doi: 10.1128/JVI.73.4.2694-2702.1999.
IkappaB-mediated inhibition of virus-induced beta interferon transcription
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- PMID:10074115
- PMCID: PMC104025
- DOI: 10.1128/JVI.73.4.2694-2702.1999
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IkappaB-mediated inhibition of virus-induced beta interferon transcription
M Algarté et al. J Virol.1999 Apr.
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Abstract
We have examined the consequences of overexpression of the IkappaBalpha and IkappaBbeta inhibitory proteins on the regulation of NF-kappaB-dependent beta interferon (IFN-beta) gene transcription in human cells after Sendai virus infection. In transient coexpression studies or in cell lines engineered to express different forms of IkappaB under tetracycline-inducible control, the IFN-beta promoter (-281 to +19) linked to the chloramphenicol acetyltransferase reporter gene was differentially inhibited in response to virus infection. IkappaBalpha exhibited a strong inhibitory effect on virus-induced IFN-beta expression, whereas IkappaBbeta exerted an inhibitory effect only at a high concentration. Despite activation of the IkappaB kinase complex by Sendai virus infection, overexpression of the double-point-mutated (S32A/S36A) dominant repressors of IkappaBalpha (TD-IkappaBalpha) completely blocked IFN-beta gene activation by Sendai virus. Endogenous IFN-beta RNA production was also inhibited in Tet-inducible TD-IkappaBalpha-expressing cells. Inhibition of IFN-beta expression directly correlated with a reduction in the binding of NF-kappaB (p50-RelA) complex to PRDII after Sendai virus infection in IkappaBalpha-expressing cells, whereas IFN-beta expression and NF-kappaB binding were only slightly reduced in IkappaBbeta-expressing cells. These experiments demonstrate a major role for IkappaBalpha in the regulation of NF-kappaB-induced IFN-beta gene activation and a minor role for IkappaBbeta in the activation process.
Figures
(A) Schematic of IκBα protein. Mutations of IκBα are as indicated: IκBα-2N, which contains the S32A/S36A double point mutation; IκBα-3C, which contains the S283A/T291A/T299A triple point mutation; IκBα-Δ4, which contains a 22-aa deletion in the PEST C-terminal domain; and the combination mutants IκBα-2N+3C and IκBα-2N+Δ4. (B) IκB expression inhibits IFN-β gene expression. The IFN-β–CAT reporter plasmid (2.5 μg) was cotransfected together with IκB expression plasmids into 293 cells by the calcium phosphate method. After 24 h the cells were infected by Sendai virus (500 HAU/ml), and after a further 24 h the cells were harvested and assayed for CAT activity by using cytoplasmic extracts (50 to 100 μg for 2 h). The ratio of IκB to IFN-β–CAT reporter is indicated on the graph. The relative CAT activity is expressed as a percentage of the activity observed after Sendai virus infection with IFN-β–CAT reporter in the absence of IκB plasmid.
Dox-inducible expression of IκB in rtTA-293 cells. (A) Cells selected for wtIκBα or TD-IκBα expression were induced with Dox for 48 h (+) or not induced (−). (B) Immunoblot analysis of IκBα-2N+3C expression after Dox induction for 0 to 96 h. (C) IκBβ induced expression by Dox for 48 h (+) or not induced (−). Whole-cell extract (20 μg) prepared from induced or uninduced rtTA-293-, wtIκBα-, IκBα-2N-, IκBα-2N+3C-, IκBα-2NΔ4-, and IκBβ-expressing cells were subjected to SDS-PAGE and transferred to nitrocellulose membrane. WtIκBα and IκBα mutants were detected with IκBα-MAD3-specific antibody (33), and wtIκBβ was detected with IκBβ-specific antibody (Santa Cruz Biotechnology).
