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Originally published In Press as doi:10.1074/jbc.M408643200 on December 15, 2004

J. Biol. Chem., Vol. 280, Issue 10, 9390-9399, March 11, 2005
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HIV-1 Tat Interactions with p300 and PCAF Transcriptional Coactivators Inhibit Histone Acetylation and Neurotrophin Signaling through CREB*

Kasuen Wong{ddagger}§, Anima Sharma{ddagger}§, Soumya Awasthi{ddagger}, Elizabeth F. Matlock{ddagger}, Lowery Rogers{ddagger}, Carine Van Lint¶||, Daniel J. Skiest**, Dennis K. Burns{ddagger}{ddagger}, and Robert Harrod{ddagger}§§

From the {ddagger}Laboratory of Molecular Virology, Department of Biological Sciences, Southern Methodist University, Dallas, Texas 75275-0376, Laboratoire de Virologie Moléculaire, Service de Chimie Biologique, Institut de Biologie et de Médecine Moléculaires, Université Libre de Bruxelles, Rue des Profs Jeener et Brachet 12, Gosselies 6041, Belgium, the **Division of Infectious Diseases, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9113, and the {ddagger}{ddagger}Division of Neuropathology, Department of Pathology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9072

Received for publication, July 29, 2004 , and in revised form, December 15, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The human immunodeficiency virus type-1 (HIV-1) infects microglia, macrophages, and astrocytes in the central nervous system (CNS) and may cause severe neurological diseases, such as AIDS-related dementias or progressive encephalopathies, as a result of CNS inflammation and neurotrophin signaling defects associated with expression of viral antigens and HIV-1 replication in the brain. The HIV Tat protein can be endocytosed by surrounding uninfected cells; interacts with transcriptional coactivators/acetyltransferases, p300/CREB-binding protein, and p300/CREB-binding protein-associated factor (PCAF); and induces neuronal apoptosis. Since nerve growth factor (NGF) receptor and brain-derived neurotrophic factor receptor signaling through CREB requires p300 and PCAF histone acetyltransferases, we sought to determine whether HIV-1 Tat coactivator interactions interfere with neurotrophin receptor signaling in neuronal cells. Here, we demonstrate that Tat-coactivator interactions inhibit NGF- and brain-derived neurotrophic factor-responsive CRE trans-activation and neurotrophin protection against apoptosis in PC12 and IMR-32 neuroblastoma cells. Purified recombinant Tat or Tat-derived synthetic peptides, spanning p300- and PCAF-binding sequences, inhibit histone H3/H4 acetylation in vitro. A Tat mutant, TatK28A/K50A, defective for binding p300 and PCAF, neither repressed NGF-responsive CRE transactivation nor inhibited histone acetylation. HIV-1 Tat interacts in PCAF complexes in post-mortem CNS tissues from donor neuro-AIDS patients, as determined by fluorescence resonance energy transfer immunoconfocal microscopy. Importantly, these findings suggest that HIV-1 Tat-coactivator interactions may contribute to neurotrophin signaling impairments and neuronal apoptosis associated with HIV-1 infections of the CNS.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Human immunodeficiency virus type 1 (HIV-1)1 infections of the CNS are associated with severe neurological disorders. In adults, between 40 and 70% of HIV-1-infected symptomatic AIDS patients develop neurological disorders (1–3). The brain, spinal cord, and peripheral nervous system are all frequently involved by HIV. Involvement of the nervous system may be due to the direct or indirect effects of HIV infection or as a result of secondary infections or malignancies (4). One of the most prevalent and clinically important primary neurological disorders is the AIDS dementia complex, a subcortical dementia, which manifests clinically as cognitive, behavioral, and motor impairment. Estimates of the prevalence of AIDS dementia range from 7 to 90% depending on the definition, but it is probably ~20–30%. Recent studies suggest that the incidence of AIDS dementia complex has not decreased to the same extent as other AIDS-related complications in the highly active antiretroviral therapy (HAART) era (5, 6). Whereas modern combination therapies, such as HAART, have been successful toward reducing the plasma burden of HIV, the majority of HAART regimen compounds do not penetrate the blood-brain or blood-nerve barriers, and HIV-1 continues to replicate in the CNS, which may serve as a reservoir for the production of HAART-resistant virus particles.

Human immunodeficiency virus type 1 principally affects the cerebral white matter, deep gray matter (e.g. basal ganglia, cingulum, and thalamus), and brainstem regions in the brain (3, 7, 8); and HIV productively infects and replicates in macrophages, microglia, and monocytes in the CNS (8–15). However, astrocytes, basal vascular endothelial cells, neurons, and oligodendrocytes may also become infected and play important roles during neuropathogenesis (9, 16–21). Advanced neurological involvement results in an overall reduction in brain mass that correlates with neuronal loss, or dropout (3, 7, 8, 9). Neuronal cell death generally coincides with high levels of HIV replication and the production of immune inflammatory factors that possess potent neurotoxic effects. HIV-1-infected macrophages, microglia, and monocytes produce significant quantities of interleukin-1{beta}, tumor necrosis factor-{alpha}, platelet-activating factor, oncostatin-M, and NO associated with immune activation (9, 10, 22–30).

In addition to inflammatory factors and neurotoxins produced by infected macrophages and microglia, HIV-1 proteins (Tat, Vpr, gp120) have been shown to cause neuronal apoptosis (30–38). HIV-1 Tat, produced by surrounding infected macrophages, microglia, or astrocytes, can interact with low density lipoprotein receptor-related protein on surfaces of neurons, which facilitates its uptake by endocytosis (39). Tat up-regulates Par-4 expression in neurons and induces apoptosis by oxidative stress associated with mitochondrial dysfunction and perturbation of Ca2+-regulated channels and glutamate receptors (31, 33, 34, 40). Accumulating evidence suggests that Tat plays an important role during neuropathogenesis, both as an intracellular and extracellular mediator of neurotoxicity (15, 31, 33, 34, 39–46).

