-tagged full length TRIM38 or TRIM38ΔRING/B-box mutant plasmid (0, 50, and 100 ng). Twenty-four hours after transfection, immunoblot analysis using the indicated antibodies was performed. (C) Effect of TRIM38ΔRING/B-box mutant on TRIF-induced IFN-β promoter activation. 293T cells were transfected with IFN-β-Luc plasmid, Flag-TRIF plasmid, together with increasing amounts plasmid expressing of Flag-tagged full length TRIM38 or TRIM38ΔRING/B-box mutant (0, 50, and 100 ng). Luciferase assays were performed 24 h after transfection.</p

Interaction of TRIM38 and TRIF.

<p>(A) TRIM38 specifically interacts with TRIF. 293T cells transfected with plasmids expressing TRIM38-Myc and Flag-TRIF, Flag-TBK1, or Flag-IKKi. After 24 h, cell lysates were immunoprecipitated using anti-Flag agarose beads. The lysates and immunoprecipitates were analyzed by immunoblot with anti-Myc and anti-Flag antibodies. (B) Endogenous interaction of TRIF with TRIM38. Hela cells were left untreated or treated with poly(I:C) for 4 h. Cell lysates were immunoprecipitated with goat anti-TRIF antibody or control goat IgG and analyzed with rabbit anti-TRIM38 or rabbit anti-TRIF antibodies. (C, D) Schematic presentations of truncation mutants of TRIF (C) and TRIM38 (D). (E) TRIF interacts with TRIM38 through its N-terminus. 293T cells were transfected with Myc-TRIM38 together with full length Flag-tagged TRIF or TRIF truncation mutants. The immunoprecipitations were performed using anti-Flag agrose beads. Immunoblot analysis was carried out similarly as in (A). (F) TRIM38 is associated with TRIF through its PRYSPRY domain. Experiments were performed as described in panel (E). The co-immunopricipitated proteins were marked by asterisks.</p

Enterovirus 71 Protease 2A^pro Targets MAVS to Inhibit Anti-Viral Type I Interferon Responses

<div><p>Enterovirus 71 (EV71) is the major causative pathogen of hand, foot, and mouth disease (HFMD). Its pathogenicity is not fully understood, but innate immune evasion is likely a key factor. Strategies to circumvent the initiation and effector phases of anti-viral innate immunity are well known; less well known is whether EV71 evades the signal transduction phase regulated by a sophisticated interplay of cellular and viral proteins. Here, we show that EV71 inhibits anti-viral type I interferon (IFN) responses by targeting the mitochondrial anti-viral signaling (MAVS) protein—a unique adaptor molecule activated upon retinoic acid induced gene-I (RIG-I) and melanoma differentiation associated gene (MDA-5) viral recognition receptor signaling—upstream of type I interferon production. MAVS was cleaved and released from mitochondria during EV71 infection. An <i>in vitro</i> cleavage assay demonstrated that the viral 2A protease (2A^pro), but not the mutant 2A^pro (2A^pro-110) containing an inactivated catalytic site, cleaved MAVS. The Protease-Glo assay revealed that MAVS was cleaved at 3 residues between the proline-rich and transmembrane domains, and the resulting fragmentation effectively inactivated downstream signaling. In addition to MAVS cleavage, we found that EV71 infection also induced morphologic and functional changes to the mitochondria. The EV71 structural protein VP1 was detected on purified mitochondria, suggesting not only a novel role for mitochondria in the EV71 replication cycle but also an explanation of how EV71-derived 2A^pro could approach MAVS. Taken together, our findings reveal a novel strategy employed by EV71 to escape host anti-viral innate immunity that complements the known EV71-mediated immune-evasion mechanisms.</p> </div

Effects of TRIM38 on TLR3-induced IFN-β signaling.

<p>(A) Expression of TRIM38 mRNA in HeLa cells treated with 100 µg/ml poly(I:C). At indicated time points, cells were harvested, and total RNA was prepared and analyzed by RT-PCR. GAPDH mRNA expression was assessed as an internal control. (B) Expression of TRIM38 protein in HeLa cells treated with 100 µg/ml poly(I:C). At indicated time points, cells were harvested and analyzed by immunoblot using the indicated antibodies. (C) Effect of TRIM38 overexpression on poly(I:C)-induced activation of IFN-β. 293T/TLR3 cells were transfected with an IFN-β-luc plasmid and TRIM38 plasmid (0, 50, 200, and 500 ng). Twenty-four hours after transfection, cells were incubated with 100 µg/ml of poly(I:C) for 4 h, and then luciferase assays were performed. (D) Effect of TRIM38 overexpression on IRF3 phosphorylation. HeLa cells were transfected with TRIM38 plasmid (0, 0.5 and 2 µg). Twenty-four hours after transfection, cells were left untreated or incubated with 100 µg/ml poly(I:C) for 4 h. Immunoblot analysis was performed using the indicated antibodies. (E, F) Effect of TRIM38 overexpression on poly(I:C)-induced transcription of IFN-β and ISG56. HeLa cells were transfected with TRIM38 plasmid (0, 0.5, and 2 µg). Twenty-four hours after transfection, cells were left untreated or incubated with 100 µg/ml of poly(I:C) for 4 h, then total RNA was extracted and quantitative real-time PCR were performed to analyze gene expression.</p

<i>In vitro</i> cleavage assay of EV71 2A^pro on MAVS and MAVS mutants expressed in HeLa cells.

