Interaction of human immunodeficiency virus type 1 Tat with the transcriptional coactivators p300 and CREB binding protein - PubMed (original) (raw)
Interaction of human immunodeficiency virus type 1 Tat with the transcriptional coactivators p300 and CREB binding protein
M O Hottiger et al. J Virol. 1998 Oct.
Abstract
Human immunodeficiency virus type 1 (HIV-1) encodes the transactivator protein Tat, which is essential for viral replication and progression to disease. Here we demonstrate that transcriptional activation by HIV-1 Tat involves p300 or the related cellular transcriptional coactivator CREB binding protein (CBP). Tat transactivation was inhibited by the 12S form of the adenovirus E1A gene product, which inhibits p300 function, and this inhibition was independent of its effect on NF-kappaB transcription. A biochemical interaction of p300 with Tat was demonstrated in vitro and in vivo by coimmunoprecipitation. The carboxy-terminal region of p300, which binds to E1A, was shown to bind specifically to the highly conserved basic domain of Tat, which also mediates binding to the Tat-responsive region RNA stem-loop structure. The ability of Tat to interact physically and functionally with this coactivator provides a mechanism to assemble a basal transcription complex which may subsequently respond to the effect of Tat on transcriptional elongation and represents a novel interaction between an RNA binding protein and a transcriptional coactivator.
Figures
FIG. 1
12S E1A wt inhibits Tat induced HIV-1 gene expression. Jurkat T-leukemia cells were transfected with HIV-CAT (3 μg) or (ΔκB) HIV-CAT (3 μg) and RSV-Tat (10 ng) (A) or in combination with RSV-12S E1A wt (5 μg) or 12S E1A Δp300 (5 μg) (B). RSV-12S E1A wt (5 μg) and 12S E1A Δp300 (5 μg) were also transfected alone with HIV-CAT (3 μg) or ΔκB-HIV CAT (3 μg) (C). A control RSV vector was included such that the same amount of expression plasmid was used in each sample. Error bars indicate standard errors of the means of three independent experiments. A 500-fold mass excess of wild-type or mutant 12S E1A did not alter the expression of RSV-CAT significantly (0.77 or 0.72% conversion, respectively).
FIG. 2
Tat interacts with p300 and CBP in vitro and in vivo. (A) p300 and CBP from Jurkat nuclear extracts were affinity purified with GST-Tat (lanes 3 and 6) or with GST as a negative control (lane 2 and 5). After three washes, bound proteins were resolved by SDS-PAGE and then subjected to Western blot analysis for p300 (left) or CBP (right). Lanes 1 and 4 represent 20 μg of the input protein. (B) Tat and p300 interact in vivo. Tat was expressed by using a CMV expression plasmid (CMV-Tat) in 293 cells and immunoprecipitated (IP) with a control rabbit IgG (lane 8) and an anti-p300 IgG (lane 9). Bound proteins were resolved by SDS-PAGE and then subjected to Western blot analysis for Tat. Lane 7 represents 25 μg of the input protein. (C) Tat interacts directly with the carboxy-terminal half of p300. In vitro-translated p300 amino-terminal half (left) and carboxy-terminal half (right) were bound to GST-Tat (lanes 12 and 15) or GST (lanes 11 and 14). Lanes 10 and 13 represent 10% of the in vitro-translated input protein.
FIG. 3
Tat binds p300 between amino acid residues 1542 and 1710. Tat was immobilized on glutathione beads from bacterial extract which express GST-Tat and incubated with radiolabeled, in vitro-translated, carboxy-terminal deletion mutants of p300 (lanes 3, 6, 9, and 12). After three washes, bound proteins were resolved by SDS-PAGE. GST was used as a negative control (lanes 2, 5, 8, and 11). Lanes 1, 4, 7, and 10 represent 10% of the input protein. In lanes 4, 6, 7, and 9, small amounts of full-length p300 due to incomplete enzymatic digestion of the DNA template were noted.
FIG. 4
Tat binds p300 through its basic domain. (A, top) The basic domain of Tat is responsible for the p300 interaction. Different GST-Tat deletion mutants were immobilized on glutathione beads from bacterial extract and incubated with radiolabeled, in vitro-translated, carboxy-terminal p300 residues 1253 to 1710 (lanes 3 to 6). GST was used as a negative control (lane 2). Lane 1 represents 10% of the input protein. (A, bottom) The basic domain of Tat specifically binds p300. GST-Tat (48-57) and GST-Tat (NBD) fusion proteins were immobilized on glutathione beads from bacterial extract and incubated with radiolabeled, in vitro-translated, carboxy-terminal p300 residues 1253 to 1710 (lanes 9 and 10). GST was used as a negative control (lane 8). Lane 7 represents 10% of the input protein. (B) Jurkat cells were transfected with (ΔκB) HIV-CAT (3 μg) and Tat (10 ng). A control RSV vector was included such that a total of 10 ng of expression plasmids was used in each sample. Values represent percent conversion over the background level. (C) Transfection of an expression vector encoding p300 stimulates Tat-induced transcriptional activation. Jurkat cells were transfected with (ΔκB) HIV-CAT (3 μg), RSV Tat (10 ng), RSV control (10 ng), and p300 (0, 1, or 3 μg). A control RSV vector was included such that equal moles of RSV vector were used in each sample. pBluescript was used to make up the mass difference. Values (+Tat) represent percent conversion over the level of transcriptional activation by Tat. Error bars indicate standard errors of the means of three independent experiments.
References
- Bannister A J, Kouzarides T. The CBP co-activator is a histone acetyltransferase. Nature. 1996;384:641–643. - PubMed
- Chrivia J C, Kwok R P, Lamb N, Hagiwara M, Montminy M R, Goodman R H. Phosphorylated CREB binds specifically to the nuclear protein CBP. Nature. 1993;365:855–859. - PubMed
- Eckner R. p300 and CBP as transcriptional regulators and targets of oncogenic events. Biol Chem. 1996;377:685–688. - PubMed
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