p73 Interacts with human immunodeficiency virus type 1 Tat in astrocytic cells and prevents its acetylation on lysine 28

Abstract

Human immunodeficiency virus type 1 (HIV-1) Tat is a potent transcriptional activator of the HIV-1 promoter and also has the ability to modulate a number of cellular regulatory circuits including apoptosis. Tat exerts its effects through interaction with viral as well as cellular proteins. Here, we studied the influence of p73, a protein that is implicated in apoptosis and cell cycle control, on Tat functions in the central nervous system. Protein interaction studies using immunoprecipitation followed by Western blot and glutathione S-transferase pull-down assays demonstrated the association of Tat with p73. Tat bound to the N-terminal region of p73 spanning amino acids 1 to 120, and this interaction required the cysteine-rich domain (amino acids 30 to 40) of Tat. Association of p73 with Tat prevented the acetylation of Tat on lysine 28 by PCAF. Functional studies including RNA interference showed that p73 inhibited Tat stimulation of the HIV-1 promoter. Furthermore, p73 prevented the interaction of Tat with cyclin T1 in vitro but not in vivo. These findings suggest possible new therapeutic approaches, using p73, for Tat-mediated AIDS pathogenesis.

FIG. 1.

Association of Tat and p73 proteins in vivo and in vitro. (A, B, and C) Cell lysates were prepared from U-87MG cells transfected with plasmids expressing different cDNA constructs as indicated above A, B, and C. Fifty micrograms was used for Western analysis to verify the expression of HA-p73 (A, lanes 1 and 2) or CFP-Tat (A, lanes 3 and 4). Approximately 300 μg of cell extract was utilized in immunoprecipitations (IP) followed by Western blot utilizing anti-Tat (α-Tat) or rabbit serum and anti-HA (α-HA) antibodies, respectively (B, lanes 4 and 5), or anti-p73, anti-HA, or rabbit serum for IP followed by anti-Tat (C, lanes 3, 4, 5, 6, and 7, respectively). In parallel, 50 μg of extracts was utilized by direct Western blot assay (B, lanes 1 to 3 and lanes 1 and 2, respectively). The arrows depict the positions of the 73- and 66-kDa p73α and p73β (B) and the 13-kDa (C) Tat. The Western or IP/Western assays were carried out according to the procedure described previously (2). (D) In vitro-translated [³⁵S]methionine-labeled wild-type p73α (lanes 1 to 3) and p73β (lanes 4 to 6) were incubated with either GST (lanes 2 and 5) or GST-Tat (lanes 3 and 6) as indicated above the lanes. The positions of the 73- and 66-kDa p73 bands bound to the GST fusion proteins are shown. (E) Stained SDS gel showing the quality and the size of GST or GST-Tat fusion proteins. MW, molecular weight (in thousands).

FIG. 2.

Identification of the Tat domain that binds to p73α or p73β. (A) Schematic representation of wild-type Tat and its deletion mutants. Binding abilities of p73α or p73β to various Tat mutants are shown on the right. B and C illustrate a representative result from GST pull-down assay obtained with p73α and p73β. Wild-type Tat or the indicated mutants fused to GST and immobilized on glutathione-Sepharose were incubated with in vitro-synthesized [³⁵S]methionine-labeled p73α or p73β proteins. After incubation for 2 h at 4°C, the bound proteins were eluted and analyzed by SDS-PAGE. (D) Stained SDS gel showing the quality and the size of GST or GST-Tat deletion mutant fusion proteins. NLS points to the nuclear localization signal domain. MW, molecular weight (in thousands).

FIG. 3.

Tat interacts with the N-terminal domain of p73. (A) Schematic representation of p73α and p73β with their important domains. (B and C) Deletion mutants of p73α used in this study as in vitro-translated proteins (B) or fused to GST (C). Binding abilities of Tat to various p73α mutants are shown on the right (B). D and E illustrate representative results from GST pull-down assay obtained with IVT [³⁵S]methionine-labeled p73α (D) or with GST-p73α (E). (F) Stained SDS gel showing the quality and the size of GST or GST-p73 (full length and deletion mutants) fusion proteins. MW, molecular weight (in thousands).

FIG. 4.

