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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Neurosci. Author manuscript; available in PMC May 14, 2013.
Published in final edited form as:
PMCID: PMC3508723
NIHMSID: NIHMS421767

HIV-1 Tat through its intact core and cysteine-rich domains inhibits Wnt/β-catenin signaling in astrocytes: Relevance to HIV neuropathogenesis

Abstract

Wnt/β-catenin is a neuroprotective pathway regulating cell fate commitment in the CNS and many vital functions of neurons and glia. Its dysregulation is linked to a number of neurodegenerative diseases. Wnt/β-catenin is also a repressor of HIV transcription in multiple cell types, including astrocytes, which are dysregulated in HIV-associated neurocognitive disorder. Given that HIV proteins can overcome host restriction factors and that perturbations of Wnt/β-catenin signaling can compromise astrocyte function, we evaluated the impact of HIV transactivator of transcription (Tat) on Wnt/β-catenin signaling in astrocytes. HIV clade B Tat, in primary progenitor-derived astrocytes (PDAs) and U87MG cells, inhibited Wnt/β-catenin signaling as demonstrated by its inhibition of active β-catenin, TOPflash reporter activity, and Axin-2 (a downstream target of Wnt/β-catenin signaling). Point mutations in either the core region (K41A) or the cysteine-rich region (C30G) of Tat abrogated its ability to inhibit β-catenin signaling. Clade C Tat, which lacks the dicysteine motif, did not alter β-catenin signaling, confirming that the dicysteine motif is critical for Tat inhibition of β-catenin signaling. Tat co-precipitated with TCF-4 (a transcription factor that partners with β-catenin) suggesting a physical interaction between these two proteins. Further, knock down of β-catenin or TCF-4 enhanced docking of Tat at the TAR region of the HIV LTR. These findings highlight a bidirectional interference between Tat and Wnt/β-catenin that negatively impacts their cognate target genes. The consequences of this interaction include alleviation of Wnt/β-catenin mediated suppression of HIV and possible astrocyte dysregulation contributing to HIV neuropathogenesis.

Introduction

HIV enters the brain during acute infection as infected leukocytes cross the blood-brain barrier and seed the CNS with virus (H. S. Fox, 2008; V. Valcour et al., 2012). Even with effective combined antiretroviral therapy (cART), it is estimated that ≥50% of HIV infected individuals experience some degree of HIV-associated neurocognitive disorder (HAND) (R. K. Heaton et al., 2010). Defining cellular and molecular mechanisms driving HIV-mediated neuropathogenesis are vital in understanding the basis for this co-morbidity and devising novel strategies targeting HIV in the CNS.

Astrocytes are infected by HIV and likely represent a significant CNS viral reservoir. HIV DNA is detected in astrocytes at a frequency that depends on their proximity to perivascular macrophages and severity of HAND (M. J. Churchill et al., 2009), though productive replication is restricted by multiple mechanisms (J. Li et al., 2002; C. L. Ong et al., 2005; J. Zhang et al., 2005). We identified the Wnt/β-catenin pathway as a potent repressor of HIV replication in astrocytes, specifically through the action of downstream effectors TCF-4 and β-catenin (D. Carroll-Anzinger et al., 2007; L. J. Henderson et al., 2012; S. D. Narasipura et al., 2012), and reported that inflammatory mediators such as IFNγ that down regulate Wnt/β-catenin signaling promote HIV productive replication in astrocytes (W. Li et al., 2011).

The HIV Transactivator of transcription (Tat) is vital for efficient transcription. Without Tat, HIV replication is repressed due to repressive chromatin architecture as well as a defect in transcription elongation. Tat induces chromatin remodeling at the HIV promoter and recruits a positive elongation complex (pTEFb) that phosphorylates RNA polymerase II, allowing for efficient transcription. Tat can be detected in the serum of HIV-infected individuals in nanogram ranges (M. O. Westendorp et al., 1995), despite the instability and relatively short half-life of Tat in culture (G. Passiatore et al., 2009). Local concentrations of Tat may be considerably higher, particularly in compartments such as the CNS where there is evidence for chronic, low-level HIV replication that drives inflammation and production of neurotoxic viral proteins (R. J. Pomerantz, 2003; F. Gonzalez-Scarano and J. Martin-Garcia, 2005).

HIV has evolved multiple mechanisms to evade host restriction factors in order to enhance viral release (J. L. Douglas et al., 2009), evade CD8+ T cell responses (A. D. Blagoveshchenskaya et al., 2002), prevent undesirable mutations (A. M. Sheehy et al., 2002), or inhibit interferon responses (N. Yan et al., 2010). We determined whether HIV employs a similar strategy to counteract inhibition by β-catenin/TCF-4. We focused on Tat because: 1) Tat enhances activity of GSK3β which would likely disrupt β-catenin signaling (S. B. Maggirwar et al., 1999; Z. Sui et al., 2006), 2) Tat is secreted and is internalized by non-infected cells (D. E. Helland et al., 1991; A. Marcuzzi et al., 1992; B. Ensoli et al., 1993; S. Debaisieux et al., 2011), and 3) Tat has a well-established role in promoting HIV neuropathogenesis by enhancing oxidative stress in target cells and inducing gliosis in astrocytes, contributing to neurodegeneration.