<p>(<b>A</b>) The cell lysates from stable cell lines expressing WT-MAVS (lanes 1&2), m-MAVS-3M (lanes 3&4), m-MAVS-209 (lanes 5&6), m-MAVS-251 (lanes 7&8), and m-MAVS-265 (lanes 9&10) were incubated with 200 ng/µL 2A^pro at 30°C for 2 h; the cell lysates were then subjected to western blot analysis to probe MAVS using an HA antibody against an HA peptide fused to the C-terminus of MAVS and MAVS mutants. Cleavage of PABP served as a readout for the enzymatic activity of 2A^pro. (<b>B</b>) Schematic diagram analyzing cleavage results from (A). The differing line thickness represents the differing extent of cleavage activity of 2A^pro on each substrate. (<b>C, D, E</b>) Time-course study of 2A^pro on WT-MAVS (C), m-MAVS-251, (D) and m-MAVS-265 (E) stably expressed in their corresponding cell lines by western blot, which was carried out with 200 ng/µL recombinant 2A^pro at 30°C for indicated time.</p

EV71 2A^pro cleaves MAVS.

<p>(<b>A, B</b>) <i>In vitro</i> dose- dependent cleavage assay of EV71 2A^pro (A) and 3C^pro (B) on MAVS using LI-COR Odyssey Dual-Color System. Increasing doses of recombinant proteases were added to the cell lysates and incubated at 37°C for 6 h (from 0–200 ng/µL, lanes 2–8); recombinant proteases (lane 1) and EV71-infected HeLa cells (lanes 9–12) served as negative and positive controls, respectively. Two antibodies recognizing different MAVS epitopes were used (E-3, 700 nm, green; AT107, 800 nm, red). An overlay of the two channels is shown in the “Merge” panel. White arrows indicate cleaved bands in EV71-infected HeLa cells and the same-size bands in 2A^pro-cleaved HeLa extracts. (<b>C</b>) Western blot analysis of MAVS in HeLa cells transfected with increasing doses of plasmids (0–4 µg) encoding EV71 3C^pro (lanes 1–4) and 3ABC (lanes 5–8) precursor protein fused with GFP. The MAVS image is an overlay of two signals from the different channels described in (A). The same cell lysates were also used to detect 3C^pro and 3ABC using an antibody against GFP; actin served as the loading control. (<b>D</b>) Western blot analysis of MAVS in BSRT7/5 cells transfected with increasing doses of pcDNA3.1-IRES-2A plasmid (lanes 1–5, 0–4 µg) and pcDNA3.1-EGFP control plasmid (4 µg). (<b>E</b>) <i>In vitro</i> dosage cleavage assay (0–200 ng/µL) of 2A^pro (lanes 2–8) and mutated 2A^pro (2A^pro-110) (lanes 10–16) on MAVS; recombinant 2A^pro (lane 1) and 2A^pro-110 (lane 9) served as negative controls. The MAVS image is an overlay of two signals from the different channels described in (A). PABP was used as a readout for the enzyme activity of 2A^pro and 2A^pro-110; actin served as a loading control. The image of 2A^pro and 2A^pro-110 (lower panel) is a direct scan of SDS-PAGE gel stained with Coomassie brilliant blue.</p

EV71 2A^pro cleaves MAVS at multiple residues.

<p>(<b>A</b>) Schematic diagram of the Protease-Glo assay of 2A^pro on MAVS extra-membrane region. (<b>B</b>) The first-round screening of 86 constructs containing the coding region for the 12-mer polypeptides covering the MAVS extra-membrane region. Luciferase assay results are shown together with gel analysis results. (<b>C</b>) The second-round screening of the 10 positive constructs selected in the first round of screening (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003231#ppat-1003231-t001" target="_blank">Table 1</a>) using a mutated pGlosensor-10F vector containing a mutation from Gly to Ala in the linker region. (<b>D</b>) Protease-Glo assay of M209, M251, and M265. These constructs were mutated from the 3 selected constructs from the second round of screening (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003231#ppat-1003231-t002" target="_blank">Table 2</a>), and each contains a point mutation (from Gly to Ala) at residue 209, 251, or 265. (<b>E</b>) <i>In vitro</i> cleavage assay of EV71 2A^pro on <i>in vitro</i> translated extra-membrane region of MAVS (MAVS-EM) and its mutants (MAVS-EM-3M) labeled with FluoroTect Green_Lys by gel analysis.</p