Tat is an 81–101-amino acid peptide that binds a uracilcontaining bulge within the stem-loop secondary structure of the Tat-activated region (TAR-RNA) in HIV-1 transcripts. Tat functions as an elongation factor and stabilizes the synthesis of full-length viral mRNAs by preventing premature termination by the TAR-RNA stem-loop (47–53). The Tat protein is acetylated on lysine residues Lys28 and Lys50/Lys51 within its TAR-RNA-binding arginine-rich motif by the transcriptional coactivators/acetyltransferases, p300/CBP, and PCAF (54–58). Acetylation of Tat on Lys28 by PCAF facilitates recruitment of the PTEF-b (cyclin T1-Cdk9) complex to the HIV-1 TAR-RNA, which results in hyperphosphorylation of heptaserines in the carboxyl terminus of RNA polymerase II to promote elongation (56, 57). Acetylation of residue Lys28 of HIV-1 Tat dissociates Tat-PCAF interactions (56, 57). Acetylation of lysines Lys50/Lys51 by p300/CBP prevents binding of acetylated Tat to TAR-RNA and is proposed to enhance HIV-1 transcriptional activation through "recycling" of the viral trans-activator (56, 57); Lys50 acetylation also creates a new binding site for PCAF and facilitates the formation of a PCAF-Tat-PTEF-b ternary complex (57). The bromodomain of PCAF specifically binds Lys50-acetylated Tat, and this interaction has been demonstrated to prevent Lys50-acetylated Tat from binding TAR-RNA (55, 57). Recruitment of p300/CBP and PCAF by Tat to the HIV-1 long terminal repeat is essential for viral replication and facilitates HIV-1 gene expression when the provirus integrates in transcriptionally silent regions of the genome (54, 56, 57, 59, 60).

In the present study, we demonstrate that HIV-1 Tat-coactivator interactions markedly inhibit neurotrophin-responsive CRE-trans-activation, histone acetylation, and NGF protection against cellular apoptosis. These findings suggest that Tat-coactivator interactions may contribute to neurotrophin signaling/transcriptional impairments and neuronal cell death associated with HIV infections of the CNS.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Transfections—Rodent PC12 pheochromocytoma cells (ATCC number CRL-1721) were cultured in 6-well plates or 60-mm2 tissue culture dishes (Nalge) coated with mouse type IV collagen (20 µg/ml in PBS; Roche Applied Science) in ATCC Ham's F-12K medium supplemented with 5% heat-inactivated fetal bovine serum, 15% horse serum (Atlanta Biologicals), 100 units/ml penicillin, and 100 µg/ml streptomycin sulfate (Invitrogen). Human IMR-32 neuroblastoma cells (ATCC number CCL-127) were cultured in collagen-treated tissue culture dishes or 6-well plates as described in ATCC 2003, Eagle's minimum essential medium supplemented with 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin sulfate. All cultures were grown under 10% CO2 at 37 °C. Mouse NGF (2.5S, grade II; Roche Applied Science) was added in certain experiments to a final concentration of 10 ng/ml.

Transfections were performed by seeding collagen-treated, 6-well tissue culture plates with ~2 x 105 cells/well and using either a calcium phosphate-based transfection system or Lipofectamine reagent (Invitrogen) as recommended in the manufacturer's protocol. The plasmids used in this study were CRE promoter-luciferase (kindly provided by Dr. N. M. Nathanson, Department of Pharmacology, University of Washington), CMV-FLAG-PCAF, CMV-FLAG-p300, CMV-adenoviral E1A 12S, CMV-E1A 12S{Delta}N (all generously provided by Dr. Y. Nakatani, Dana Farber Cancer Institute, Harvard Medical School), RSV-HIV-1 Tat, RSV-HIV-1 TatK28A, RSV-HIV-1 TatK50A, RSV-HIV-1 TatK28A/K50A, pGEX-GST-HIV-1 Tat, and pGEX-GST-HIV-1 TatK28A/K50A (described in Ref. 56). Cells were harvested by scraping, washed twice with 500 µl of PBS, resuspended in 80 µl of SDS lysis buffer (Promega), and lysed by rapid freeze-thawing. Samples were centrifuged at 14,000 rpm at 4 °C, and protein concentrations were determined using the Bradford microassay and spectrophotometric analyses at 595 nm. Luciferase assays were performed on 50 µg of total cellular proteins for each sample using a Berthold Lumat LB 9507 luminometer. Results shown are representative of duplicate or triplicate experiments.

Fluorescence Resonance Energy Transfer (FRET) Immuno-laser Confocal Microscopy—Formalin-fixed post-mortem CNS tissue (cingulum; thalamus) thin sections from uninfected or HIV-1-infected donor neuro-AIDS patients clinically diagnosed with primary encephalopathies were analyzed by FRET immuno-laser confocal microscopy to detect HIV-1 Tat-PCAF interactions. Briefly, the slides were fixed in 0.2% gluteraldehyde, 1% formaldehyde in PBS and washed three times with PBS, and nonspecific antigens were blocked by incubation with 3% (w/v) bovine serum albumin, 0.5% (v/v) Tween 20 in PBS for 1 h at room temperature. An anti-PCAF goat polyclonal antibody (Santa Cruz Biotechnology) and an anti-HIV-1 Tat rabbit polyclonal antibody (Advanced Bioscience Laboratories) were diluted (1:1000) in BLOTTO buffer (50 mM Tris-HCl, pH 8.0, 2 mM CaCl2, 80 mM NaCl, 0.2% (v/v) IGEPAL CA-630, 0.02% (w/v) sodium azide, 5% (w/v) nonfat dry milk), and slides were incubated in primary antibodies with agitation for 2 h at room temperature. A weakly emitting FITC-conjugated anti-goat secondary antibody (donor) was used to detect PCAF, and a Cy3-conjugated anti-rabbit secondary antibody (FRET acceptor) was used to detect HIV-1 Tat in infected CNS tissue sections. FRET immuno-laser confocal microscopy was performed using a Nikon C1 confocal system mounted on a Nikon TE2000-U inverted fluorescence microscope.

Annexin-V-FITC Staining and Fluorescence-activated Cell Sorting— For apoptosis analyses, RSV-HIV-1 Tat (or Tat mutant)-transfected PC12 cells were prestimulated with NGF (10 ng/ml) for 8 h prior to serum deprivation for 48 h. Cells were harvested, washed twice with PBS, and resuspended in 200 µl of 1x annexin buffer and stained with 5 µl of annexin-V-FITC (BD Biosciences) for 10 min. Five hundred microliters of 1x annexin-buffer were added to each sample, and fluorescence flow cytometric analyses were performed using a BD Biosciences FACSCaliber instrument.

