Inhibition of histone deacetylases induces bovine leukemia virus expression in vitro and in vivo

Abstract

Packaging into nucleosomes results in a global transcriptional repression as a consequence of exclusion of sequence-specific factors. This inhibition can be relieved by using inhibitors of histone deacetylases, acetylation being a major characteristic of transcriptionally active chromatin. Paradoxically, the expression of only approximately 2% of the total cellular genes is modulated by histone hyperacetylation. To unravel the potential role of this transcriptional control on BLV expression, we tested the effect of two highly specific inhibitors of deacetylases, trichostatin A (TSA) and trapoxin (TPX). Our results demonstrate that treatment with TSA efficiently enhanced long terminal repeat-directed gene expression of integrated reporter constructs in heterologous D17 stable cell lines. To further examine the biological relevance of these observations made in vitro, we analyzed ex vivo-isolated peripheral blood mononuclear cells (PBMCs) from bovine leukemia virus (BLV)-infected sheep. TSA deacetylase inhibitor induced a drastic increase in viral expression at levels comparable to those induced by treatment with phorbol-12-myristate 13-acetate and ionomycin, the most efficient activators of BLV expression known to date. TSA acted directly on BLV-infected B lymphocytes to increase viral expression and does not seem to require T-cell cooperation. Inhibition of deacetylation after treatment with TSA or TPX also significantly increased viral expression in PBMCs from cattle, the natural host for BLV. Together, our results show that BLV gene expression is, like that of a very small fraction of cellular genes, also regulated by deacetylation.

FIG. 1.

Response of the BLV promoter to TSA in cell culture. D17-LTR WT cells harboring a stably integrated LTR-WT plasmid were cultured in the absence (lane 1) or in the presence (lane 2) of TSA, and the luciferase activities were measured after 48 h. The average values from three independent experiments are presented. Error bars, standard deviations; ∗∗, highly statistically significant (P ≤ 0.01).

FIG. 2.

Modulation of p24 expression by TSA in cells isolated from infected sheep. (A) PBMCs were isolated from the circulating blood of sheep 8, purified by Percoll gradient, and cultivated in the absence (lane 1) or in the presence (lane 2) of TSA or PMA-ionomycin (lane 3) for 48 h. The major capsid protein p24 was titrated in the cell culture supernatants by ELISA. The supernatants from BLV-infected FLK cells (lane 4) and D17 fibroblasts (lane 5) were used as positive and negative controls, respectively. (B) p24 expression in PBMCs from sheep. The data, presented as optical densities (means ± standard deviations [error bars]), are from three independent experiments. The statistical evaluation was performed according to Student's t test. ∗∗, highly statistically significant (P ≤ 0.01).

FIG. 3.

Detection of apoptosis in B lymphocytes from infected sheep. PBMCs were cultured for 48 h in the absence or in the presence of TSA or PMA-ionomycin and labeled with monoclonal antibody 1H4 directed against surface IgMs and a FITC-coupled conjugate. The cells were then fixed, stained with PI, and analyzed by dual-immunofluorescence flow cytometry. Doublets were excluded using the scatter gating method (R1 [not shown]). Results from a representative experiment corresponding to cultures established from sheep 8 are presented as dot plots. The quadrant (R2) indicates the apoptotic B cells within the PBMC cultures.

FIG. 4.

Activation of BLV expression in B lymphocytes from infected sheep by TSA. T cells were depleted from PBMCs isolated from the sheep 8, and the selected B lymphocytes were cultured for 48 h in the absence (lane 1) or in the presence (lane 2) of TSA or PMA-ionomycin (lane 3). The BLV p24 antigen was titrated in the cell culture supernatants by ELISA, and the supernatants from BLV-infected FLK cells (lane 4) and D17 fibroblasts (lane 5) were used as positive and negative controls, respectively. The data presented correspond to the mean values from three independent experiments (error bars, standard deviations). The statistical evaluation was performed according to Student's t test. ∗∗, highly statistically significant (P ≤ 0.01).

FIG. 5.

Modulation of p24 expression in infected cows by TSA and TPX. PBMCs were isolated from the circulating blood of BLV-infected cattle (cow 01), purified by Histopaque gradient, and cultivated for 48 h in the absence (lane 1) or in the presence of TSA (lane 2), TPX (lane 3), or PMA-ionomycin (lane 4). The major capsid protein p24 in the cell culture supernatants was titrated by ELISA. The supernatants from BLV-infected FLK cells (lane 5) and D17 fibroblasts (lane 6) were used as positive and negative controls, respectively. The data, presented as mean optical densities (means ± standard deviations [error bars]), are from three independent experiments. The statistical evaluation was performed according to Student's t test. ∗∗, highly statistically significant (P ≤ 0.01).

FIG. 6.

Detection of apoptosis in B lymphocytes from BLV-infected cows. PBMCs isolated from three cows (cows 01, 51, and 57) were cultured for 48 h in the presence or in the absence of TSA, TPX, or PMA-ionomycin and labeled with the monoclonal antibody 1H4 IgM and a FITC-conjugated secondary antibody. The cells were then fixed, stained with PI, and analyzed by dual-immunofluorescence flow cytometry. Doublets were excluded using the scatter gating method (R1 [not shown]). Shown are representative experiments corresponding to PBMC cultures isolated from cow 01, presented as dot plots. The quadrants (R2) indicate the apoptotic B cells within the PBMC cultures.