Materials and Methods

Cell culture

U87MG and U251MG astroglioma cell lines were obtained from the NIH AIDS Research and Reference Reagent Program (Frederick, MD) and the American Type Culture Collection (ATCC, Manassas, VA), respectively. They were propagated in Dulbecco’s modified eagle’s medium (DMEM, Gibco Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated fetal bovine serum (HI-FBS) (Sigma, St. Louis, MO) and 1% penicillin-streptomycin (Gibco Invitrogen) in 5% CO2 humidified atmosphere at 37°C. Progenitor-derived astrocytes (PDAs) were generated from neural progenitor cells as previously described (S. Lamba et al., 2009). Briefly, progenitor cells (provided by Dr. Eugene Major, NINDS, NIH, MD) were seeded on poly-D-lysine coated T-75 tissue culture flasks at 2×106 cells/flask and maintained in progenitor medium consisting of neurobasal media (Gibco Invitrogen) supplemented with 0.5% bovine albumin (Sigma), neurosurvival factor (NSF) (Lonza, Walkersville, MD), N2 components (Gibco Invitrogen), 25 ng/ml fibroblast growth factor (bFGF), 20 ng/ml epidermal growth factor (EGF) (R & D Systems, Minneapolis MN), 50 μg/ml gentamicin (Lonza, Walkersville, MD) and 2 mM L-glutamine (Gibco Invitrogen). To induce differentiation, progenitor medium was replaced with PDA medium containing DMEM supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine and 50 μg/ml gentamicin. Cultures were >90% positive for glial fibrillary acidic protein (GFAP) after 30 days of differentiation. Both adherent primary cells and cell lines were removed by treatment with 1mM EDTA for 5 min with gentle scraping or pipetting multiple times.

Flow cytometry

Intracellular staining of cells to detect β-catenin by flow cytometry was performed as previously described (W. Li et al., 2011), using an antibody that specifically detects active β-catenin (ABC) that is not phosphorylated at Ser37 and Thr41 (US Biological, Massachusetts, MA). Data were acquired in an LSR II flow cytometer (BD Biosciences) and analyzed with FlowJo software (Tree Star, Ashland, OR).

Plasmid transfections

Transfections were performed with indicated plasmids using TransIT-LT1 reagent as per manufacturer’s instruction (Mirus Bio LLC, Madison, WI). Cells were approximately 60–70% confluent at the time of transfections. The TOPflash construct (Millipore, Billerica, MA) to detect Wnt/β-catenin signaling contains multiple TCF/LEF binding sites tied to a luciferase reporter. Renilla construct is an internal control for transfection efficiency and contains cDNA encoding Renilla luciferase under the control of a CMV promoter for constitutive expression. pCDNA3.1 vector (Invitrogen) was included throughout to equalize DNA added per transfection condition. Tat expression constructs Tat101 (Addgene plasmid #14654), TatK41A (Addgene plasmid #14665) and TatC30G (Addgene plasmid #14656) were provided courtesy of Dr. Matija Peterlin. The following reagents were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: pSV2tat72 (Tat72) from Dr. Alan Frankel; and pCTatBL43.CS (wild-type clade C) from Dr. Udaykumar Ranga. The TatΔ49–59 has been previously described (W. Li et al., 2008) and was a kind gift from Dr. Avindra Nath (NINDS,NIH).

Dual luciferase reporter assay

24h after transfection, culture medium was removed, cells were gently washed with PBS once and 100 μl of passive lysis buffer was added and incubated at 37 °C for 10–12 min. Cells were lysed by pipetting up and down several times, spun at 5000 rpm for 4 min to remove debris and 10–20 μl was used to assay for luciferase activity using dual luciferase reporter assay (Promega) in a single injector luminometer. Total protein concentration was estimated using Pierce BCA protein assay kit (Thermo Scientific). Relative light units were normalized to μg/ml of protein or to co-transfected Renilla luciferase, as indicated. Graphs were plotted from data obtained as a mean of three independent experiments with standard deviation as error bars.

