Positive and negative regulation of the innate antiviral response and beta interferon gene expression by deacetylation - PubMed (original) (raw)

Positive and negative regulation of the innate antiviral response and beta interferon gene expression by deacetylation

Inna Nusinzon et al. Mol Cell Biol. 2006 Apr.

Abstract

Beta interferon (IFN-beta) gene expression in response to virus infection relies on the dynamic assembly of a multiprotein enhanceosome complex that is initiated by the activation of two inducible transcription factors, interferon regulatory factor 3 (IRF3) and NF-kappaB. Virus or double-stranded RNA-induced activation of IFN-beta gene expression is prevented by the addition of protein deacetylase inhibitors. The isolated IRF-responsive positive regulatory domain was found to require deacetylation for its activity, but IRF3 protein activation leading to its nuclear translocation and DNA binding was not impaired by deacetylase inhibition. In contrast, NF-kappaB activity was not affected by deacetylase inhibitors. RNA interference indicated that several deacetylase enzymes, including histone deacetylase 1 (HDAC1), HDAC8, and HDAC6, influence IFN-beta gene expression with opposing effects. While HDAC1 and HDAC8 repress IFN-beta expression, HDAC6 acts as a coactivator essential for enhancer activity. Virus replication is enhanced in HDAC6-depleted cells, demonstrating HDAC6 is an essential component of innate antiviral immunity.

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Figures

FIG. 1.

FIG. 1.

Deacetylase activity is required for innate antiviral defense. A. Duplicate wells of 2fTGH cells were infected with dilutions of Sendai virus to achieve MOIs of 62.5, 12.5, 2.5, 0.5, 0.1, and 0 PFU/cell. Simultaneously with inoculation, cells were treated with TSA at the indicated concentrations. Cells were stained and analyzed 24 h postinfection. B. 2fTGH cells were infected with Sendai virus (MOI, 10 PFU/cell) for the indicated times in the presence or absence of TSA. RT-PCR was carried out using primers for IFN-β or the control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). C. 2fTGH cells were pretreated with dsRNA for 8 h with or without TSA and then infected with VSV at MOIs of 50, 5, 0.5, 0.05, 0.005, and 0 PFU/cell. Cells were stained and analyzed 18 h postinfection. D. 2fTGH cells were treated with dsRNA, TSA, and cycloheximide (CHX) as indicated, and RT-PCR for IFN-β and GAPDH was performed. E. (Top) HeLa cells were treated with dsRNA and TSA as indicated, and ChIP assays were performed using antiserum specific for RNA Pol II. Coprecipitated DNA was amplified by PCR using primers specific for the IFN-β promoter. (Bottom) Phosphorimaging was used for quantitative analysis, and values were normalized to the input.

FIG. 2.

FIG. 2.

Deacetylase activity is required for IFN-β enhanceosome activity. A. Luciferase assays were performed in 2fTGH cells using the indicated reporter genes, and cells were treated with dsRNA and TSA for the indicated times. B. 2fTGH cells were treated with dsRNA and TSA as indicated, and nuclear and cytoplasmic fractions were separated followed by SDS-PAGE and Western blotting with antibodies for IRF3. C. 2fTGH cells were treated with dsRNA and TSA as indicated, nuclei were purified, and nuclear lysates were incubated with a biotinylated double-stranded oligonucleotide containing IRF3 binding sites. IRF3 was eluted from DNA and visualized by SDS-PAGE and Western blotting. Asterisks indicate the IRF3 band. Bound, IRF3 that was bound to oligonucleotide; total, total nuclear IRF3; c, control containing no oligonucleotide.

FIG. 3.

FIG. 3.

HDAC1 and HDAC8 inhibit IFN-β expression. A. Luciferase assays with the indicated reporters were performed in the presence of siRNA for HDAC1 (siHD1) or scrambled control (Con). At 48 h after transfection with siRNA and reporter, cells were treated with dsRNA for the indicated times. B. Two clones stably expressing RNA interference vectors specific for HDAC1 were analyzed by Western blotting. wt, wild-type cells. C. The stable clones were treated with dsRNA as indicated, and RT-PCR was performed for IFN-β or glyceraldehyde-3-phosphate dehydrogenase (GAPDH). WT, wild-type cells. D. Similar to the experiment in panel A, except siRNA specific for HDAC8 was used.

FIG. 4.

FIG. 4.

HDAC6 is required for IFN-β expression and innate antiviral immunity. A. Luciferase assays with the indicated reporters were performed in the presence of siRNA for HDAC6 (siHD6) or scrambled control (Con). At 48 h after transfection with siRNA and reporter, cells were treated with dsRNA for the indicated times. B. (Top) Western blot for HDAC6 in 2fTGH cells transfected with HDAC6-specific siRNA. (Bottom) 2fTGH cells transfected with siRNA were treated with dsRNA, and RT-PCR was performed for IFN-β and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). WB, Western blot. C. 2fTGH cells were transfected with siRNA and 48 h later were treated with dsRNA (8 h) or Sendai virus (5 h) or left untreated (unt) and then infected with VSV-GFP for 18 h. D. (Top) Luciferase assay similar to the experiment shown in panel A, but with A549 cells (control) or an HDAC6-silenced A549 stable cell line (HD6 KD). (Bottom) Western blot for HDAC6 in the A549 cells above. E. Similar to the experiment in panel C, but with A549 cells (Con) or a HDAC6-silenced A549 stable cell line (HD6 KD).

FIG. 5.

FIG. 5.

HDACs are activators and repressors of IFN-β. A. HDAC6 activates IRF3, while HDAC1 and HDAC8 repress both IRF3 and NF-κB. Together, these HDACs strike a balance for optimal activation of IFN-β. B. TSA inhibits all HDAC activity, thereby blocking IFN-β activation. C. siRNA specific for either HDAC1 or HDAC8 relieves the inhibitory actions of these proteins, resulting in a heightened transcriptional response. D. siRNA specific for HDAC6 removes the activating effect of this deacetylase, causing lower IFN-β expression.

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