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, 20 (21), 8254-63

Inhibition of p300 Histone Acetyltransferase by Viral Interferon Regulatory Factor

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Inhibition of p300 Histone Acetyltransferase by Viral Interferon Regulatory Factor

M Li et al. Mol Cell Biol.

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) has been consistently identified in Kaposi's sarcomas, body cavity-based lymphomas, and some forms of Castleman's disease. The K9 open reading frame of KSHV encodes a viral interferon regulatory factor (vIRF) which functions as a repressor for cellular interferon-mediated signal transduction and as an oncogene to induce cell growth transformation. We demonstrate that KSHV vIRF directly interacts with cellular transcriptional coactivator p300 and displaces p300/CBP-associated factor from p300 complexes. This interaction inhibits the histone acetyltransferase activity of p300, resulting in drastic reduction of nucleosomal histone acetylation and alteration of chromatin structure. As a consequence, vIRF expression markedly alters cellular cytokine expression, which is regulated by acetylation of nucleosomal histones. These results demonstrate that KSHV vIRF interacts with and inhibits the p300 transcriptional coactivator to circumvent the host antiviral immune response and to induce a global alteration of cellular gene expression. These studies also illustrate how a cellular gene captured by a herpesvirus has evolved several functions that suit the needs of the virus.

Figures

FIG. 1
FIG. 1
Interaction of vIRF with p300. (A) In vivo interaction of vIRF with p300. COS-1 cells were transfected with expression vector (lane 1) or the flag-tagged vIRF expression vector (lane 2). After 48 h, cell extracts were used for immunoprecipitations (I.P.) with an antiflag antibody, followed by immunoblotting assay (I.B.) with an anti-p300 antibody. vIRF expression in transfected COS-1 cells was determined by immunoblotting with an antiflag antibody (bottom panel). (B) In vivo interaction of vIRF with p300 in KSHV-infected BCBL-1 cells. Lysates of BJAB (lane 1) and TPA-induced BCBL-1 (lane 2) cells were used for immunoprecipitation with an anti-p300 antibody, followed by immunoblotting with an anti-vIRF antibody. A whole-cell lysate was used to show vIRF expression (bottom panel). (C) In vitro interaction of vIRF with p300. flag-tagged p300 and vIRF proteins were purified using an antiflag antibody column, eluted by a competing peptide, and dialyzed overnight. After 30 min of incubation of the purified flag-p300 and flag-vIRF proteins (lane 2) or the purified flag-vIRF protein alone (lane 3), an anti-p300 antibody was used to precipitate p300 complexes. Immune complexes were resolved by SDS–8% PAGE, followed by immunoblotting with an antiflag antibody. A mixture of purified flag p300 and flag vIRF protein (lane 1) was used as a control.
FIG. 2
FIG. 2
Mutational analysis of vIRF binding to p300. (A) A summary of vIRF deletion mutants. Amino-terminally flag-tagged vIRF mutants were cloned into the pcDNA3.1 vector to allow for their expression in COS-1 cells. (B) Mutational analysis of vIRF binding to p300. COS-1 cells were transfected with wt vIRF and its mutants (mt1, mt2, and mt3). Cell lysates were used for immunoprecipitation (I.P.) with an anti-p300 antibody, followed by immunoblotting (I.B.) with an antiflag antibody to detect vIRF (lanes 6 to 10). The expression level of wt vIRF and its mutants was demonstrated by immunoblotting with an antiflag antibody (lanes 1 to 5). Lanes 1 and 6, vector alone; lanes 2 and 7, wt vIRF; lanes 3 and 8, vIRF mt1; lanes 4 and 9, vIRF mt2; lanes 5 and 10, vIRF mt3. Asterisks indicate the heavy chain of immunoglobulin and background, and arrows indicate wt vIRF and vIRF mt2 protein.
FIG. 3
FIG. 3