Effect of p73-Tat association on Tat function. (A and E) U-87MG cells were transfected with 0.1 μg of the reporter plasmids (LTR or Bax-Luc) alone or in combination with 0.5 μg of Tat, p73α, or p73β expression plasmids, separately or combined, as indicated. Total amounts of transfected DNA were maintained constant by the addition of empty control vector. Cell extracts were prepared 48 h after transfection, and luciferase activity was determined. The data represent the mean value of three separate transfection experiments. Protein expression as well as equal protein loading were examined by Western blot assays using anti-p73, anti-Tat, and anti-Grb2 antibodies. (B) HL3T1 cells were transfected with 1.0 μg of Tat, p73α, or p73β expression plasmids, separately or combined, as indicated. Interaction of p73 with HIV LTR DNA was demonstrated by ChIP assays. The primers used in these experiments are described in Materials and Methods. Anti-p73 (lane 4), preimmune serum (lane 3), and no antibody (lane 2) were used in these experiments. NMS, normal mouse serum. (C) HL3T1 cells were transfected with siRNA-p73 (lanes 4 to 6) and/or CMV-p73 (lanes 2, 3, 5, and 6). Approximately 50 μg of extracts was utilized in the Western blot assay utilizing anti-p73 or anti-Grb2 antibody, respectively. The arrows depict the positions of the different proteins. Cells transfected only with pcDNA3 were utilized as negative controls (lanes 1 and 4). (D) HL3T1 cells were transfected with 0.5 μg of Tat expression plasmid in the presence and absence of siRNA p73. Total amounts of transfected DNA were maintained constant by the addition of empty control vector. Cell extracts were prepared 48 h after transfection, and CAT activity was determined. The data represent the mean values of three separate transfection experiments.

FIG. 5.

Effect of p73 on Tat acetylation. (A to D) Full-length (wild-type [wt]) or mutant (mut.) Tat and/or p73 (full-length or deletion mutant) proteins were incubated with GST (A to C, lanes 1, 3, and 5, and D, lanes 1 and 4) or GST-HAT (from p300) (A and C, lanes 2, 4, and 6, and B, lanes 2, 3, 5, and 6) or GST-HAT (from PCAF) (B, lanes 2, 4, and 6) and [¹⁴C]acetyl coenzyme A. Acetylated (Ac) products were resolved by 15% SDS-PAGE, dried, and exposed to X-ray film.

FIG. 6.

Functional interplay between p73 and Tat mutants. U-87MG cells were transfected with 0.1 μg of the reporter LTR-Luc plasmid alone or in combination with 0.5 μg of Tat (full length or mutants), p73α, or p73β expression plasmids, separately or combined, as indicated. Total amounts of transfected DNA were maintained constant by the addition of empty control vector. The data represent the mean values of three separate transfection experiments. Cell extracts were prepared 48 h after transfection, and luciferase activity was determined. Protein expression as well as equal protein loading were examined by Western blot assays using anti-p73 and anti-Tat antibodies.

FIG. 7.

Physical interaction between Tat/p73 and PCAF or cyclin T1. Cell lysates were prepared from U-87MG cells transfected with plasmids expressing different cDNA constructs as indicated above each panel. Approximately 300 μg of cell extract was utilized in immunoprecipitations (I.P.) with anti-Flag, anti-Tat, anti-p73, anti-cyclin T1, and anti-p300 antibodies or with rabbit preimmune serum (nuclear import signal [NIS]) (A and D, lanes 7, and B and C, lanes 8) or Sepharose beads (A and D, lanes 8, and B and C, lanes 9). Western analysis was performed using anti-Tat, anti-p73, and anti-cyclin T1 antibodies. In parallel, 50 μg of extracts was utilized by direct Western blot assay (A and B, lanes 1 to 3, and C and D, lanes 1 and 2). The arrow depicts the position of the detected proteins.

FIG. 8.

In vitro interaction between Tat and p73 in the presence of cyclin T1. Wild-type Tat or the indicated mutants fused to GST and immobilized on glutathione-Sepharose were incubated with in vitro-synthesized [³⁵S]methionine-labeled p73α, p73β, ΔN-p73α, ΔN-p73β, or cyclin T1 proteins in various combinations. After incubation for 2 h at 4°C, the bound proteins were eluted and analyzed by SDS-PAGE.

FIG. 9.

Subcellular localization of Tat and/or p73. CFP-Tat-transfected cells demonstrate the nuclear subcellular localization of the HIV transactivator protein (blue); in these Tat positive cells, p73 is also localized with the same nuclear distribution (green). In cell cultures transfected with a CFP-tagged empty vector, the location of the vector is exclusively cytoplasmic (−Tat panel, blue). The DsRed1 plasmid (red) was used to mark the mitochondria. Immunolabeling shows the nuclear localization of Tat or p73 (Tat+mito and p73+mito panels). For all panels, original magnification is ×400.