Activation of the IκB kinase complex by Sendai virus infection. (A) Strain 293 cells were either left untreated (lanes 1 and 4), treated with TNF-α (10 ng/ml) for 10 min (lanes 2 and 5), or infected with Sendai virus (80 HAU/ml) for 8 h (lanes 3 and 6). The IκB kinase complex was immunoprecipitated from whole-cell extracts (200 μg) with the anti-IKKα antibody H-744 (Santa Cruz) and assayed for IκBα phosphorylation by using recombinant wild-type GST-IκBα (aa 1 to 55) (lanes 1 to 3) or mutant GST-IκBα (aa 1 to 55; S32/36A) substrate (lanes 4 to 6). (B) Kinetic analysis of IκB kinase activation after Sendai virus infection. Strain 293 cells were infected with Sendai virus for different times as indicated, and the kinase activity was measured as described above. (C) Phosphorylation and degradation of IκBα in response to Sendai virus infection. Whole-cell extracts (20 μg) from 293 cells infected with Sendai virus (80 HAU/ml) for different times as indicated were subjected to SDS-PAGE and transferred to nitrocellulose membrane. IκBα was detected with a monoclonal anti-IκBα antibody. The positions of IκBα and phosphorylated IκBα are indicated by the arrows.
mRNA expression of IFN-β in IκBα- and IκBβ-expressing cells after Sendai virus infection. (A) Total RNA (5 μg) prepared from cells 0 to 48 h after Sendai virus infection was used for RNase protection analysis with the human CK3 cytokine template set of the RiboQuant Multi-Probe RPA kit. Cells lines are indicated at the top of each panel. Panels: A, rtTA-293 cells; B, wtIκBα-expressing cells; C, IκBα-2N-expressing cells; D, IκBβ-expressing cells. Where indicated, the cells were pretreated with Dox 48 h prior to Sendai virus infection. As a control, the level of GAPDH is shown.
Quantification of IFN-β mRNA expression. Quantification of RNase protection autoradiographs was obtained by normalizing values to the GAPDH and L32 (housekeeping gene) mRNA levels and plotting the values as IFN-β/GAPDH mRNA ratios. Panels: A, rtTA-expressing cells; B, wtIκBα-expressing cells; C, IκBα-2N-expressing cells; D, IκBβ-expressing cells. Lightly shaded columns, no Dox addition; darkly shaded columns, Dox addition (1 μg/ml) for 48 h.
Inhibition of IFN-β promoter activity in IκB-expressing 293 cells. IFN-β–CAT reporter plasmid (2.5 μg) was transfected into rtTA-293- and IκB-expressing 293 cells by the calcium phosphate method. After 24 h, cells were infected with Sendai virus (500 HAU/ml) for 90 min as indicated. Cultures were harvested at 48 h posttransfection, and 100 μg of the cytoplasmic extracts was prepared from the various cell lines and assayed for CAT activity for 4 h. As indicated, the cells were treated or not treated with Dox 48 h before transfection. The CAT activity observed with extracts of rtTA cells infected by Sendai virus (20 to 30% acetylation) was taken as the 100% value.
Inhibition of IFN-β promoter activity in IκB-expressing 293 cells. IFN-β–CAT reporter plasmid (2.5 μg) was transfected into rtTA-293- and IκB-expressing 293 cells by the calcium phosphate method. After 24 h, cells were infected with Sendai virus (500 HAU/ml) for 90 min as indicated. Cultures were harvested at 48 h posttransfection, and 100 μg of the cytoplasmic extracts was prepared from the various cell lines and assayed for CAT activity for 4 h. As indicated, the cells were treated or not treated with Dox 48 h before transfection. The CAT activity observed with extracts of rtTA cells infected by Sendai virus (20 to 30% acetylation) was taken as the 100% value.
Analysis of PRDII DNA binding activity in IκB-expressing cells. Nuclear extracts were prepared from rtTA-293-expressing (A), wtIκBα-expressing (B), IκBα-2N-expressing (C), and IκBβ-expressing (D) cells. Cells were infected by Sendai virus (500 HAU/ml) and harvested at the times indicated. Cells were treated with Dox at 48 h prior to Sendai virus infection. Nuclear extracts were incubated in the presence of 5 μg of poly(dI-dC) for 20 min prior to the addition of radiolabelled PRDII probe. For supershift experiments, NF-κB-specific antisera (31) were preincubated in presence of electrophoretic mobility shift assay buffer and poly(dI-dC) prior to the addition of the nuclear extracts.
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