Protein Purification—GST-HIV-1 Tat and GST-TatK28A/K50A fusion proteins were induced in Escherichia coli BL21 bacteria containing appropriate pGEX expression constructs using 100 µM isopropyl 1-thio-{beta}-D-galactopyranoside (Invitrogen) in Luria-Bertani broth overnight with 100 µg/ml ampicillin at 37 °C. Cells were pelleted by centrifugation, washed, and resuspended in 5 ml of PBS containing the protease inhibitors, anti-pain dihydrochloride, bestatin, chymostatin, leupeptin, and pepstatin (50 ng/ml each; Roche Applied Science). Bacteria were lysed by repeated sonication over an ice bath, using a Bronson sonic dismembrator equipped with a microtip and operated at 70% duty cycle. Lysates were precleared by centrifugation and were incubated for2hat 4 °C with 500 µl of a 50% mixture of glutathione-Sepharose 4B (Amersham Biosciences) equilibrated in PBS (plus protease inhibitors). Following incubation, matrices were pelleted by centrifugation at 2500 rpm for 5 min at 4 °C and washed twice with PBS (plus protease inhibitors). Bound GST fusion proteins were eluted in 10 mM reduced glutathione (Sigma) in PBS (plus protease inhibitors), dialyzed overnight at 4 °C against 25 mM Hepes, pH 7.9, 5 mM KCl, 0.5 mM MgCl2, 0.5 mM EDTA, 0.25 mM dithiothreitol, and 10% glycerol; fractions were stored in aliquots at -80 °C.

Radioactive in Vitro HAT Assays—Radioactive acetylation assays were performed in vitro by preincubating 5 units of recombinant HAT-active p3001195–1707 or PCAF351–832 (Upstate Biotechnology, Inc., Lake Placid, NY) with GST-HIV-1 Tat or GST-HIV-1 TatK28A/K50A proteins (25, 50, or 100 ng) in acetylation buffer containing 50 mM Tris-HCl, pH 8.0, 1 mM DTT, 0.1 mM EDTA, 50 mg/ml bovine serum albumin, 10% glycerol, and 5 ml of [acetyl-1-14C]acetyl-coenzyme A (51.60 mCi/mmol, NEC-313; PerkinElmer Life Sciences) on ice for 10 min. Purified core histones H2A, H2B, H3, and H4 (Upstate Biotechnology) were added, and samples were incubated at 37 °C for 10 min; then reactions were quenched by the addition of 6 µl of 5x SDS-PAGE loading buffer, and acetylated products were resolved by electrophoresis through 4–20% Tris-glycine gels and visualized by autoradiography/fluorography using ENHANCE reagent (PerkinElmer Life Sciences). For certain experiments, increasing amounts (50, 100, and 200 ng) of synthetic peptides, Tat23–43, TatK/A23–43, Tat41–61, and TatKK/AA41–61 (Biosynthesis), were added to HAT assays (61).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
HIV-1 Tat-Coactivator Interactions Inhibit Neurotrophin-responsive CRE Trans-activation—Neurotrophins are essential for neuronal survival, differentiation, injury repair and synapse formation (Fig. 1A) (62–66). However, NGF has been shown to synergistically trans-activate the HIV-1 LTR with Tat to promote viral replication (67, 68), and NGF is an autocrine/paracrine survival factor for HIV-1-infected macrophages and monocytes (Fig. 1A) (69–71). Neurotrophins also significantly counter the neurotoxic effects of inflammatory factors, such as tumor necrosis factor-{alpha}, interleukin-1{beta}, RANTES, and NO, produced by CNS immune cells (microglia, macrophages, and monocytes) in response to viral antigens or HIV replication (Fig. 1A). NGF and BDNF receptor signaling induce CREBSer133 phosphorylation and transcriptionally activate CRE enhancers within promoters of neuronal survival genes (72–75). Since Ser133-phospho-CREB requires the coactivators/acetyltransferases, p300/CBP and PCAF, for transcriptional activation (76–79), we investigated whether interactions between HIV-1 Tat and transcriptional coactivators might interfere with neurotrophin-signaling/transcription through CREB in neuronally derived cells. The HIV-1 Tat protein is acetylated on lysine residues Lys28 and Lys50/Lys51 by p300/CBP and PCAF (Fig. 1B). Importantly, we have previously demonstrated that interactions between Tat and the HAT domains of p300/CBP and PCAF repress p53-dependent trans-activation and tumor suppressor functions in IMR-32 human neuroblastoma cells, and HIV-1 Tat-HAT domain binding significantly prevents PCAF-mediated acetylation of p53 on Lys320 in vitro and in vivo in response to UV irradiation (61).



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  		FIG. 1.
HIV-1 Tat-coactivator interactions inhibit neurotrophin signaling through CREB. A, neurotrophins produced by neurons or HIV-1-infected macrophages, microglia and monocytes protect against cellular apoptosis and stimulate HIV-1 replication by trans-activating elements within the viral LTR. Inflammatory factors (tumor necrosis factor-{alpha}, interleukin-1{beta}, TGF-{beta}, and NO) and chemokines (RANTES and MIP-1{alpha}/{beta}) possess neurotoxicity and cause neuronal cell death; these factors may also induce immune cellular proliferation and stimulate HIV-1 replication in the CNS. B, diagram showing functionally overlapping binding sites for HIV-1 Tat and the adenoviral E1A 12S factor within the catalytic acetyltransferase domains of p300/CBP and PCAF. C, PC12 cells were co-transfected with a CRE promoter-luciferase reporter construct (0.5 µg) and increasing amounts of either RSV-HIV-1 Tat, RSV-HIV-1 TatK28A/K50A, or pcDNA3.1-GFP (0.5, 1.0, and 1.5 µg) (56). Transfected cells were stimulated with NGF or BDNF (D) at 10 ng/ml, and relative luciferase activities were determined using equivalent amounts of total proteins. Expression levels of HIV-1 Tat, TatK28A/K50A, and actin were determined by immunoblotting. E, IMR-32 human neuroblastoma cells were transfected as in D and stimulated with BDNF (10 ng/ml). Error bars represent S.D. values (n = 2).