Inhibition of p300 HAT activity by vIRF. Recombinant baculovirus containing the flag-tagged p300, vIRF, or v-cyclin was used to purify each protein from insect cells. Purified p300 protein (30 nM) was mixed with [3H]acetyl-CoA and histone H4 serving as substrates in the presence of increasing nanomolar amounts of vIRF or v-cyclin as indicated at the bottom of panel A. After 5 min, p300 HAT activity was measured by immunoblotting with an antibody specific for acetylated histone H4 (A) and quantitating radioactivity of 3H-labeled histone H4 (B). In lane 7, p300 protein was first mixed with the substrates for 5 min, followed by incubation with vIRF protein (150 nM) for an additional 25 min. The bottom panel of panel A shows the amount of histone H4 protein used in each reaction, detected by an anti-H4 antibody. The values in panel B represent the averages of three independent experiments.
FIG. 4
FIG. 4
Competition of vIRF with P/CAF for binding to p300. (A) The displacement of P/CAF from p300 complexes by vIRF. COS-1 cells were transfected with 0.5 μg of an expression vector containing the flag-tagged P/CAF gene together with an increasing amount of expression vector containing the flag-tagged vIRF gene as indicated at the bottom of the figure. At 48 h posttransfection, p300 complexes were precipitated with an anti-p300 antibody coupled to agarose beads, resolved by SDS-PAGE, transferred onto a nitrocellulose membrane, and reacted with an antiflag antibody. (B) Expression of P/CAF and vIRF. Whole-cell lysates of transfected COS-1 cells were used to determine the level of P/CAF and vIRF by immunoblotting with an antiflag antibody. I.B., immunoblotting; I.P., immunoprecipitation.
FIG. 5
FIG. 5
Cell cycle analysis of vIRF-expressing cells. Exponentially growing cells were stained for chromosomal DNA with PI and were analyzed on a FACScan flow cytometer. Results presented are representative of five individual experiments using two independently established HS27 and NIH 3T3 cell lines.
FIG. 6
FIG. 6
Confocal microscopic analysis of chromosomal DNA staining. (A) Reduction of chromosomal DNA staining by the stable expression of vIRF. HS27/cDNA3 and HS27/vIRF cells were stained with To-Pro 3 dye or antihistone antibody. Stained cells were examined under the Leica confocal immunofluorescence microscope. (B) Reduction of chromosomal DNA staining by the transient expression of vIRF. HS27 cells were transfected with an expression vector containing flag-tagged vIRF. At 48 h posttransfection, HS27 cells were stained with antiflag antibody (left panel) and To-Pro 1 dye (right panel). Stained cells were examined under the Leica confocal immunofluorescence microscope. Comparison of two cells in this panel shows that the left one had vIRF expression, as shown in positive green immunofluorescence with antiflag antibody, whereas the right one did not have vIRF expression, as shown in negative immunofluorescence staining with antiflag antibody. (C) Reduction of chromosomal DNA staining by vIRF expression in BCBL-1 cells. BCBL-1 cells were stimulated with TPA for 48 h, fixed with 1% paraformaldehyde, and stained with anti-vIRF antibody (left panel) and To-Pro 3 dye (right panel). Stained BCBL-1 cells were examined under the Leica confocal immunofluorescence microscope. Among the cells in this panel, the BCBL-1 cell at left had vIRF expression, as shown in positive green immunofluorescence with anti-vIRF antibody, whereas the other cells did not have vIRF expression, as shown in negative immunofluorescence staining with anti-vIRF antibody.
FIG. 6
FIG. 6
Confocal microscopic analysis of chromosomal DNA staining. (A) Reduction of chromosomal DNA staining by the stable expression of vIRF. HS27/cDNA3 and HS27/vIRF cells were stained with To-Pro 3 dye or antihistone antibody. Stained cells were examined under the Leica confocal immunofluorescence microscope. (B) Reduction of chromosomal DNA staining by the transient expression of vIRF. HS27 cells were transfected with an expression vector containing flag-tagged vIRF. At 48 h posttransfection, HS27 cells were stained with antiflag antibody (left panel) and To-Pro 1 dye (right panel). Stained cells were examined under the Leica confocal immunofluorescence