Protein immunoprecipitation and western blot

For standard western blot, PDA were treated with recombinant Tat (rTat; 10ng/ml) for 12 h, then lysed using standard RIPA buffer and prepared for western blot using antibody directed against active (hypo-phosphorylated) β-catenin (Sigma). For immunoprecipitation, PDA cultures were first crosslinked with 1.0% formaldehyde (Sigma) to preserve protein-protein interactions, then lysed with RIPA buffer. Lysates were then pre-cleared by incubation with magnetic A/G beads (Thermo Scientific) and a non-targeting mouse IgG (Cell Signaling) for 1h. Lysates were then incubated with magnetic A/G beads and with either IgG control or antibody against hemagglutinin (HA)-tag (Abcam) overnight at 4°C with rotation. Beads were washed extensively with Tris-buffered saline (TBS) supplemented with 2M urea, followed by elution with low pH buffer (pH 2.9; GE Healthcare). Additionally, to minimize non-specific interference from detection of antibody heavy chain in western blotting, HA-tag antibodies and their cognate control antibodies were crosslinked to magnetic A/G beads by incubation with 200mM triethanolamine (GE Healthcare) containing 50mM dimethyl pimelimidate dihydrochloride (DMP; Sigma) prior to immunoprecipitation. For western blotting, lysates or immunoprecipitated samples were separated on a 10% SDS-PAGE gel, transferred to a nitrocellulose membrane, blocked with Superblock (Thermo Scientific)containing 0.1% Tween 20 (T20) for 1 h, incubated with the indicated antibody overnight at 4°C (total β-catenin, Sigma, 1:5,000; TCF-4, Cell Signaling, 1:1,000) in superblock-0.1% T20. Membranes were washed extensively with Tris-buffered saline with Tween-20 (TBST) and incubated with secondary antibody conjugated to horseradish peroxidase (HRP; 1:50,000 in Superblock-0.1% Tween 20) for 45 min at RT. Membranes were again washed extensively in TBST and developed with SuperSignal West Femto maximum sensitivity substrate according to manufacturer’s instructions (Thermo Scientific). WB for GFAP was performed using Mouse monoclonal anti-GFAP antibody (Cell Signaling; Danvers, MA) and secondary HRP- linked anti-mouse antibody (Thermoscientific, Rockford, IL). GLT-1 WB used xxxx. Glutamine Synthetase WB used Rabbit anti-glutamine synthetase antibody (Thermoscientific) and secondary HRP- linked anti-Rabbit antibody (Cell Signaling).

Glutamate uptake assay

Glutamate uptake in PDAs was measured using a glutamate assay kit purchased from Biovision Inc (Milpitas, CA). Briefly, PDAs were treated with the GLT-1 inhibitor DL-TBOA (Tocris Bioscience, Bristol, UK) at 100μM or with vehicle (DMSO). At 1 hr post treatment, PDAs were spiked with glutamate at a final concentration of 1.2mM. At 10 min post glutamate addition, 10ml of supernatant was used to measure the amount of glutamate using a colorimetric and spectrophotometry at λ = 450 nm method.

Quantitative real-time PCR and RT-PCR

RNA was isolated from progenitor-derived astrocytes (PDA) using the RNeasy Mini kit (Qiagen, Germantown, MD). Subsequently, cDNA was synthesized using Quantitect reverse transcription kit (Qiagen). Real-time RT-PCR was performed using a Quantitect SYBR green PCR kit (Qiagen) in a 7500 Real Time PCR System (Applied Biosystems, Foster City, CA) using 7500 software v2.0.1. Melting curve analysis was performed to ensure the amplification of a single product. Primers used were: Axin2-F-5′-ACAACAGCATTGTCTCCAAGCAGC and Axin2-R-5′-GCGCCTGGTCAAACATGATGGAAT; and GAPDH-F-5′-CTTCAACGACCACTTTGT and GAPDH-R-5′-TGGTCCAGGGGTCTTACT. For chromatin immunoprecipitation, primers used were TAR1-F-5′-AGCTTTCTACAAGGGACTTTCCGC and TAR1-R-5′-ATTGAGGCTTAAGCAGTGGGTTCC. Fold change was calculated by relative quantification using the comparative CT method with GAPDH (RT-PCR) or non-targeting IgG (ChIP) as control. Relative quantification (RQ) = 2−ΔΔCt, where ΔCt=CtTarget − CtControl, and ΔΔCt=ΔCttreated − ΔCtuntreated.

Chromatin immunoprecipitation assay (ChIP)

ChIP assays were performed from U87MG astrocytoma cells using the Magna ChIP A/G kit (Millipore). Cells were seeded to 60–70% confluency, treated as described in the figure legends, and then processed for ChIP beginning with cross-linking proteins to DNA by 1.0% formaldehyde. Chromatin was sonicated six times for 10 s each, generating DNA fragments of about 500–1000 base pairs. The sonicated supernatants containing the DNA were diluted with ChIP dilution buffer (0.01% SDS, 1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris–HCl pH 8.1, and 167 mM NaCl) to a total volume of 500 μl + 2.5 μl protease inhibitor cocktail (PIC), and precleared by rotating for 1 h at 4 °C with magnetic protein A/G beads and isotype control antibody (Cell Signaling; Danvers, MA). The lysate was transferred to a fresh tube. Supernatant (5 μl) was reserved for input and then 5 μg of control IgG or anti-hemagglutinin tag (HA) were added to the reaction mixture with magnetic protein A/G beads. After extensive washes with low salt, high salt, LiCl and TE buffers, the immune complexes were treated with ChIP elution buffer (NaCl, SDS, TRIS hydrochloride and proteinase K) at 62°C for 2 hours, followed by 10 minutes at 95°C to reverse crosslinking. Afterwards, DNA was precipitated with ethanol solution and isolated by spin column purification. Purified DNA was then amplified by real-time PCR using primer pairs that flank the transcription initiation site of the HIV-1 LTR, as described above. Data were analyzed according to the comparative CT method and were normalized to IgG control and reported as fold change in binding relative to control siRNA.

Statistical analysis

Descriptive statistics and graphical analyses were used as appropriate. Group means were compared by ANOVA and post-hoc tests, when the data was distributed normally. When the data was not normally distributed, nonparametric analysis was performed. All tests assumed a two-sided significance level of 0.05.