 
To determine whether HIV-1 Tat-coactivator interactions interfere with neurotrophin receptor signaling/transcription, PC12 cells were co-transfected with a synthetic CRE-containing promoter-luciferase reporter construct and increasing amounts of RSV-HIV-1 Tat, RSV-HIV-1 TatK28A/K50A, or pcDNA 3.1-GFP as a negative control. Numerous laboratories have confirmed that the HIV-1 Tat mutant, TatK28A/K50A, is defective for interactions with p300/CBP and PCAF (56, 61), and we have shown that this mutant retains its ability to enter nuclei of IMR-32 cells (which is important, since the K50A mutation disrupts the nuclear localization signal) (61). Following transfection, the cells were stimulated by treatment with NGF (10 ng/ml; Roche Applied Science) for 8 h. Results in Fig. 1C demonstrate that NGF stimulation significantly activates CREB-dependent transcription (10-fold) in PC12 cells. Expression of increasing amounts of HIV-1 Tat inhibited NGF-responsive CRE trans-activation in a dose-dependent manner (Fig. 1C). The coactivator binding-defective TatK28A/K50A mutant, however, did not influence NGF-responsive CRE trans-activation; nor did the GFP control (Fig. 1C). Expression of the wild type Tat and TatK28A/K50A mutant proteins were detected by immunoblotting using an anti-HIV-1 Tat rabbit polyclonal antibody (56). Actin levels are shown as a control for approximately equivalent protein loading (Fig. 1C).

We next tested whether HIV-1 Tat-coactivator interactions inhibit CREB-dependent transcription induced by BDNF receptor signaling. PC12 and human IMR-32 neuroblastoma cells were co-transfected as described and stimulated by treatment with BDNF (10 ng/ml; Sigma) for 8 h. As shown in Fig. 1, D and E, BDNF stimulation activates CREB-dependent transcription in PC12 and IMR-32 cells. HIV-1 Tat repressed BDNF-responsive CRE trans-activation in a dose-dependent manner in both cell types (Fig. 1, D and E). The TatK28A/K50A mutant, which is defective for p300/CBP and PCAF binding, did not significantly alter BDNF-responsive CRE transcriptional activation; nor did the GFP control (Fig. 1, D and E). HIV-1 Tat, TatK28A/K50A, and actin proteins were detected in transfected cells by immunoblotting (Fig. 1D, lower panels). These results suggest that Tat coactivator/acetyltransferase interactions interfere with neurotrophin receptor signaling and CREB-dependent transcription in neuronally derived cells.

HIV-1 Tat-HAT Domain Interactions Inhibit Histone Acetylation by Coactivators PCAF and p300—The HIV-1 Tat protein is acetylated on lysine residues Lys28/Lys50 by PCAF and on Lys50/Lys51 by p300 (Fig. 2A) (54–57). Acetylation on residues Lys50/Lys51 of Tat prevents binding to TAR-RNA in vitro (55, 56), and acetylation on Lys28 by PCAF enhances Tat-cylin T1-Cdk9 complex formation (56, 57). Since we have previously demonstrated that Tat-HAT domain interactions prevent coactivator-mediated acetylation of the tumor suppressor p53 (61), we investigated whether Tat-coactivator binding might inhibit histone acetylation by p300 and PCAF in radioactive acetylation assays using [14C]acetyl-coenzyme A (acetyl-CoA). Full-length recombinant GST-Tat or the coactivator binding-defective mutant, GST-TatK28A/K50A, were preincubated with purified HAT-active PCAF351–832 and p3001195–1707 (Upstate Biotechnology) in the presence of [14C]acetyl-coA prior to the addition of recombinant core histones H2A, H2B, H3, and H4 (Upstate Biotechnology). In certain experiments, Tat-derived synthetic peptides, spanning the PCAF-interacting domain (Tat23–43) or p300-interacting domain (Tat41–61) or peptides that contain alanine substitution mutations (TatK/A23–43 and TatKK/AA41–61; Biosynthesis) that abolish coactivator binding were used instead of full-length Tat protein (56, 61). As shown in Fig. 2B, increasing amounts of GST-Tat (25, 50, and 100 ng) inhibited PCAF351–832-mediated acetylation of histones H3/H4 in a dose-dependent manner. The GST-TatK28A/K50A mutant protein did not significantly affect H3/H4 acetylation by PCAF (Fig. 2B). Similarly, the purified GST-Tat protein prevented histone H3/H4 acetylation by recombinant p3001195–1707, whereas the GST-TatK28A/K50A mutant did not alter histone acetylation (Fig. 2B). A synthetic peptide, spanning the PCAF-binding domain of HIV-1 Tat, Tat23–43, markedly inhibited histone H3/H4-acetylation by PCAF351–832 (Fig. 2C). The Tat41–61 peptide, which contains lysine Lys50, which is acetylated by PCAF (54), also inhibited histone H3/H4 acetylation by recombinant PCAF351–832 (Fig. 2C). The alanine substitution mutant peptides, TatK/A23–43 and TatKK/AA41–61, did not significantly influence histone acetylation (Fig. 2C). Histone H3/H4-acetylation by p3001195–1707 HAT activity was also inhibited by both Tat23–43 and Tat41–61 synthetic peptides but not by the mutant TatK/A23–43 and TatKK/AA41–61 peptides (Fig. 2D). The fact that the Tat23–43 synthetic peptide inhibits p3001195–1707-mediated histone acetylation is interesting, since this peptide contains no known acetylation sites for the p300 HAT. It is possible that p300 also binds to residues in the vicinity of residue Lys28 of Tat, especially since PCAF is known to acetylate Lys50, which interacts with p300 (54–57). Intriguingly, under these assay conditions, we only observed acetylation of Tat23–43 in experiments using PCAF351–832 (Fig. 2E); no acetylation of the GST-Tat protein by either PCAF351–832 or p3001195–1707 was observed, and it is likely that efficient Tat acetylation requires interactions with full-length PCAF or p300. Acetylation of histones H2A/H2B by PCAF351–832 was also inhibited by the Tat23–43 peptide (Fig. 2E), although H2A/H2B acetylation was generally weak (these histones are poor substrates for acetylation) and was not observed in all experiments. Collectively, these results suggest that interaction between HIV-1 Tat and the catalytic HAT domains of transcriptional coactivators, p300 and PCAF, inhibits histone acetylation, consistent with the ability of Tat to repress neurotrophin-responsive CRE transactivation in neuronally derived cells.



