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, 74 (8), 3781-92

The IRF-3 Transcription Factor Mediates Sendai Virus-Induced Apoptosis

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The IRF-3 Transcription Factor Mediates Sendai Virus-Induced Apoptosis

C Heylbroeck et al. J Virol.

Abstract

Virus infection of target cells can result in different biological outcomes: lytic infection, cellular transformation, or cell death by apoptosis. Cells respond to virus infection by the activation of specific transcription factors involved in cytokine gene regulation and cell growth control. The ubiquitously expressed interferon regulatory factor 3 (IRF-3) transcription factor is directly activated following virus infection through posttranslational modification. Phosphorylation of specific C-terminal serine residues results in IRF-3 dimerization, nuclear translocation, and activation of DNA-binding and transactivation potential. Once activated, IRF-3 transcriptionally up regulates alpha/beta interferon genes, the chemokine RANTES, and potentially other genes that inhibit viral infection. We previously generated constitutively active [IRF-3(5D)] and dominant negative (IRF-3 DeltaN) forms of IRF-3 that control target gene expression. In an effort to characterize the growth regulatory properties of IRF-3, we observed that IRF-3 is a mediator of paramyxovirus-induced apoptosis. Expression of the constitutively active form of IRF-3 is toxic, preventing the establishment of stably transfected cells. By using a tetracycline-inducible system, we show that induction of IRF-3(5D) alone is sufficient to induce apoptosis in human embryonic kidney 293 and human Jurkat T cells as measured by DNA laddering, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling assay, and analysis of DNA content by flow cytometry. Wild-type IRF-3 expression augments paramyxovirus-induced apoptosis, while expression of IRF-3 DeltaN blocks virus-induced apoptosis. In addition, we demonstrate an important role of caspases 8, 9, and 3 in IRF-3-induced apoptosis. These results suggest that IRF-3, in addition to potently activating cytokine genes, regulates apoptotic signalling following virus infection.