Results

HIV-1 Tat diminishes β-catenin signaling

We previously demonstrated that astrocytes exhibit robust β-catenin signaling, which mediates inhibition of HIV transcription (D. Carroll-Anzinger et al., 2007; L. J. Henderson et al., 2012; W. Li et al., 2011; S. D. Narasipura et al., 2012). To determine whether HIV has evolved a microbial adaptation mechanism to counteract the effects of β-catenin/TCF-4 on its replication, we evaluated the impact of HIV Tat on β-catenin signaling. We monitored β-catenin dependent signaling using TOPflash construct which contains multiple TCF/LEF binding sites linked to minimal thymidine kinase (TK) promoter and luciferase gene. We performed these experiments in progenitor-derived astrocytes (PDAs) as well as astrocytic cell lines (U87MG, U251MG) to show that results are consistent between cell types and are not limited to fetal astrocytes. PDAs exhibit prototypical characteristics of astrocytes including expression of GFAP, glutamate transporter 1/EAAT2, glutamate synthase, and are capable of glutamate uptake in a standard glutamate uptake assay (Fig. 1a–d). Transfection with a Tat expression plasmid encoding full-length (101aa) clade B Tat reduced the activity of the β-catenin reporter TOPflash by 59% in PDA (Fig. 2a) and 41% in the astrocytoma cell line U87MG (Fig. 2b). Similarly, treatment with recombinant Tat protein (rTat; 86aa form) reduced TOPflash activity by 45% in PDA (Fig. 2a) and 60% in U87MG (Fig. 2b). To confirm that Tat reduces canonical Wnt signaling, PDA were treated with rTat for 1–48 h, then RNA was isolated to detect mRNA levels of Wnt target gene Axin-2. As shown in Fig. 2c, rTat significantly decreased axin2 mRNA at 6 and 12 hours post-treatment in PDAs. These results indicate that both endogenously expressed and exogenously added HIV Tat can down-regulate β-catenin-dependent signaling in primary progenitor-derived astrocytes and in an astrocytic cell line.

Fig 1
Prototypical characteristics of astrocytes exhibited by human progenitor-derived astrocytes (PDAs)
Fig 2
Tat inhibits β-catenin-dependent signaling in astrocytes

Astrocytes exhibit robust expression of the central mediator of this pathway, β-catenin (Fig. 3a). To determine the effect of Tat on β-catenin, we monitored active β-catenin level by flow cytometry in response to Tat treatment. Treatment with recombinant Tat (clade B, 86aa form) at 10ng/ml was sufficient to diminish active β-catenin by 20% in two astrocytoma cell lines, U87MG (Fig. 3d) and U251MG (Fig. 3e). Due to the difficulty of preparing primary astrocytes for flow cytometry, PDA β-catenin levels were evaluated by western blot. β-catenin levels were significantly reduced in Tat-treated PDA (Fig. 3b,c).

Fig 3
Tat diminishes active β-catenin in astrocytes

Tat effects on β-catenin signaling are independent of Tat exon 2 and its basic domain

Tat exists in multiple forms, including a full-length (101aa) form translated from two exons, a 72aa splice variant that is encoded by the first exon, and an 86aa form produced by certain laboratory passaged HIV strains. Tat length can vary depending on the HIV isolate (W. Li et al., 2009). Additionally, intracellular Tat is cleaved at the C-terminus by calpains (G. Passiatore et al., 2009). The functional domains of Tat are summarized in Fig. 4a. Within exon 1 (1–48aa) is the minimal HIV transactivation domain, which promotes Tat binding to the transactivation responsive (TAR) region to accelerate the rate of HIV transcription. Translocation of Tat into the nucleus is mediated by the nuclear localization signal (NLS) located within the basic domain (aa49–59). The second exon of Tat (aa73–101) contributes to optimal transactivation (W. Li et al., 2009) and may be involved in internalization (M. Ma and A. Nath, 1997), but its other functions are largely undefined. To identify domain(s) of Tat that are involved in down-regulation of β-catenin signaling, we transfected PDA with TOPflash reporter with or without an expression plasmid encoding the 72aa form of Tat. Tat (72aa) reduced β-catenin dependent signaling by approximately 67% in PDA (Fig. 4b), indicating that residues 73–101 are not involved in the ability of Tat to down-regulate β-catenin signaling. We next sought to determine the impact of Tat localization on its ability to down regulate β-catenin signaling. Evidence from several groups indicates that the basic region of Tat (aa48–59) is vital for nuclear import, and that mutation or deletion of arginine residues within this domain results in cytoplasmic localization of Tat (J. Hauber et al., 1989; M. J. Orsini and C. M. Debouck, 1996; L. W. Meredith et al., 2009). Transfection of PDA with a Tat construct deleted in the basic domain (Tat cDNA Δ48–56) reduced TOPflash activity by approximately 78% (Fig. 4c), indicating that the basic domain is not required for Tat-mediated inhibition of β-catenin signaling.