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  		FIG. 2.
HIV-1 Tat interacts with the acetyltransferase domains of PCAF and p300 and inhibits histone acetylation. A, amino acid sequences of wild type TatExon1 and synthetic Tat-derived peptides, Tat23–43 and Tat41–61, used in radioactive HAT assays. The positions of coactivator binding-defective mutations K28A and K50A/K51A are indicated (56). B, GST-HIV-1 Tat (25, 50, and 100 ng) inhibits PCAF351–832 and p3001195–1707-mediated histone H3/H4 acetylation in vitro. Relative input levels of GST-HIV-1 Tat and GST-HIV-1 TatK28A/K50A detected by immunoblotting using a goat polyclonal anti-GST antibody (Amersham Biosciences) are shown at top. The synthetic peptides, Tat23–43 and Tat41–61 (50, 100, and 200 ng), inhibit PCAF351–832 (C) and p3001195–1707 (D) HAT activities. E, weak acetylation of the Tat23–43 peptide by PCAF351–832 was observed in certain experiments.

 
HIV-1 Tat and Adenoviral E1A 12S Similarly Inhibit NGF-responsive CRE Trans-activation—The adenoviral E1A 12S protein has been demonstrated to bind the HAT domain of p300/CBP and directly inhibit coactivator-mediated histone acetylation and repress transcriptional activation (61, 80, 81). Further, the adenoviral E1A 12S protein has been shown to interact with PCAF, preventing its association with p300 (82). To determine whether the kinetics of HIV-1 Tat- and adenoviral E1A 12S-mediated transcriptional repression are similar in neuronally derived cells, PC12 cells were co-transfected with a synthetic CRE-containing promoter-luciferase reporter plasmid, RSV-HIV-1 Tat, RSV-HIV-1 TatK28A/K50A, CMV-E1A 12S, or CMV-E1A 12S{Delta}N, an adenoviral E1A 12S mutant deleted for amino-terminal residues essential for coactivator binding (83). Following transfection, cells were stimulated by treatment with NGF (10 ng/ml) for 8 h, and luciferase activities were measured using equivalent amounts of total cellular proteins. Results in Fig. 3A demonstrate that expression of the adenoviral E1A 12S protein significantly repressed NGF-responsive transcriptional activation through CREB in a dose-dependent manner. The E1A 12S{Delta}N coactivator binding-defective mutant, however, did not alter NGF receptor signaling/transcription in PC12 cells (Fig. 3A). Expression of HIV-1 Tat, but not HIV-1 TatK28A/K50A, inhibited NGF-responsive CRE trans-activation to a similar extent as observed for the adenoviral E1A 12S factor (Fig. 3A), suggesting that both HIV-1 Tat and E1A 12S proteins interfere with coactivator functions to inhibit cellular signaling/transcription pathways.



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  		FIG. 3.
HIV-1 Tat and adenoviral E1A 12S proteins inhibit NGF-responsive CRE trans-activation to similar degrees. A, IMR-32 human neuroblastoma cells were co-transfected with a CRE-promoter-luciferase reporter construct and either RSV-HIV-1 Tat, RSV-HIV-1 TatK28A/K50A, CMV-E1A 12S, or CMV-E1A 12S{Delta}N (0.5, 1.0, and 1.5 µg) and were stimulated with NGF (10 ng/ml) for 8 h. Relative luciferase activities were determined using equivalent amounts of total cellular proteins. B, PC12 cells were co-transfected as in A with a CRE promoter-luciferase reporter construct and either a constant amount (0.5 µg) of RSV-HIV-1 Tat in the presence of increasing CMV-E1A 12S (0.5 and 1.5 µg) or constant CMV-E1A 12S (0.5 µg) and increasing RSV-HIV-1 Tat (0.5 and 1.5 µg). Error bars represent S.D. values (n = 2).

 
To determine whether HIV-1 Tat and adenoviral E1A 12S proteins affect identical cellular targets to repress neurotrophin-induced CRE-trans-activation, we next co-transfected PC12 cells with a CRE-containing promoter-luciferase reporter construct and a constant amount of RSV-HIV-1 Tat (0.5 µg) in the presence of increasing CMV-E1A 12S plasmid (0.5, 1.5 µg) or a constant amount of CMV-E1A 12S in the presence of increasing RSV-HIV-1 Tat. Transfected PC12 cultures were stimulated with NGF (10 ng/ml) for 8 h, and relative luciferase activities were determined as in Fig. 3A. Nerve growth factor stimulation induced ~51-fold transcriptional activation from the CRE-luc reporter construct (Fig. 3B). Both HIV-1 Tat and E1A 12S repressed NGF-responsive CREB-dependent transcription at similar levels when transfected alone at 0.5 µg (Fig. 3B). Surprisingly, co-transfection of constant RSV-HIV-1 Tat with increasing CMV-E1A 12S did not result in significantly greater transcriptional repression, and, indeed, co-transfection of constant CMV-E1A 12S in the presence of increasing RSV-HIV-1 Tat produced an antagonistic effect that countered Tat-mediated transcriptional repression (Fig. 3B). There are several possible complicated explanations for these results. HIV-1 Tat or E1A 12S could influence the other's transcriptional interactions, either directly or indirectly, by affecting cellular signaling pathways or by influencing various protein-binding partners. Thus, it is likely that HIV-1 Tat and adenoviral E1A 12S repress neurotrophin-responsive transcription through multiple and/or distinct interactions with cellular factors.

