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Originally published online as doi:10.1189/jlb.0306189 on July 14, 2006

Published online before print July 14, 2006
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(Journal of Leukocyte Biology. 2006;80:659-667.)
© 2006 by Society for Leukocyte Biology

Inhibition of HIV-1 replication by RNA interference of p53 expression

Eduardo Pauls, Jordi Senserrich, Bonaventura Clotet and Jose A. Esté1

Retrovirology Laboratory irsiCaixa, Hospital Universitari Germans Trias i Pujol, Universitat Autònoma de Barcelona, Spain

1 Correspondence: Fundació irsiCaixa, Laboratori de Retrovirologia, Hospital Universitari Germans Trias i Pujol, Ctra. Del Canyet s/n, Badalona, Barcelona 08916, Spain. E-mail: jaeste{at}irsicaixa.es


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ABSTRACT
 
p53 expression and activation have been associated to faster human immunodeficiency virus (HIV) disease progression, most probably by inducing CD4+ T cell death but also through its cooperative effect in the control of viral gene transcription by viral regulatory proteins. Here, we show that RNA interference of p53 in HIV-1 reporter (HeLa P4-R5 MAGI) and lymphoid (SupT1) cell lines blocked HIV-1 and Tat-induced transcription from the HIV-1 promoter and HIV-1 replication in acutely infected cells, suggesting a cooperative role of p53 in HIV-1 transcription. Contrary to SupT1 cells, which encode several mutations on the p53 DNA binding domain, death of HIV-1-induced syncytia was reduced in cocultures of HeLa P4-R5 MAGI with persistently infected HIV-1 cells. To our knowledge, this is the first demonstration of the effect of the loss of function of p53 in HIV-1 replication, which is independent on its classical DNA binding activity. Our results suggest two independent roles for p53 in HIV-1 infection: cooperation in HIV long-terminal repeat transcription and virus-induced cell death.

Key Words: transcription • apoptosis • transcription • shRNA • silencing


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INTRODUCTION
 
The replication of the human immunodeficiency virus type 1 (HIV-1) in lymphocytes correlates with the degree of activation of the infected cell. Multiple host factors are cause or consequence of HIV-1-induced cell activation, leading to increased virus replication and accelerated T cell death. Among them, the tumor-promoting protein p53 has been shown to be activated by HIV-1 infection [1 ]. The association of p53 and HIV-1 proteins has been described, suggesting a complex net of reciprocal interactions. Overexpression of mutant p53 appears to cooperate in HIV-1 long-terminal repeat (LTR) transcriptional activity [2 , 3 ]. p53 binds to a site within the binding region of the HIV-1 LTR [4 ] and contributes to the tumor necrosis factor-induced, LTR-dependent transcription [5 , 6 ]. In addition, the interaction of p53 with viral factors such as Nef [7 ] or its effect on HIV-1 reverse transcriptase (RT) function [8 , 9 ] has been postulated as alternative mechanisms of p53 involvement in HIV replication. More recently, p53 has been proposed as an essential determinant of HIV envelope-induced cell death [10 , 11 ]. Presentation of HIV-1 envelope to CD4+ CXC chemokine receptor 4 (CXCR4)+ cells induces a cascade of events leading to Bax-mediated/Bcl-2-inhibited loss of mitochondrial membrane potential, apoptosis-inducing factor release, cytochrome c release, caspase activation, nuclear activation, and cell death [11 ]. Phosphorylation of p53 on serine 15 and serine 46 precedes Bax up-regulation [10 , 12 ]. This molecular sequence of events has been studied in cocultures of HIV-1-persistently infected cells with CD4+ target cells; that is, the role of p53 activation in cell death has been shown in syncytia induced by HIV-1 envelope.

RNA interference (RNAi) has become a valid method to evaluate the role of viral and cellular genes in HIV infection [13 ]. We and others [14 15 16 17 ] have shown that RNAi may be used to silence the expression of viral and cellular genes required for HIV-1 infection (most recently reviewed in refs. [18 , 19 ]). In turn, RNAi has been successfully applied to evaluate the loss of function of p53 in cell culture [20 21 22 ]. Thus, RNAi supposes an alternative to overexpression experiments or double-negative approaches to clarify the role of p53 in HIV-1 infection. Here, we have constructed short hairpin RNA (shRNA)-expressing plasmids to generate stable, p53 down-regulated HeLa HIV reporter (HeLa P4-R5 MAGI) and SupT1 cells. We have found that stable expression of shRNA p53 and silencing of p53 led to a marked reduction in HIV-1 or Tat-induced transcription, which may be independent of the role of p53 in HIV-1 envelope-induced cell death.