Fig 4
Tat effects on β-catenin are independent of exon 2 and the basic region

The ability of Tat to down-regulate β-catenin signaling is dependent on its intact core and cysteine-rich domains

We next sought to evaluate whether the core region (aa38–48) or cysteine-rich region (aa22–37) of Tat are involved in inhibition of β-catenin signaling. Introducing a point mutation into the core domain (K41A) or a mutation at Cys30 to glycine (C30G) abrogated the ability of Tat to down regulate β-catenin signaling in PDAs (Fig. 5a). The Tat C30G construct enhanced β-catenin activity by approximately 2-fold in PDAs (Fig. 5a). While Tat C30G also abrogated Tat inhibition of β-catenin signaling in U87MG, it did not increase TOPflash activity above baseline (Fig. 5b). The K41A Tat construct reduced TOPflash activity in U87MG by 16.6% (Fig. 5b) which, while statistically significant, is unlikely to be biologically relevant. These findings suggest that there are differences in domain requirements for Tat-mediated down regulation of β-catenin between cell lines and primary cells. Nonetheless, these data demonstrate that the intact core and cysteine-rich domains of Tat are important for its ability to inhibit β-catenin signaling in primary progenitor derived astrocytes.

Fig 5
Down-regulation of β-catenin signaling by Tat requires intact core and cysteine- rich domains

Tat from HIV clade C does not down-regulate β-catenin signaling

Clade B Tat contains a dicysteine motif (C30C31) at positions 30–31 that is absent in clade C (C30S31) but is highly conserved in non-C clades. We observed that a point mutation at position 30 abolished the ability of Tat to inhibit β-catenin signaling (Fig. 5). We next evaluated whether clade C Tat, which contains a cysteine residue at position 30 only, inhibits β-catenin signaling. Transfection of PDA with a Tat expression plasmid derived from clade C did not reduce β-catenin signaling (Fig. 6), suggesting that the dicysteine motif is important for inhibition of β-catenin signaling.

Fig 6
Clade C Tat does not inhibit TOPflash activity in astrocytes

Knock down of TCF-4 and β-catenin increase docking of Tat at the TAR region of the HIV LTR

We previously showed that knock down of TCF-4 or β-catenin increases the docking and processivity of RNA polymerase II (Pol II) on the HIV LTR in astrocytes (S. D. Narasipura et al., 2012). Tat-mediated transactivation of the LTR is required for efficient transcription by Pol II. Therefore, we evaluated the consequences of Tat/β-catenin/TCF-4 interaction on HIV transcription. We first established whether Tat/TCF-4/β-catenin directly associate with each other in vivo. As shown in Fig. 7a, Tat co-precipitated with TCF-4 but not β-catenin. To determine the impact of TCF-4/β-catenin on Tat binding to the TAR element, U87MG were knocked down for β-catenin or TCF-4 and transfected with LTR and Tat constructs, followed by chromatin immunoprecipitation to detect Tat. Knock down of β-catenin or TCF-4 increased binding of Tat at the TAR region by 2.3-fold and 3.5-fold, respectively (Fig. 7b). Collectively, these results indicate that TCF-4 associates with Tat and that TCF-4 and β-catenin inhibit the ability of Tat to transactivate the HIV LTR.

Fig 7
Knock down of β-catenin or TCF-4 enhances docking of Tat on TAR region of the HIV LTR

Discussion

We provide evidence here to show that HIV Tat inhibits Wnt/β-catenin signaling in astrocytes. Robust expression of Wnt/β-catenin is an important regulator of the extent of HIV productive replication in astrocytes (D. Carroll-Anzinger et al., 2007; W. Li et al., 2011; L. J. Henderson et al., 2012; S. D. Narasipura et al., 2012). Inflammatory mediators such as IFNγ enhance HIV productive replication in astrocytes by antagonizing β-catenin signaling through a stat-3 dependent mechanism (W. Li et al., 2011). Wnt/β-catenin is thus a host restriction factor for HIV in astrocytes. HIV has many mechanisms to overcome host restriction factors, such as the action of Vif on APOBEC3G (A. M. Sheehy et al., 2002) or Vpu on tetherin (J. L. Douglas et al., 2009). Tat inhibition of β-catenin signaling represents a mechanism for viral evasion of the suppressive effect of β-catenin signaling on HIV transcription. β-catenin/TCF-4 inhibit both basal and Tat-mediated transactivation of the HIV LTR (L. J. Henderson et al., 2012; S. D. Narasipura et al., 2012). We recently identified several TCF-4 binding sites on the HIV promoter (L. J. Henderson et al., 2012). We further demonstrated that β-catenin signaling inhibits HIV basal transcription by inducing the formation of a repressive complex composed of TCF-4, β-catenin and the nuclear matrix binding protein SMAR1 on the LTR at −143 nt from the +1 transcription initiation site. The complex pulls the HIV DNA into the nuclear matrix and render it inaccessible to transcription machinery (L. J. Henderson et al., 2012). Althoughβ-catenin/TCF-4 signaling also inhibits Tat-mediated transactivation of the HIV LTR, the mechanism does not involve the −143 site (L. J. Henderson et al., 2012). Rather, we show here that Tat binding to TCF-4 sequesters Tat away from the TAR element, a required association for accelerated rate of transcription. Likewise, Tat binding to TCF-4 sequesters TCF-4 away from its cognate target genes such as Axin-2. We were unable to demonstrate an association between Tat and β-catenin, which may indicate that Tat itself does not directly bind to β-catenin, but does not rule out that β-catenin through its association with TCF-4 may be part of this Tat binding complex.