Neuronal Repression of CRE Trans-activation by HIV-1 Tat Is Dependent on PCAF—In order to determine whether the repressive effects of HIV-1 Tat upon NGF-responsive transcriptional activation through CREB are due to inhibition of PCAF and/or p300 HATs, PC12 cells were co-transfected as described with a CRE-promoter luciferase plasmid and 1.5 µg of RSV-HIV-1 Tat in the presence of increasing amounts of CMV-PCAF or CMV-p300 expression constructs (1.5, 2.5, and 5.0 µg). The transfected cultures were stimulated with NGF (10 ng/ml) for 8 h, and the percentage of restored neurotrophin-responsive CRE transcriptional activity was quantified using equivalent amounts of total cellular proteins. Interestingly, as shown in Fig. 4, overexpression of p300 restored ~10% of NGF-responsive CRE trans-activation at the highest concentration tested (5.0 µg), and p300 slightly countered the inhibitory effects of Tat. Ectopic PCAF overexpression, however, restored 28% of CRE transcriptional activation at 2.5 µg and restored nearly 35% of NGF-responsive CRE trans-activation at the highest concentration tested (5.0 µg), indicative that HIV-1 Tat primarily inhibits PCAF coactivator/acetyltransferase functions to repress neurotrophin receptor signaling/transcription in neuronally derived cells (61).



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  		FIG. 4.
Repression of NGF-responsive CRE trans-activation by HIV-1 Tat is dependent upon PCAF. PC12 cells were co-transfected with a CRE promoter-luciferase reporter plasmid, RSV-HIV-1 Tat (1.5 µg), and increasing amounts (1.5, 2.5, and 5.0 µg) of CMV-PCAF or CMV-p300 expression constructs prior to NGF stimulation for 8 h. Relative luciferase activities were measured using equivalent amounts of total cellular proteins. Error bars represent S.D. values (n = 2).

 
HIV-1 Tat Interacts with PCAF Complexes in CNS Tissues from Neuro-AIDS Patients—In order to determine whether Tat-PCAF interactions may contribute to neurotrophin signaling impairments during HIV-1 neuropathogenesis, we performed FRET immunoconfocal microscopy to analyze Tat-PCAF protein interactions in formalin-fixed post-mortem CNS tissue sections (cingulum; thalamus) from four donor HIV-1-infected neuro-AIDS patients clinically diagnosed with primary encephalopathies. A post-mortem CNS tissue sample from an uninfected individual was also analyzed as a negative control for antibody staining. FRET imaging is a widely accepted sensitive method to quantitatively analyze protein-protein interactions when sample amounts are limiting (as for CNS biopsy material). The CNS tissue sections were fixed and permeabilized, and nonspecific antigens were blocked prior to immunostaining. PCAF was detected using an anti-PCAF goat polyclonal antibody (Santa Cruz Biotechnology) and a weakly emitting FITC-conjugated anti-goat secondary antibody (FRET donor; Jackson Immunoresearch Laboratories). The Tat protein FRET signal was detected using an anti-HIV-1 Tat rabbit polyclonal antibody (Advanced Bioscience Laboratories) and a Cy3-conjugated anti-rabbit secondary antibody (FRET acceptor; Jackson Immunoresearch Laboratories). The PCAF transcriptional coactivator was detected in uninfected control as well as in HIV-1-infected CNS tissues; no significant HIV-1 Tat-specific FRET signal was observed in the uninfected control, and the background was adjusted to this level (Fig. 5A). In HIV-1-infected CNS tissues, numerous HIV-1 Tat-expressing cells were detected that produced a strong Cy3-FRET-signal as a result of the close proximity between PCAF and HIV-1 Tat proteins (Fig. 5A). It is important to note that, in HIV-1-infected CNS tissue sections, PCAF-Tat protein complexes were visualized in the cytoplasm as well as in nuclei (Fig. 5B). Therefore, the subcellular distribution of PCAF may be regulated by signaling in various cell types of the CNS. The FRET signal derived from HIV-1 Tat-PCAF interactions was observed through progressive Z-stack focal planes of a single HIV-1-infected cell, and the most intense FRET signal was observed in sections representing the central region of the nucleus (Fig. 5C). These results suggest that HIV-1 Tat and the PCAF coactivator interact in infected CNS tissues from neuro-AIDS patients, and Tat-PCAF complexes may contribute to neurotrophin signaling impairments associated with cognitive deficiencies during AIDS-related dementia.



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  		FIG. 5.
FRET immunofluorescence-confocal microscopy analyses of HIV-1 Tat-PCAF interactions in post-mortem CNS tissues of neuro-AIDS patients. Wide field FRET immunofluorescence-laser confocal microscopy analyses of normal control brain tissue (A, top panel) and a representative HIV-1-infected CNS tissue section from a donor neuro-AIDS patient 11406 (A, bottom panel). B, FRET immunoconfocal microscopy analyses of HIV-1 Tat-PCAF interactions in post mortem CNS tissues from three HIV-1-infected patients (11406, 7674, and 13689) clinically diagnosed with primary encephalopathies. C, three-dimensional FRET analyses of HIV-1 Tat-PCAF interactions in different focal planes of an HIV-1-infected cell in CNS tissue from patient 11406. Phase contrast is shown in merged FRET images for reference.

