Abstract
Viral infection or lipopolysaccharide (LPS) treatment induces expression of a large array of genes, the products of which play a critical role in host antipathogen immunity and inflammation. We have previously reported that the expression of ubiquitin-specific protease 25 (USP25) is significantly up-regulated after viral infection or LPS treatment, and this is essential for innate immune signaling. However, the mechanism behind this phenomenon is unclear. In this study, we found that viral infection-induced up-regulation of Usp25 is diminished in cells lacking interferon regulatory factor 7 (IRF7) or interferon α receptor 1 (IFNAR1) but not p65. Sendai virus- or type I interferon-induced up-regulation of Usp25 requires de novo protein synthesis of IRF7. Furthermore, IRF7 directly binds to the two conserved IRF binding sites on the USP25 promoter to drive transcription of Usp25, and mutation of these two sites abolished Sendai virus-induced IRF7-mediated activation of the USP25 promoter. Our study has uncovered a previously unknown mechanism by which viral infection or LPS induces up-regulation of USP25.
Highlights
Host pattern recognition receptors recognize pathogen-associated molecular patterns and initiate a series of signaling cascades that lead to activation of transcription factors including NF-B and interferon regulatory factor 3 (IRF3)2 [1,2,3]
We found that reconstitution of interferon regulatory factor 7 (IRF7) into Irf3Ϫ/ϪIrf7Ϫ/Ϫ MEFs promoted SeV-induced up-regulation of Usp25 more robustly than did reconstitution of IRF3 (Fig. 1D)
These data suggest that IRF7 and IRF3 but not p65 are essential transcription factors for virus-induced up-regulation of ubiquitinspecific protease 25 (USP25)
Summary
Mice—Ifnar1ϩ/Ϫ mice were purchased from The Jackson Laboratory and maintained and crossed to obtain Ifnar1ϩ/ϩ and Ifnar1Ϫ/Ϫ littermates in the specific pathogen-free facility of Wuhan University. Rabbit anti-USP25 was described previously [35] and kindly provided by Gemma Marfany (University de Barcelona, Barcelona, Spain). Twenty-four hours after transfection, cells were lysed in passive lysis buffer, and the firefly and Renilla luciferase activities were determined using a Dual-Luciferase reporter assay kit (Promega). The cells were washed three times with PBS and harvested in chromatin immunoprecipitation (ChIP) lysis buffer (50 mM Tris1⁄7HCl, pH 8.0, 1% SDS, 5 mM EDTA) followed by sonication until the sizes of DNA were 400 – 600 bp. The purified DNA was assayed by quantitative PCR with an CFX Connect system with a SYBR Green One Step Real-Time PCR kit. Statistical Analysis—Differences between experimental and control groups were determined by Prism software with twoway analysis of variance and Bonferroni test. p values less than 0.05 were considered statistically significant
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