Based on the data presented here and others recently published (W. Li et al., 2011; L. J. Henderson et al., 2012; S. D. Narasipura et al., 2012), we suggest a model of bi-directional inhibition in which β-catenin and TCF-4 repress basal and Tat transactivation of the HIV LTR through distinct mechanisms and are in turn antagonized by HIV Tat (Fig. 8). Under basal LTR activity (without significant Tat level), the TCF-4/β-catenin/SMAR1 complex is associated with the HIV LTR and transcription is low or silent. Low levels of Tat may be produced but are primarily retained in the cytoplasm by association with TCF-4. When β-catenin signaling is disrupted, for example by pro-inflammatory mediators (IFNγ) or any other signal that down regulates the β-catenin pathway, this complex is disrupted and LTR activity increases. If this spike in promoter activity is sufficient to allow for Tat concentrations to reach a threshold level, Tat will a) allow for efficient viral replication and spread of HIV virions in the CNS; and b) antagonize β-catenin signaling through mutual binding/inhibition with TCF-4 and enhanced degradation of β-catenin to maintain a permissive state for HIV replication. Therefore, Tat-mediated effects on the β-catenin pathway would not only increase HIV replication in infected cells, but could potentially cause dysregulation of uninfected cells and enhance susceptibility of bystanders to HIV infection as well.

Fig. 8
Model of Tat/β-catenin interaction in astrocytes

In support of our proposed model, the ability of Tat to down regulate β-catenin signaling is independent of its nuclear localization signal. TatΔ49–59, which lacks a nuclear localization sequence in the basic domain (R. Truant and B. R. Cullen, 1999) and is sequestered in the cytoplasm of transfected cells (W. Li et al., 2008), also reduced TOPflash activity in primary astrocytes even more effectively than wild-type Tat. These findings indicate that the nuclear localization of Tat is not required for its effect on β-catenin signaling and point to a mechanism of mutual inhibition in which both Tat and TCF-4 are sequestered in the cytoplasm away from their transcriptional co-factors leading to reduced expression of β-catenin (Axin-2) and HIV targets (Fig. 7). A mutation in the core domain (K41A) abrogated the ability of Tat to down-regulate β-catenin signaling. Further, mutation of Cys30 to glycine abrogated the ability of Tat to diminish TOPflash activity. This finding prompted us to examine the relevance of the cysteine-rich region of Tat (aa 22–37) in Tat-mediated inhibition of β-catenin signaling. This region is strongly associated with neurotoxicity (A. Nath et al., 1996; M. Mishra et al., 2008). Specifically, a dicysteine motif (C30C31) that is highly conserved in almost all HIV clades is involved in dysregulation and/or apoptosis of neurons (M. Mishra et al., 2008). An intact dicysteine motif is required for Tat-induced activation of the N-methyl-D-aspartate (NMDA) receptor that can lead to excitotoxicity of neurons (W. Li et al., 2008). Interestingly, the dicysteine motif is absent in clade C Tat, which contains a serine substitution at position 31 (C30S31). Correspondingly, clade C Tat has severely attenuated neurotoxicity in vitro (M. Mishra et al., 2008), though there is not yet strong evidence that points to decreased incidence of HIV-associated dementia or other neurocognitive disorders in regions where clade C predominates (A. A. Constantino et al., 2011; J. A. Joska et al., 2011). Clade C Tat did not reduce TOPflash activity in astrocytes, which indicates that the dicysteine motif is involved in inhibiting β-catenin signaling.

There are several possible alternative mechanisms of Tat interface with components of the β-catenin pathway leading to signaling inhibition. Tat binds to low density lipoprotein (LRP) (Y. Liu et al., 2000; E. A. Eugenin et al., 2007), a co-receptor for the Wnt/β-catenin pathway, and leads to its internalization which will then sequester it away from Wnt ligands that initiate β-catenin signaling. Tat also enhances the activity of GSK3β, albeit in neurons (S. B. Maggirwar et al., 1999; Z. Sui et al., 2006), which leads to the phosphorylation of β-catenin that tags it for proteosomal degradation. Indeed, given that levels of active β-catenin are reduced at 12 hours post-Tat treatment, it is likely that increased degradation of β-catenin due to phosphorylation by GSK3β is at least partly responsible for reduction in signaling.

Collectively, these findings highlight the β-catenin pathway as an important restriction factor for HIV replication in astrocytes and identify a mechanism of viral adaptation (Tat) to diminish β-catenin signaling in these cells. Investigating the interplay between HIV and the β-catenin pathway in the presence or absence of Tat will lead to greater understanding of the mechanisms that regulate HIV in astrocytes and the biologic consequences of perturbed β-catenin signaling that may contribute to the role of astrocytes in HIV-associated neuropathogenesis.

Acknowledgments

This work was supported by National Institutes of Health R01 NS060632 to LA, F31 NS071999 to LJH, and the Chicago Developmental Center for AIDS Research (D-CFAR, P30 AI 082151) supported by NIAID, NCI, NIMH, NIDA, NICHD, NHLBI, and NCCAM.

