doi: 10.1073/pnas.0900818106. Epub 2009 Apr 28.
ISG56 is a negative-feedback regulator of virus-triggered signaling and cellular antiviral response
Affiliations
- PMID:19416887
- PMCID: PMC2683125
- DOI: 10.1073/pnas.0900818106
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ISG56 is a negative-feedback regulator of virus-triggered signaling and cellular antiviral response
Ying Li et al. Proc Natl Acad Sci U S A..
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Abstract
IFN-stimulated gene 56 (ISG56) is one of the first identified proteins induced by viruses and type I IFNs. In this study, we identified ISG56 as a virus-induced protein associated with MITA, an adapter protein involved in virus-triggered induction of type I IFNs. Overexpression of ISG56 inhibited Sendai virus-triggered activation of IRF3, NF-kappaB, and the IFN-beta promoter, whereas knockdown of ISG56 had opposite effects. Consistently, overexpression of ISG56 reversed cytoplasmic poly(I:C)-induced inhibition of vesicular stomatitis virus (VSV) replication, whereas knockdown of ISG56 inhibited VSV replication. Competitive coimmunoprecipitation experiments indicated that ISG56 disrupted the interactions between MITA and VISA or TBK1, two components in the virus-triggered IFN signaling pathways. These results suggest that ISG56 is a mediator of negative-feedback regulation of virus-triggered induction of type I IFNs and cellular antiviral responses.
Conflict of interest statement
The authors declare no conflict of interest.
Figures
ISG56 and ISG54 interact with MITA. (A) ISG56 and ISG54 interact with MITA but not TBK1 or IRF3. 293 cells (2 × 106) were transfected with the indicated plasmids (5 μg each). Coimmunoprecipitation and immunoblot analysis were performed with the indicated antibodies (Upper). Expression of the transfected proteins was analyzed by immunoblots with anti-HA and anti-Flag (Lower). (B) Endogenous ISG56 and ISG54 are associated with MITA. 293 cells (2 × 107) were infected with SeV for the indicated times or left uninfected. Immunoprecipitation and immunoblot analysis were performed with the indicated antibodies.
ISG56 inhibits virus-induced signaling. (A) ISG56 and ISG54 inhibit SeV-induced activation of the IFN-β promoter in a dose-dependent manner. 293 cells (1 × 105) were transfected with an IFN-β promoter reporter (0.1 μg) and increased amount of ISG56 or ISG54 expression plasmids. Eighteen hours after transfection, cells were left uninfected or infected with SeV for 12 h before luciferase assays were performed. (B) ISG56 inhibits SeV-induced expression of IFN-β mRNA. 293 cells (2 × 105) were transfected control or ISG56 expression plasmid (1 μg). Eighteen hours after transfection, cells were left uninfected or infected with SeV for 12 h before real-time PCRs were performed. (C) ISG56 and ISG54 inhibit SeV-induced ISRE activation in a dose-dependent manner. Experiments were performed similarly as inA except that an ISRE reporter was used. (D andE) ISG56 and ISG54 inhibit SeV-induced dimerization (D) and phosphorylation (E) of IRF3. 293 cells were transfected with the indicated plasmids for 24 h. Cells were then left uninfected or infected with SeV for 8 h (D) or 6 h (E) before native or SDS/PAGE and immunoblots with the indicated antibodies were performed. (F) Effects of ISG56 and ISG54 on SeV-induced activation of NF-κB. The experiments were similarly performed as inA. (G) Effects of ISG56 and ISG54 on SeV-induced degradation of IκBα. 293 cells (2 × 105) were transfected with the indicated expression plasmids (1 μg each). Eighteen hours after transfection, cells were left untreated or infected with SeV for the indicated times before immunoblot analysis was performed. Shown in the lower panels is TNF-induced IκBα degradation. (H) ISG56 inhibits SeV-induced activation of the IFN-β promoter in human primary macrophages. The experiments were similarly performed as inA except that the cells were transfected with the Nucleofactor method. (I) Effects of ISG56 and ISG54 on cytoplasmic poly(I:C)-induced activation of the IFN-β promoter. 293 cells (1 × 105) were transfected with the indicated plasmids (0.5 μg each) for 18 h. Cells were then further transfected with poly(I:C) (1 μg) or left untransfected for 24 h before luciferase assays were performed. (J) Effects of ISG56 and ISG54 on EGF-induced activation of Elk. 293 cells (1 × 105) were transfected with a pFR-luc reporter (0.2 μg) and an Elk reporter (0.02 μg) together with the indicated expression plasmids (0.5 μg each). Eighteen hours after transfection, cells were treated with EGF (50 ng/ml) for 12 h before luciferase assays were performed.