Figures

FIG. 1
FIG. 1
Constitutively active IRF-3 is toxic to cells. 293 cells were transfected with 10 μg of control pEGFPC1 vector (A), pEGFPC1-wtIRF-3 (B), or pEGFPC1-IRF-3(5D) (C). Beginning 36 h after transfection, cells were selected in media containing G418 (400 μg/ml); after 2 weeks of selection, cells were fixed in the plate with ice-cold methanol and were stained with Giemsa. Expression of the GFP fusion proteins was monitored by fluorescence microscopy.
FIG. 2
FIG. 2
Inducible expression of IRF-3 and IRF-3(5D). Whole-cell extracts (20 μg) were prepared from rtTA-293 (A and B) and rtTA-Jurkat (C and D) cells. rtTA-293 wtIRF-3 (A), rtTA-293 IRF-3(5D) (B), rtTA-Jurkat wtIRF-3 (C), and rtTA-Jurkat IRF-3(5D) (D) cells were induced with DOX (1 μg/ml) for 0 to 48 h and were analyzed for IRF-3 expression by immunoblot analysis. IRF-3(5D) protein migrated more slowly than endogenous IRF-3 protein on SDS-PAGE at a position similar to phosphorylated IRF-3 protein.
FIG. 3
FIG. 3
Constitutively active IRF-3 induces apoptosis. TUNEL staining of IRF-3(5D)-expressing 293 (A) and Jurkat cells (C). rtTA-293 IRF-3(5D) and rtTA-Jurkat IRF-3(5D) cells were left untreated or induced with DOX for 48 (293) or 72 h (Jurkat). Cells were then stained by the TUNEL method (green filter) and with Hoechst dye to visualize all nuclei (blue filter) as described in Materials and Methods. (B) Kinetics of DNA fragmentation in 293 IRF-3(5D)-expressing cells. Plates of rtTA-, wtIRF-3-, and IRF-3(5D)-expressing 293 cells were induced with DOX (1 μg/ml) for 0 to 5 days. DNA was isolated from each sample and was analyzed by agarose gel electrophoresis as described in Materials and Methods. (D) Kinetics of IRF-3(5D)-induced apoptosis in Jurkat T cells. wtIRF-3- and IRF-3(5D)-expressing Jurkat T cells were induced with DOX (1 μg/ml) for 0 to 72 h as indicated. Cells (5 × 106) were fixed and stained with propidium iodide. Cellular DNA content was analyzed by flow cytometry.
FIG. 3
FIG. 3
Constitutively active IRF-3 induces apoptosis. TUNEL staining of IRF-3(5D)-expressing 293 (A) and Jurkat cells (C). rtTA-293 IRF-3(5D) and rtTA-Jurkat IRF-3(5D) cells were left untreated or induced with DOX for 48 (293) or 72 h (Jurkat). Cells were then stained by the TUNEL method (green filter) and with Hoechst dye to visualize all nuclei (blue filter) as described in Materials and Methods. (B) Kinetics of DNA fragmentation in 293 IRF-3(5D)-expressing cells. Plates of rtTA-, wtIRF-3-, and IRF-3(5D)-expressing 293 cells were induced with DOX (1 μg/ml) for 0 to 5 days. DNA was isolated from each sample and was analyzed by agarose gel electrophoresis as described in Materials and Methods. (D) Kinetics of IRF-3(5D)-induced apoptosis in Jurkat T cells. wtIRF-3- and IRF-3(5D)-expressing Jurkat T cells were induced with DOX (1 μg/ml) for 0 to 72 h as indicated. Cells (5 × 106) were fixed and stained with propidium iodide. Cellular DNA content was analyzed by flow cytometry.
FIG. 3
FIG. 3
Constitutively active IRF-3 induces apoptosis. TUNEL staining of IRF-3(5D)-expressing 293 (A) and Jurkat cells (C). rtTA-293 IRF-3(5D) and rtTA-Jurkat IRF-3(5D) cells were left untreated or induced with DOX for 48 (293) or 72 h (Jurkat). Cells were then stained by the TUNEL method (green filter) and with Hoechst dye to visualize all nuclei (blue filter) as described in Materials and Methods. (B) Kinetics of DNA fragmentation in 293 IRF-3(5D)-expressing cells. Plates of rtTA-, wtIRF-3-, and IRF-3(5D)-expressing 293 cells were induced with DOX (1 μg/ml) for 0 to 5 days. DNA was isolated from each sample and was analyzed by agarose gel electrophoresis as described in Materials and Methods. (D) Kinetics of IRF-3(5D)-induced apoptosis in Jurkat T cells. wtIRF-3- and IRF-3(5D)-expressing Jurkat T cells were induced with DOX (1 μg/ml) for 0 to 72 h as indicated. Cells (5 × 106) were fixed and stained with propidium iodide. Cellular DNA content was analyzed by flow cytometry.
FIG. 4
FIG. 4
IRF-3 potentiates virus-induced apoptosis. rtTA-Jurkat (A and B), rtTA-Jurkat wtIRF-3 (C and D), and rtTA-Jurkat IRF-3(5D) (E to H) were cultured in the presence (A to D, G, and H) or absence of DOX (1 μg/ml) (E and F). After 12 h, cells were either left untreated (A, C, E, and G) or were infected with Sendai virus (80 HAU/ml) for 72 h (B, D, F, and H). Cells (5 × 106) were fixed and stained with propidium iodide. Cellular DNA content was analyzed by flow cytometry.
FIG. 5
FIG. 5
Inhibition of virus-induced apoptosis. Control 293 and 293 IRF-3 ΔN-expressing cells were left untreated or were infected with Sendai virus (80 HAU/ml) for 24, 48, and 72 h. rtTA-293 wtIRF-3 were cultured in the presence or absence of DOX (1 μg/ml) as indicated. After 24 h, cells were either left untreated or were infected with Sendai virus as described above. The number of apoptotic cells was determined by TUNEL staining as described in Materials and Methods.