LITERATURE CITED

  • Blagoveshchenskaya AD, Thomas L, Feliciangeli SF, Hung CH, Thomas G. HIV-1 Nef downregulates MHC-I by a PACS-1-and PI3K-regulated ARF6 endocytic pathway. Cell. 2002;111:853–866. [PubMed]
  • Carroll-Anzinger D, Kumar A, Adarichev V, Kashanchi F, Al-Harthi L. Human immunodeficiency virus-restricted replication in astrocytes and the ability of gamma interferon to modulate this restriction are regulated by a downstream effector of the Wnt signaling pathway. J Virol. 2007;81:5864–5871. [PMC free article] [PubMed]
  • Churchill MJ, Wesselingh SL, Cowley D, Pardo CA, McArthur JC, Brew BJ, Gorry PR. Extensive astrocyte infection is prominent in human immunodeficiency virus-associated dementia. Ann Neurol. 2009;66:253–258. [PubMed]
  • Constantino AA, Huang Y, Zhang H, Wood C, Zheng JC. HIV-1 clade B and C isolates exhibit differential replication: relevance to macrophage-mediated neurotoxicity. Neurotox Res. 2011;20:277–288. [PMC free article] [PubMed]
  • Debaisieux S, Rayne F, Yezid H, Beaumelle B. The Insand outs of HIV-1 Tat. Traffic. 2011;13(3):355–363. [PubMed]
  • Douglas JL, Viswanathan K, McCarroll MN, Gustin JK, Fruh K, Moses AV. Vpu directs the degradation of the human immunodeficiency virus restriction factor BST-2/Tetherin via a {beta}TrCP-dependent mechanism. J Virol. 2009;83:7931–7947. [PMC free article] [PubMed]
  • Ensoli B, Buonaguro L, Barillari G, Fiorelli V, Gendelman R, Morgan RA, Wingfield P, Gallo RC. Release, uptake, and effects of extracellular human immunodeficiency virus type 1 Tat protein on cell growth and viral transactivation. J Virol. 1993;67:277–287. [PMC free article] [PubMed]
  • Eugenin EA, King JE, Nath A, Calderon TM, Zukin RS, Bennett MV, Berman JW. HIV-tat induces formation of an LRP-PSD-95-NMDAR-nNOS complex that promotes apoptosis in neurons and astrocytes. Proc Natl Acad Sci U S A. 2007;104:3438–3443. [PMC free article] [PubMed]
  • Fox HS. Virus-host interaction in the simian immunodeficiency virus-infected brain. J Neurovirol. 2008;14:286–291. [PMC free article] [PubMed]
  • Gonzalez-Scarano F, Martin-Garcia J. The neuropathogenesis of AIDS. Nat Rev Immunol. 2005;5:69–81. [PubMed]
  • Hauber J, Malim MH, Cullen BR. Mutational analysis of the conserved basic domain of human immunodeficiency virus tat protein. J Virol. 1989;63:1181–1187. [PMC free article] [PubMed]
  • Heaton RK, et al. HIV-associated neurocognitive disorders persist in the era of potent antiretroviral therapy: CHARTER Study. Neurology. 2010;75:2087–2096. [PMC free article] [PubMed]
  • Helland DE, Welles JL, Caputo A, Haseltine WA. Transcellular transactivation by the human immunodeficiency virus type 1 tat protein. J Virol. 1991;65:4547–4549. [PMC free article] [PubMed]
  • Henderson LJ, Narasipura SD, Adarichev V, Kashanchi F, Al-Harthi L. J Virol. 2012. Identification of novel TCF-4 binding sites on the HIV LTR which associate with TCF-4, b-catenin and SMAR1 to repress HIV transcription. In Press. [PMC free article] [PubMed]
  • Joska JA, Westgarth-Taylor J, Myer L, Hoare J, Thomas KG, Combrinck M, Paul RH, Stein DJ, Flisher AJ. Characterization of HIV-Associated Neurocognitive Disorders among individuals starting antiretroviral therapy in South Africa. AIDS Behav. 2011;15:1197–1203. [PubMed]
  • Lamba S, Ravichandran V, Major EO. Glial cell type-specific subcellular localization of 14-3-3 zeta: an implication for JCV tropism. Glia. 2009;57:971–977. [PubMed]
  • Li J, Liu Y, Park IW, He JJ. Expression of exogenous Sam68, the 68-kilodalton SRC-associated protein in mitosis, is able to alleviate impaired Rev function in astrocytes. J Virol. 2002;76:4526–4535. [PMC free article] [PubMed]
  • Li W, Li G, Steiner J, Nath A. Role of Tat protein in HIV neuropathogenesis. Neurotox Res. 2009;16:205–220. [PubMed]
  • Li W, Henderson LJ, Major EO, Al-Harthi L. IFN-gamma mediates enhancement of HIV replication in astrocytes by inducing an antagonist of the beta-catenin pathway (DKK1) in a STAT 3-dependent manner. J Immunol. 2011;186:6771–6778. [PMC free article] [PubMed]
  • Li W, Huang Y, Reid R, Steiner J, Malpica-Llanos T, Darden TA, Shankar SK, Mahadevan A, Satishchandra P, Nath A. NMDA receptor activation by HIV-Tat protein is clade dependent. J Neurosci. 2008;28:12190–12198. [PubMed]