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MATERIALS AND METHODS
 
Vector construction
Oligonucleotides encoding shRNAs directed against mRNA of p53, HIV-1 Rev, and green fluorescent protein (GFP) were designed based on sequences previously published [21 , 23 , 24 ] and purchased from Sigma-Aldrich (Madrid, Spain). A shRNA encoding for an irrelevant gene was designed and used as a control (CD40). shRNA antiluciferase-encoding oligonucleotide was provided by Clontech (Madrid, Spain). Oligonucleotides were annealed and cloned into a pSIREN-RetroQ retroviral vector (Clontech) downstream of the pU6 promoter, according to the manufacturer’s instructions. mRNA target sequences are summarized in Table 1 . pSIREN plasmid expressing a modified GFP (ZsGreen, Clontech) or puromycin resistance was used as indicated in different cases.


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  		Table 1. mRNA Target Sequences of shRNAs

Generation of cell lines stably expressing shRNAs
HeLa P4-R5 MAGI [AIDS Reagent Program, National Institutes of Health (NIH), Bethesda, MD] cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% heat-inactivated fetal calf serum (FCS; Innogenetics, Barcelona, Spain) and 1 µgr/ml puromycin (Sigma-Aldrich). Lymphoid SupT1 cells were cultured in RPMI supplemented with 10% FCS and selection antibiotics when required. To increase cell transfection, we packaged pSIREN vectors into retroviral particles pseudotyped with the envelope of the vesicular stomatitis virus (VSV)-G as described before [20 ]. Briefly, 293GP2 packaging cells (Clontech) were cotransfected with 10 µg pSIREN vector and 10 µg VSV-G by calcium phosphate precipitation. After 72 h, filtered and concentrated viral supernatants were used to infect HeLa P4-R5 MAGI and SupT1 cells. HeLa P4-R5 MAGI were cloned and selected depending on GFP expression. SupT1 cells were selected with 2 µg/ml puromycin for 3 weeks.

Cocultures between HIV-infected and uninfected cells
Target, uninfected HeLa P4-R5 MAGI or SupT1 cells were cultured in 96-well plates with uninfected or HIV-1 (NL4-3) persistently infected MOLT-4/CC chemokine receptor 5 (CCRS)+CXCR4+ cells for 24 h in DMEM or RPMI, respectively, containing 10% FCS in the presence or the absence of the indicated drugs. Experiments were performed at least in triplicate wells. Cocultures were evaluated for syncytia formation by contrast-phase microscopy and Hoechst 33342 nuclei staining.

Western blot
HeLa P4-R5 MAGI or SupT1 cells (2–5x106) were harvested from exponential growing cultures, washed with phosphate-buffered saline (PBS), and lysed in chilled hypotonic buffer (NaCl 150 mM, Triton X-100 1%, EDTA 1 mM, and dithiothreitol, 1 mM, in Tris 25 mM, pH 7.5, freshly supplemented with a cocktail of protease inhibitors, phenylmethylsulfonyl fluoride, 100 mM, and okadaic acid, 1 mM). After 10 min on ice, cell debris was removed by centrifugation, and the total cell homogenates were stored at –20°C until used. Samples were run using Nu-PAGE 4–12% gels (Invitrogen, Barcelona, Spain) and blotted onto Protan nitrocellulose membranes (Schleicher and Schuell BioScience, Dassel, Germany). The membranes were blocked with PBS-Tween 0.5% nonfat milk 5% (blocking solution) for 2 h at room temperature and incubated overnight with mouse monoclonal antibodies (mAb) against human p53 (Santa Cruz Biotechnology, CA, diluted 1/1000 in blocking solution) or actin (Santa Cruz Biotechnology, 1/2000 in blocking solution) at 4°C. After washing, the membranes were incubated with biotinylated mAb (Invitrogen, 1/2000 in blocking solution) for 1 h at room temperature, washed again, and incubated with streptavidin-horseradish peroxidase (HRP) mAb (Invitrogen, 1/2000 in blocking solution) for 1 h at room temperature and then revealed with ECL-Plus solution (Amersham Biosciences, Cerdanyola, Spain). In some cases, anti-mouse–HRP mAb (1/1000 in blocking solution) were used directly for 1 h at room temperature and revealed as described above.