 
Recombinant GST-HIV-1 Tat, but Not GST-TatK28A/K50A, Represses NGF-induced Expression of Endogenous Bcl-2—Exogenous recombinant HIV-1 Tat protein has been shown to become endocytosed (41) and enter nuclei of PC12 cells (84). Therefore, in order to determine whether HIV-1 Tat coactivator (PCAF; p300) interactions influence NGF-induction of CREB-dependent gene expression in neuronal cells, we investigated the effects of purified recombinant GST-HIV-1 Tat or GST-TatK28A/K50A mutant proteins upon NGF-responsive induction of endogenous Bcl-2 expression. Riccio et al. (74) have demonstrated that neurotrophin signaling increases Bcl-2 protein levels in synaptic neurons, which are significantly dependent upon activation of CREB and correlate with induction of neuronal survival pathways. Results in Fig. 6A demonstrate that purified recombinant GST-HIV-1 Tat and GST-TatK28A/K50A proteins efficiently entered nuclei of PC12 cultures following a 3-h preincubation period, as determined by immunofluorescence microscopy using a goat polyclonal anti-GST antibody. The GST protein control was not internalized by PC12 cells (Fig. 6A). PC12 cultures were then stimulated by treatment with NGF (10 ng/ml) for 12 h, and immunoblotting was performed to determine the effects of HIV-1 Tat-coactivator interactions upon neurotrophin-responsive induction of endogenous Bcl-2 (Fig. 6B). Actin expression was measured as a control for comparable protein loading (Fig. 6B, lower panel). Nerve growth factor stimulation induced Bcl-2 expression in PC12 cells (Fig. 6B, top panel). Preincubation with increasing amounts of recombinant GST-HIV-1 Tat significantly inhibited NGF-responsive induction of Bcl-2 in a dose-dependent manner (Fig. 6B). Importantly, the GST-TatK28A/K50A mutant protein, which is defective for binding PCAF and p300 transcriptional coactivators (56, 61), did not affect NGF induction of endogenous Bcl-2 (Fig. 6B), and the GST control also had no effect upon Bcl-2 expression (Fig. 6B). These results suggest that HIV-1 Tat-coactivator interactions inhibit NGF induction of CREB-dependent neuroprotective factors, such as Bcl-2, and could contribute to neurotrophin signaling impairments and neuronal apoptosis in HIV-1-infected neuro-AIDS patients with primary encephalopathies. Recently, Bergonzini et al. (85) reported that HIV-1 Tat inhibits neuronal differentiation and NGF-responsive Id1 expression in stimulated PC12 cells. Our results complement these findings and allude to a molecular mechanism for inhibition of neurotrophin-mediated transcription as a result of HIV-1 Tat-coactivator interactions.



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  		FIG. 6.
HIV-1 Tat inhibits NGF protection against serum deprivation-associated neuronal apoptosis. A, PC12 cells were preincubated with purified recombinant GST control, GST-HIV-1 Tat, or GST-TatK28A/K50A proteins (33.3 ng/ml) prior to stimulation with NGF (10 ng/ml) for 12 h. Immunofluorescence microscopy was performed using a goat polyclonal anti-GST antibody (Amersham Biosciences) to detect GST-HIV-1 Tat fusion proteins. Phase contrast and 4',6-diamidino-2-phenylindole nuclear stained images are shown for reference. B, PC12 cells (2 x 105) were preincubated with increasing amounts (50 and 100 ng) of purified recombinant GST control, GST-HIV-1 Tat, or GST-TatK28A/K50A proteins for 3 h prior to stimulation with NGF (10 ng/ml) in collagen-coated 6-well tissue culture plates as in A. Whole cell extracts were prepared by freeze-thawing, and immunoblotting was performed to detect endogenous Bcl-2 and actin proteins. C, PC12 cells were transfected with RSV-HIV-1 Tat, RSV-HIV-1 TatK28A/K50A, or a C{beta}S empty vector control (1.5 µg) (56); following transfection, the cultures were serum-starved for 48 h either in the absence or presence of NGF stimulation (10 ng/ml). The percentages of apoptotic cells in each sample were measured by annexin-V-FITC staining (BD Biosciences) and flow cytometric analyses. D, PC12 cells were transfected as in C with RSV-HIV-1 TatK28A or RSV-HIV-1 TatK50A (1.5 µg), and cultures were serum-starved in the presence of NGF stimulation. The numbers 1 and 2 indicate that cells were either immediately stimulated with NGF (1) or were treated with NGF (2) following transfection.

 
HIV-1 Tat-Coactivator Interactions Inhibit NGF Protection against Neuronal Apoptosis—Neurotrophin signaling plays an essential role in protecting neurons against apoptosis induced by inflammatory factors (e.g. tumor necrosis factor-{alpha}, interleukin-1{beta}, NO) through CREB-dependent transcriptional activation of antiapoptotic genes, such as bcl-2 (72–75). To determine whether HIV-1 Tat-coactivator interactions might inhibit NGF-mediated protection against neuronal cell death, PC12 cells were transfected with RSV-HIV-1 Tat, RSV-TatK28A, RSV-TatK50A, RSV-TatK28A/K50A, or a C{beta}S empty vector control. The cultures were either immediately treated with NGF (10 ng/ml) or were stimulated with NGF following transfection. Then the cells were washed twice with serum-free medium and cultured for 48 h in the absence of serum to observe the influence of HIV-1 Tat-coactivator interactions upon NGF-mediated protection against serum deprivation-associated apoptosis. Cells were harvested, and percentages of apoptotic cells were determined by staining with annexin-V-FITC (BD Biosciences) and quantified by flow cytometric analyses using a FACSCaliber instrument (BD Biosciences). Serum deprivation induced significant apoptosis in C{beta}S vector-transfected untreated cells (53.86 and 50.82% annexin-V-positive), whereas prestimulation with NGF markedly protected against serum deprivation-associated cell death (32.80 and 18.63% annexin-V-positive) (Fig. 6, C and D). Prestimulation of PC12 cells with NGF prior to expression of HIV-1 Tat or the TatK28A/K50A mutant resulted in significant protection against apoptosis (wild type Tat: 34.80%; TatK28A/K50A: 30.03% annexin-V-positive) (Fig. 6C). However, expression of wild type HIV-1 Tat inhibited NGF-protective effects against serum deprivation-associated apoptosis (51.15% annexin-V-positive) (Fig. 6C). The coactivator binding-defective mutant, TatK28A/K50A, did not affect NGF protection against cell death when expressed prior to NGF stimulation (25.53% annexin-V-positive) (Fig. 6C). We next examined two HIV-1 Tat mutants, TatK28A and TatK50A, containing single amino acid substitutions for lysine residues Lys28 and Lys50 acetylated by PCAF or p300 (54–57). As shown in Fig. 6D, expression of either TatK28A or TatK50A prior to NGF stimulation did not significantly affect NGF-protective effects against neuronal apoptosis. These results are interesting, since we had observed that HIV-1 Tat repressive effects upon NGF-responsive CRE trans-activation were principally mediated through the transcriptional coactivator PCAF (see Fig. 4). We had also found that the synthetic peptides Tat23–43 and Tat41–61 were both capable of inhibiting histone H3/H4-acetylation catalyzed by PCAF351–832 (see Fig. 2). Results demonstrating that mutation of either Lys28 or Lys50 abrogates the inhibitory influence of HIV-1 Tat upon NGF protection against apoptosis suggest that both sites are probably necessary for PCAF interactions with Tat in vivo (Fig. 6D).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
HIV-1 has infected more than 40 million individuals globally, causing more than 21 million deaths since the beginning of the AIDS pandemic (8, 86). Whereas sub-Saharan Africa represents the area most devastated by AIDS-related illnesses, the Joint United Nations Programme on HIV/AIDS has recognized the potential for future epidemics in highly populated China and India (87, 88). Thus, HIV-1 is likely to increasingly become an emerging global health problem as the incidence of AIDS-related illnesses and new infections continues to rise.

