27 research outputs found
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 NH<sub>4</sub>Cl for 16 h before immunoblot analysis was performed.</p
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
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
Identification of a Nonstructural DNA-Binding Protein (DBP) as an Antigen with Diagnostic Potential for Human Adenovirus
<div><p>Background</p><p>Human adenoviruses (HAdVs) have been implicated as important agents in a wide range of human illnesses. To date, 58 distinct HAdV serotypes have been identified and can be grouped into six species. For the immunological diagnosis of adenoviruses, the hexon protein, a structural protein, has been used. The potential of other HAdV proteins has not been fully addressed.</p> <p>Methodology/Principal Findings</p><p>In this study, a nonstructural antigenic protein, the DNA binding protein (DBP) of human adenovirus 5 and 35 (Ad5, Ad35) - was identified using immunoproteomic technology. The expression of Ad5 and Ad35 DBP in insect cells could be detected by rhesus monkey serum antibodies and healthy adult human serum positive for Ad5 and Ad35. Recombinant DBPs elicited high titer antibodies in mice. Their conserved domain displayed immunological cross-reactions with heterologous DBP antibodies in Western blot assays. DBP-IgM ELISA showed higher sensitivity adenovirus IgM detection than the commercial Adenovirus IgM Human ELISA Kit. A Western blot method developed based on Ad5 DBP was highly consistent with (χ<sup>2</sup> =  44.9, <i>P</i><0.01) the Western blot assay for the hexon protein in the detection of IgG, but proved even more sensitive.</p> <p>Conclusions/Significance</p><p>The HAdV nonstructural protein DBP is an antigenic protein that could serve as an alternative common antigen for adenovirus diagnosis.</p> </div
Comparison of DBP-IgG Western blot and hexon Western blot.
<p>χ<sup>2</sup>  =  44.9, <i>P</i><0.01. The sensitivity and specificity of the DBP Western blot versus the hexon Western blot was 99% and 66.7%, respectively. Calculation formula: sensitivity  =  TP/(TP+FN), specificity  =  TN/(FP+TN), where TP is the number of true positives, FN is the number of false negatives, FP is the number of false positives, and TN is the number of true negatives <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056708#pone.0056708-Loong1" target="_blank">[36]</a>.</p
Antigenicity of Ad5 and Ad35 DBP.
<p>The Ad5 and Ad35 DBP were expressed in insect cells. Western blot analysis were performed to determine the antigenicity of Ad5 and Ad35 DBP using corresponding anti-DBP serum (1:1,000 dilutions) derived from rhesus monkeys (A and B) and anti-Ad5 and Ad35 serum (1∶400 dilutions) derived from healthy adult volunteers (C and D).</p
Comparison of DBP-IgM ELISA and commercial Adenovirus IgM Human ELISA Kit in the diagnosis of HAdVs infection related to number of days after onset of fever.
<p>a. Number of positive samples.</p><p>b. Percentage of positive samples.</p