Flow cytometry and proliferative assay
HeLa P4-R5 MAGI cells were treated with trypsin-free Versene 1:5000 (Invitrogen) and detached before antibody staining as described before [25 ]. Briefly, HeLa P4-R5 MAGI and SupT1-transduced cells were stained with the following mAb: CD4–peridinin chlorophyll complex protein and CXCR4–phycoerythrin (BD Biosciences, Madrid, Spain) for 20 min, washed twice in PBS, resuspended in PBS containing 1% formaldehyde, and analyzed in a FACSCalibur flow cytometer (BD Biosciences).

For cell proliferation assays, HeLa P4-R5 MAGI and SupT1 cells were seeded at a rate of 2 x 104 cells in 48-well plates, and cells were detached when required and counted with fluorescent bead counts (Citognos, Salamanca, Spain) during the indicated times.

Interferon-{alpha} (IFN-{alpha}) and inflammatory cytokine quantification
IFN-{alpha} in the supernatants of HeLa P4-R5 MAGI and SupT1 cells was measured with an IFN-{alpha} enzyme-linked immunosorbent assay kit (5–500 pg/ml; R&D Systems, Minneapolis, MN), according to the manufacturer’s instructions. Inflammatory cytokines were measured by flow cytometry by cytometric bead array (BD Biosciences).

Quantitative RT-polymerase chain reaction (qRT-PCR)
Total cellular RNA was purified from HeLa P4-R5 MAGI or SupT1 cells by a RNeasy mini kit (Qiagen, Valencia, CA), as recommended by the manufacturer. Relative levels of p53 and 2',5'-oligoadenylate synthetase (OAS1) mRNA were measured by two-step qRT-PCR and normalized to glyceraldehyde 3-phosphate dehydrogenase (GADPH) mRNA expression in an ABI PRISM 7000 (Applied Biosystems, Foster City, CA). Primers and DNA probes were purchased from Applied Biosystems (assay-on-demand).

Transfection
HeLa P4-R5 MAGI cells (1x106) were seeded in six-well plates the day before transfection. Indicated amounts of HIV-1 Tat protein expressing plasmid (pcTat) were transfected with Lipofectamine 2000 reagent (Invitrogen) following the manufacturer’s instructions. Twenty-four hours later, cells were detached, and a fraction of cells (1/10) was washed and lysed with 50 µl buffer containing 25 mM Tris-HCl, pH 6.8, and 0.05% IGEPAL (Sigma-Aldrich). Lysates were stored at –80°C until used in a ß-galactosidase (ß-gal) detection assay, qRT-PCR, or Western blot.

Viral stocks
HIV-1 X4 tropic strain NL4-3 was grown in SupT1 cells. Medium was recovered, aliquoted, and stored at –80°C until use. High titer of HIV envelope pseudotyped was produced in 293T cells by cotransfection of 9 µg HIV-1 NL4-3 expression plasmid lacking functional envelope (pNL4-3.Luc.R–E–, NIH AIDS Reagent Program) together with 9 µg Env gp160 expression plasmid pHenv using the CalPhos transfection system (Clontech). Supernatants containing virus were harvested 72 h post-transfection, filtered, and stored at –80°C until use. Cell-free viral stock was estimated using an enzyme-linked immunoassay for antigen HIV-p24 detection (INNOTESTTM HIV-antigen, Innogenetics).

HIV infection and replication
HeLa P4-R5 MAGI cells (5x103) were seeded in a 96-well plate and infected with 12 ng/ml X4 HIV-1 strain NL4-3. After 5 days of infection, cells were lysed with 30 µl lysis buffer (Tris-HCl, 0.05% IGEPAL) and kept frozen (–80°C) until levels of viral replication were determined by ß-gal assay [26 ].