Neurotrophins are produced by neurons, macrophages, microglia, monocytes, and astrocytes in the CNS and are essential for neuronal differentiation, synapse formation, protection against apoptosis, and neuronal injury repair (63–65). NGF binds to the high affinity p140TrkA receptor on surfaces of target cells and activates phosphatidylinositol-3-kinase and mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 signaling cascades, convergent upon the nuclear Ca2+-dependent kinases, RSK2 and MSK-1/2 (64, 72–75, 89). NGF stimulation results in Ser133 phosphorylation of CREB and transcriptional activation from CRE-containing cellular promoters (73, 75). Phosphorylated CREB (Ser133-phospho-CREB) recruits the coactivators/acetyltransferases, p300/CBP and PCAF, to the promoters of neurotrophin-responsive genes to activate transcription (76, 77, 83, 90). p300/CBP and PCAF acetylate lysine residues within histones H2A, H2B, H3, and H4 (p300/CBP preferentially acetylate H4, whereas PCAF preferentially acetylates H3) to facilitate displacement of nucleosomes from genetic promoter sequences (91). The transcriptional coactivators, p300/CBP and PCAF, also interact with and recruit basal transcription factors, TFIIB and RNA polymerase II, to promote assembly of initiation complexes (92, 93).

The roles of neurotrophins and neurotrophin-receptor signaling, either as protective (antiapoptotic) factors or potentiators of viral replication, during HIV neuropathogenesis are not completely understood. Messenger RNAs coding for NGF and basic fibroblast growth factor are increased in brain tissues from AIDS-dementia patients, but these neurotrophic factors do not sufficiently protect against neuronal cell death associated with CNS HIV infections (94). BDNF receptor (p145TrkB) expression is also increased in post-mortem CNS tissues from neuro-AIDS patients (95). NGF functions as an autocrine/paracrine survival factor produced by HIV-1-infected macrophages and monocytes (69, 70). HIV-1-infected macrophages and monocytes produce significantly elevated levels of soluble NGF and express both high affinity (p140TrkA) and low affinity (p75NTR) NGF receptors on their surfaces (69). Indeed, a monoclonal antibody against NGF prevented HIV-1 replication and CD4+ T-cell loss in severe combined immunodeficient mice engrafted with human peripheral blood lymphocytes, supporting an essential role for NGF during HIV-1 pathogenesis resulting in immune suppression (70). Nerve growth factor protects HIV-1-infected macrophages and monocytes against apoptosis through NF-{kappa}B-dependent signaling/transcription pathways (71). Thus, whereas HIV-1-infected neuro-AIDS patients with primary encephalopathies exhibit neurotrophin-signaling/transcriptional impairments, resulting in pronounced neuronal apoptosis and cognitive defects, NGF signaling increases the survival and persistence of HIV-infected macrophages and monocytes in the CNS.

We have demonstrated that interactions between HIV-1 Tat and the transcriptional coactivators, p300/CBP and PCAF, inhibit neurotrophin-responsive CRE trans-activation and histone H3/H4 acetylation and block NGF protective effects against serum deprivation-associated neuronal apoptosis. We also observed that HIV-1 Tat-coactivator interactions significantly repress CREB-dependent induction of endogenous Bcl-2 in NGF-stimulated neuronal cells. Our results suggest that Tat-PCAF interactions, in particular, mediate the repressive effects of HIV-1 Tat upon neurotrophin-induced transcriptional activation through CREB. Tat-PCAF nuclear complexes were visualized in post-mortem CNS tissues from HIV-1-infected neuro-AIDS patients diagnosed with primary viral encephalopathies, using FRET immunofluorescence-confocal microscopy. Collectively, these findings indicate that HIV-1-infections in the CNS may perturb neurotrophin signaling and neuroprotective pathways by inhibiting p300 and PCAF transcriptional coactivator functions.


   FOOTNOTES
 
* This work was supported in part by the Department of Biological Sciences, Southern Methodist University. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ These authors contributed equally to this work. Back

|| Maître de Recherches from the Fonds National de la Recherche Scientifique (Belgium) and supported by the Fonds National de la Recherche Scientifique, Televie, Free University of Brussels (ARC), Internationale Brachet Stiftung, CGRI-INSERM cooperation, the Theyskens-Mineur Foundation, Region Wallone-Commission Europeenne FEDER, and the Agence Nationale de Recherches sur le SIDA (ANRS, France). Back

§§ To whom correspondence should be addressed. Tel.: 214-768-3864; Fax: 214-768-3955; E-mail: rharrod{at}mail.smu.edu.

1 The abbreviations used are: HIV-1, human immunodeficiency virus type-1; CRE, cyclic AMP-responsive element; CREB, cyclic AMP-responsive element-binding protein; p300/CBP, p300/CREB-binding protein; PCAF, p300/CBP-associated factor; NGF, nerve growth factor; BDNF, brain-derived neurotrophic factor; CNS, central nervous system; HAART, highly active antiretroviral therapy; TAR, Tat-activated region; PBS, phosphate-buffered saline; CMV, cytomegalovirus; FRET, fluorescence resonance energy transfer; FITC, fluorescein isothiocyanate; GST, glutathione S-transferase; HAT, histone acetyltransferase; RANTES, regulated on activation normal T cell expressed and secreted; GFP, green fluorescent protein. Back


   ACKNOWLEDGMENTS
 
We thank Carolyn K. Harrod for assistance in preparing the manuscript. We acknowledge Dr. G. Franchini (NCI, National Institutes of Health (NIH)) for generous support and Dr. J. Nacsa (NCI, NIH) for technical assistance.



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