microscope. Comparison of two cells in this panel shows that the left one had vIRF expression, as shown in positive green immunofluorescence with antiflag antibody, whereas the right one did not have vIRF expression, as shown in negative immunofluorescence staining with antiflag antibody. (C) Reduction of chromosomal DNA staining by vIRF expression in BCBL-1 cells. BCBL-1 cells were stimulated with TPA for 48 h, fixed with 1% paraformaldehyde, and stained with anti-vIRF antibody (left panel) and To-Pro 3 dye (right panel). Stained BCBL-1 cells were examined under the Leica confocal immunofluorescence microscope. Among the cells in this panel, the BCBL-1 cell at left had vIRF expression, as shown in positive green immunofluorescence with anti-vIRF antibody, whereas the other cells did not have vIRF expression, as shown in negative immunofluorescence staining with anti-vIRF antibody.
FIG. 7
FIG. 7
Alteration of in vivo histone H3 and H4 acetylation by vIRF expression or butyric acid treatment. Identical amounts of proteins from HS27/cDNA3 cells (lanes 1 and 3) and HS27/vIRF cells (lanes 2 and 4) treated with butyric acid overnight (lanes 3 and 4) or mock treated (lanes 1 and 2) were used for immunoblotting analysis with antibodies specific for the acetylated histone H3 (A) or H4 (B). Arrows indicate acetylated histones H3 (Ac-H3) and H4 (Ac-H4). Numbers at left of each panel show sizes in kilodaltons.
FIG. 8
FIG. 8
Immunofluorescence test of in vivo histone H3 and H4 acetylation. HS27/cDNA3 and HS27/vIRF cells were stained with antibodies which specifically reacted with the acetylated forms of histones H3 and H4. Cells were visualized with Nomarski optics. Immunofluorescence testing was performed with a Leica confocal immunofluorescence microscope.
FIG. 9
FIG. 9
Enhanced DNA staining of vIRF-expressing cells by butyric acid treatment. HS27/cDNA3 and HS27/vIRF cells were treated with butyric acid (5 mM) overnight or mock treated. Cells were stained with To-Pro 1 dye for 1 min and examined under the Leica confocal microscope.
FIG. 10
FIG. 10
Recovery of histone acetylation by overexpression of wt p300 HAT. HS27/vIRF cells were transfected with wt p300 (lane 3) or p300 ΔHAT mutant (lane 4). HS27/cDNA3 (lane 1) and HS27/vIRF (lane 2) were included as controls. Identical amounts of proteins from cell lysates were used for immunoblotting analysis with an antibody specific for the acetylated histone H4. The bottom panel shows the amount of cellular histone H4 protein in each lane, detected by an anti-H4 antibody. Arrows indicate the acetylated form of histone H4 (Ac-H4) or total histone H4 (H4). Numbers at left are molecular masses in kilodaltons.
FIG. 11
FIG. 11
Inhibition of cellular MIF gene expression by vIRF. (A) Inhibition of cellular MIF gene expression by vIRF. Total RNA was extracted from NIH 3T3/cDNA3 and NIH 3T3/vIRF cells and used for RNase protection assays with the in vitro-transcribed 32P-labeled MIF or GAPDH probe. The protected fragments were resolved by electrophoresis on a 5% acrylamide–urea gel, and autoradiograms were developed in a Fuji Phospho Imager. Detailed procedures are described in Materials and Methods. (B) Alteration of the promoter activity of MIF by vIRF. MIF-luc reporter plasmid (0.25 μg) and pGKβgal reporter plasmid (0.25 μg) were transfected into NIH 3T3 cells together with 0.25 μg of vIRF, wt p300, or p300 ΔHAT expression vector as indicated. Luciferase was measured at 48 h posttransfection, and luciferase values were normalized by β-galactosidase activity. Values for luciferase activity represent the averages of three independent experiments. Lysates of transfected NIH 3T3 cells were used to determine the level of p300 and p300 ΔHAT mutant by immunoblotting with an anti-p300 antibody. Lane 1, NIH 3T3 cells transfected with vector alone; lane 2, NIH 3T3 cells transfected with vIRF; lane 3, NIH 3T3 cells transfected with vIRF and p300; lane 4, NIH 3T3 cells transfected with vIRF and p300 ΔHAT.

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