FIG. 6
FIG. 6
IFN release is not implicated in IRF-3-induced apoptosis. (A) Control 293 and 293 IRF-3 ΔN-expressing cells were left untreated or were infected with Sendai virus (80 HAU/ml) for 24, 48, and 72 h in the presence or absence of IFN-α (400 IU/ml) or neutralizing antibody for alpha/beta interferon (1/100) (Sigma) as indicated. Viability was measured by using an MTT assay as described in Materials and Methods. Symbols: ■, 293; □, 293 plus IFN-α; ▵, 293 plus anti-IFN-α; ●, 293 IRF-3 ΔN. (B) TUNEL staining of Jurkat cells. The rtTA-Jurkat cells were either left untreated, were infected with Sendai virus (80 HAU/ml), or were treated with IFN-α (400 IU/ml) for 72 h; anti-IFN-α antibody was added with Sendai virus. The number of apoptotic cells was determined by TUNEL as described in Materials and Methods. (C) RPA of IFN-β and IFN-γ mRNA production. The rtTA-, wtIRF-3-, and IRF-3(5D)-expressing 293 and Jurkat cells were cultured in the presence or absence of DOX, as indicated, for 24 h. Cells were then either left untreated or were infected with Sendai virus for 72 h. Total RNA was isolated from each sample and was analyzed by RNase protection analysis by using the human CK-3 RPA kit (Pharmingen), according to manufacturer's instructions.
FIG. 6
FIG. 6
IFN release is not implicated in IRF-3-induced apoptosis. (A) Control 293 and 293 IRF-3 ΔN-expressing cells were left untreated or were infected with Sendai virus (80 HAU/ml) for 24, 48, and 72 h in the presence or absence of IFN-α (400 IU/ml) or neutralizing antibody for alpha/beta interferon (1/100) (Sigma) as indicated. Viability was measured by using an MTT assay as described in Materials and Methods. Symbols: ■, 293; □, 293 plus IFN-α; ▵, 293 plus anti-IFN-α; ●, 293 IRF-3 ΔN. (B) TUNEL staining of Jurkat cells. The rtTA-Jurkat cells were either left untreated, were infected with Sendai virus (80 HAU/ml), or were treated with IFN-α (400 IU/ml) for 72 h; anti-IFN-α antibody was added with Sendai virus. The number of apoptotic cells was determined by TUNEL as described in Materials and Methods. (C) RPA of IFN-β and IFN-γ mRNA production. The rtTA-, wtIRF-3-, and IRF-3(5D)-expressing 293 and Jurkat cells were cultured in the presence or absence of DOX, as indicated, for 24 h. Cells were then either left untreated or were infected with Sendai virus for 72 h. Total RNA was isolated from each sample and was analyzed by RNase protection analysis by using the human CK-3 RPA kit (Pharmingen), according to manufacturer's instructions.
FIG. 7
FIG. 7
CPP-32 activation in virus-infected and IRF-3(5D)-expressing cells. (A) Whole-cell extracts from 293 and DOX-induced rtTA-wtIRF-3-, and IRF-3(5D)-expressing 293 cells infected with Sendai virus (80 HAU/ml) or treated with DOX for the times indicated were subjected to SDS-PAGE and were transferred to nitrocellulose membrane. (B) Whole-cell extracts from untreated or DOX-induced rtTA-, wtIRF-3-, or IRF-3(5D)-expressing Jurkat cells infected with Sendai virus (80 HAU/ml) or induced with DOX for the times indicated were subjected to SDS-PAGE and were transferred to nitrocellulose membrane. CPP-32 and its cleavage products were detected by immunoblot analysis by using a polyclonal CPP-32 antibody (a gift from P. R. Sekaly).
FIG. 8
FIG. 8
Caspase 8 and caspase 9 are involved in IRF-3-dependent activation of CPP-32/caspase-3. (A) rtTA-Jurkat IRF-3(5D) cells were cultured in the presence of 4 μg of APO-1-3 per ml or 50 μM Etoposide for 48 h in the continuous presence of caspase blockers zVAD, zIETH, and zLEHD (200 μM), as indicated. Viability was evaluated by using trypan blue exclusion. (B) wtIRF-3-expressing Jurkat cells were treated for 48 h with DOX (5 μg/ml) to stimulate IRF-3 production and were then infected with Sendai virus (400 HAU/ml/106 cells) for 24, 48, or 72 h in the continuous presence of caspase blockers zVAD, zIETH, and zLEHD (200 μM). Viability was evaluated by using trypan blue exclusion. (C) IRF-3(5D)-expressing Jurkat cells were treated with DOX (5 μg/ml) for 24, 48, or 72 h to stimulate IRF-3(5D) production. DOX treatment was accomplished in the continuous presence of caspase blockers zVAD, zIETH, and zLEHD (200 μM). Viability was evaluated by using trypan blue exclusion. Symbols in B and C: ■, Sendai virus; ▴, Sendai virus plus zLEHD; ⧫, Sendai virus plus zIETD; ●, Sendai virus plus zVAD. (D) IRF-3(5D)-expressing Jurkat cells were treated with DOX (5 μg/ml) for 24, 48, and 72 h; whole-cell extracts were prepared at different times after treatment with DOX and were incubated with fluorogenic substrates for caspase 8 (■) and caspase 9 (▴) as described in Materials and Methods. Caspase activity is represented by the fluorescence ratio between DOX-induced versus noninduced cells.

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