Effects of RNAi-mediated knockdown of ISG56 and ISG54 on virus-induced signaling. (A) Effects of ISG56 and ISG54 RNAi plasmids on expression of ISG56 and ISG54. In the upper panels, 293 cells (2 × 105) were transfected with the indicated expression (0.5 μg each) and RNAi plasmids (1 μg each) for 24 h before immunoblot analysis was performed with anti-Flag. In the lower two panels, 293 cells (2 × 105) were transfected with the indicated RNAi plasmids (1 μg each) for 24 h, and then infected with SeV for 10 h before immunoblot analysis was performed with the indicated antibodies. (B) Effects of ISG56 and ISG54 RNAi on SeV-induced ISRE activation. 293 cells (1 × 105) were transfected with the indicated RNAi plasmids (0.5 μg each) for 24 h, and then infected with SeV or left uninfected for 12 h before reporter assays were performed. (C) Effects of ISG56 and ISG54 RNAi on SeV-induced NF-κB activation. The experiments were similarly performed as inB except that NF-κB reporter plasmid was used. (D) Effects of ISG56 and ISG54 RNAi on SeV-induced activation of the IFN-β promoter. 293 cells (1 × 105) were transfected with the indicated amount of RNAi plasmids. An empty vector was added to ensure that each transfection received the same amount of total DNA. The reporter assays were similarly performed as inB. (E) Effects of ISG56 and ISG54 RNAi on SeV-induced expression of downstream genes. 293 cells (2 × 105) were transfected with the indicated RNAi plasmid (1 μg each). Twenty-four hours after transfection, cells were left uninfected or infected with SeV for 12 h before RT-PCR was performed. (F andG) Effects of ISG56 RNAi on SeV-induced activation of the IFN-β promoter in human primary macrophages (F) or DCs (G). The experiments were similarly performed as inB except the cells were transfected with the Nucleofactor method. (H) Effects of ISG56 and ISG54 RNAi on cytoplasmic poly(I:C)-induced activation of the IFN-β promoter. 293 cells (1 × 105) were transfected with the indicated RNAi plasmids (0.5 μg each). Twenty-four hours later, cells were further transfected with poly(I:C) (1 μg) or left untransfected and luciferase assays were performed 24 h later.
Roles of ISG56 and ISG54 in cellular antiviral response. (A) Overexpression of ISG56 increases VSV replication. 293 cells (1 × 105) were transfected with the indicated expression plasmids (0.5 μg each). Eighteen hours later, cells were further transfected with poly(I:C) (1 μg) or left untransfected. Twenty-four hours after transfection, cells were infected with VSV [multiplicity of infection (moi), 0.1], and the supernatants were harvested at 24 h postinfection. Supernatants were analyzed for VSV production with standard plaque assays. Graphs show mean ± SD (n = 3). (B) Knockdown of ISG56 inhibits VSV replication. Plaque assays were performed as inA except that a control or ISG56/54 RNAi plasmid (no. 1) (1 μg) was transfected. Graphs show mean ± SD (n = 3).
ISG56 disrupts the interaction of MITA with VISA or TBK1. (A) ISG56 disrupts the association of MITA with VISA or TBK1 in a dose-dependent manner. 293 cells (2 × 106) were transfected with the indicated plasmids (5 μg each) and an increase amount of ISG56 expression plasmids. Coimmunoprecipitation and immunoblot analysis were performed as in Fig. 1A. (B) ISG56 does not disrupt MDA5–VISA or TRIF–TBK1 interactions. 293 cells were transfected with the indicated plasmids and the coimmunoprecipitation and immunoblot analysis were performed as in Fig. 1A. (C) Kinetics of MITA–VISA and MITA–TBK1 associations after viral infection. 293 cells were left uninfected or infected with SeV for the indicated times. Immunoprecipitation and immunoblot analysis were performed with the indicated antibodies.
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