  • Liu Y, Jones M, Hingtgen CM, Bu G, Laribee N, Tanzi RE, Moir RD, Nath A, He JJ. Uptake of HIV-1 tat protein mediated by low-density lipoprotein receptor-related protein disrupts the neuronal metabolic balance of the receptor ligands. Nat Med. 2000;6:1380–1387. [PubMed]
  • Ma M, Nath A. Molecular determinants for cellular uptake of Tat protein of human immunodeficiency virus type 1 in brain cells. J Virol. 1997;71:2495–2499. [PMC free article] [PubMed]
  • Maggirwar SB, Tong N, Ramirez S, Gelbard HA, Dewhurst S. HIV-1 Tat-mediated activation of glycogen synthase kinase-3beta contributes to Tat-mediated neurotoxicity. J Neurochem. 1999;73:578–586. [PubMed]
  • Marcuzzi A, Weinberger J, Weinberger OK. Transcellular activation of the human immunodeficiency virus type 1 long terminal repeat in cocultured lymphocytes. J Virol. 1992;66:4228–4232. [PMC free article] [PubMed]
  • Meredith LW, Sivakumaran H, Major L, Suhrbier A, Harrich D. Potent inhibition of HIV-1 replication by a Tat mutant. PLoS One. 2009;4:e7769. [PMC free article] [PubMed]
  • Mishra M, Vetrivel S, Siddappa NB, Ranga U, Seth P. Clade-specific differences in neurotoxicity of human immunodeficiency virus-1 B and C Tat of human neurons: significance of dicysteine C30C31 motif. Ann Neurol. 2008;63:366–376. [PubMed]
  • Narasipura SD, Henderson LJ, Fu S, Kashanchi F, Al-Harthi L. Role of {beta}-catenin and TCF/LEF family members in transcriptional activity of HIV in astrocytes. J Virol. 2012;86:1911–1921. [PMC free article] [PubMed]
  • Nath A, Psooy K, Martin C, Knudsen B, Magnuson DS, Haughey N, Geiger JD. Identification of a human immunodeficiency virus type 1 Tat epitope that is neuroexcitatory and neurotoxic. J Virol. 1996;70:1475–1480. [PMC free article] [PubMed]
  • Ong CL, Thorpe JC, Gorry PR, Bannwarth S, Jaworowski A, Howard JL, Chung S, Campbell S, Christensen HS, Clerzius G, Mouland AJ, Gatignol A, Purcell DF. Low TRBP levels support an innate human immunodeficiency virus type 1 resistance in astrocytes by enhancing the PKR antiviral response. J Virol. 2005;79:12763–12772. [PMC free article] [PubMed]
  • Orsini MJ, Debouck CM. Inhibition of human immunodeficiency virus type 1 and type 2 Tat function by transdominant Tat protein localized to both the nucleus and cytoplasm. J Virol. 1996;70:8055–8063. [PMC free article] [PubMed]
  • Passiatore G, Rom S, Eletto D, Peruzzi F. HIV-1 Tat C-terminus is cleaved by calpain 1: implication for Tat-mediated neurotoxicity. Biochim Biophys Acta. 2009;1793:378–387. [PMC free article] [PubMed]
  • Pomerantz RJ. Reservoirs, sanctuaries, and residual disease: the hiding spots of HIV-1. HIV Clin Trials. 2003;4:137–143. [PubMed]
  • Sheehy AM, Gaddis NC, Choi JD, Malim MH. Isolation of a human gene that inhibits HIV-1 infection and is suppressed by the viral Vif protein. Nature. 2002;418:646–650. [PubMed]
  • Sui Z, Sniderhan LF, Fan S, Kazmierczak K, Reisinger E, Kovacs AD, Potash MJ, Dewhurst S, Gelbard HA, Maggirwar SB. Human immunodeficiency virus-encoded Tat activates glycogen synthase kinase-3beta to antagonize nuclear factor-kappaB survival pathway in neurons. Eur J Neurosci. 2006;23:2623–2634. [PubMed]
  • Truant R, Cullen BR. The arginine-rich domains present in human immunodeficiency virus type 1 Tat and Rev function as direct import in beta-dependent nuclear localization signals. Mol Cell Biol. 1999;19:1210–1217. [PMC free article] [PubMed]
  • Valcour V, Chalermchai T, Sailasuta N, Marovich M, Lerdlum S, Suttichom D, Suwanwela NC, Jagodzinski L, Michael N, Spudich S, van Griensven F, de Souza M, Kim J, Ananworanich J. Central Nervous System Viral Invasion and Inflammation During Acute HIV Infection. J Infect Dis 2012 [PMC free article] [PubMed]
  • Westendorp MO, Frank R, Ochsenbauer C, Stricker K, Dhein J, Walczak H, Debatin KM, Krammer PH. Sensitization of T cells to CD95-mediated apoptosis by HIV-1 Tat and gp120. Nature. 1995;375:497–500. [PubMed]
  • Yan N, Regalado-Magdos AD, Stiggelbout B, Lee-Kirsch MA, Lieberman J. The cytosolic exonuclease TREX1 inhibits the innate immune response to human immunodeficiency virus type 1. Nat Immunol. 2010;11:1005–1013. [PMC free article] [PubMed]
  • Zhang J, Liu Y, Henao J, Rugeles MT, Li J, Chen T, He JJ. Requirement of an additional Sam68 domain for inhibition of human immunodeficiency virus type 1 replication by Sam68 dominant negative mutants lacking the nuclear localization signal. Gene. 2005;363:67–76. [PubMed]
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