SupT1 cells were seeded at a rate of 2 x 104 cells in a 96-well plate and infected with HIV-1 X4 tropic strain NL4-3. Cells were infected at a dose of 5000 pg/ml HIV-1 Gag p24 protein in the presence or absence of HIV inhibitors: 1 µg/ml RT inhibitor azidothymidine (AZT; Sigma-Aldrich) and 1 µg/ml CXCR4 antagonist AMD3100 [27 ]. After a 6-day infection, p24 antigen in the supernatant was measured as described before. Cells were fixed, permeabilized (Fix and Perm, Caltag, Burlingame, CA), and stained with KC57 anti-HIV-CA p24 antigen mAb (Coulter, Barcelona, Spain) and analyzed in a FACSCalibur flow cytometer (BD Biosciences).

ß-gal detection assay
ß-gal activity in 30 µl cell extracts was quantified by a colorimetric assay as described elsewhere [26 ]. Absorbance (450 nm) was divided by mock-transfected sample absorbance levels to obtain fold induction.

Single-cycle infection assays
SupT1 cells were seeded at a rate of 1 x 106 cells in a 24-well plate and infected with a replicative, deficient HIV-1 x4 tropic strain NL4-3, which encodes the luciferase gene instead of Env. Cells were inoculated with viral stocks (6000 pg/ml p24 of the HIVenv-HIV), and 72 h later, cells were washed twice in PBS, concentrated, and stored at –80°C for the luciferase assay. Luciferase activity was measured using the Bright-GloTM luciferase assay system (Promega, Hospitalet, Spain) on a plate-reading luminometer (Fluoroskan Ascent FL, Thermo, Waltham, MA).

Statistical analysis
Experimental data were analyzed with the Student’s t-test (parametric, two-tailed). Results are given as the mean ± SEM of at least three experiments.

GenBank accession number
The GenBank accession number for the p53 sequence of SupT1 cells is DQ263704.


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RESULTS
 
Retrovirus-mediated expression of shRNAs suppresses p53 expression in the HeLa P4-R5 MAGI cell line
RNAi technology is an alternative approach to overexpression experiments or double-mutant, negative constructions to study the role of cellular factors. Thus, we could study the effect of down-regulating p53 expression in cell lines commonly used to evaluate HIV replication. To silence p53 in a HeLa P4-R5 MAGI cell line, we cloned previously, widely used shRNA targeting p53 [21 ] and a control shRNA into a retroviral expression plasmid. Both constructions were packaged into VSV-pseudotyped retroviral particles and then used to transduce HeLa P4-R5 MAGI cells. Cells expressing shRNAs were cloned and selected on expression of the reporter GFP. Two clones expressing different levels of reporter gene were selected (HeLashp53 c1 and HeLashp53 c2) in addition to wild-type HeLa P4-R5 MAGI and cells expressing an irrelevant shRNA (HeLashCD40; Fig. 1A ). We selected two clones to prevent possible side-effects, depending on the level of expression of the vector. We confirmed p53 down-regulation by measuring relative levels of p53 mRNA by real-time PCR (Fig. 1B) . p53 protein was determined by Western blot (Fig. 1C) , showing low levels of p53 as compared with parental cells (HeLa wild-type) or cells expressing control shRNA. No significant differences were observed between both clones expressing shRNAs against p53, and p53 suppression was stable for more than 3 months. Our results confirm the specificity of the shRNA used to target p53 gene expression [28 , 29 ].


Figure 1
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  		Figure 1. Stable interference of p53 in HeLa P4-R5 MAGI cells, which were transduced with a retroviral vector expressing shRNAs against p53 and CD40. (A) Cells were cloned and selected by expression of the reporter fluorescent protein (GFP). Mean fluorescence intensity (MFI) values determined by flow cytometry are shown for each clone selected. (B) Level of p53 mRNA was measured by real-time qPCR and normalized to GADPH mRNA. (C) Western blot analysis of p53 expression. (D) Proliferation of HeLa cells [•, HeLashp53 c1; {diamondsuit}, HeLashp53 c2; {blacksquare}, HeLa wild-type (wt); and {blacktriangleup}, HeLashCD40].

Nevertheless, to discard nonspecific effects as a result of IFN production [30 ], HeLa P4-R5 MAGI cells expressing shRNAs were tested for IFN-{alpha}, inflammatory cytokines, and OAS1 expression, and no differences were noted (data not shown). Expression of shRNAp53 did not have an effect on cell proliferation (Fig. 1D) , CD4 receptor expression, or CXCR4 coreceptor expression as measured by flow cytometry (data not shown).

Generation of a p53-negative lymphoid cell line
SupT1 is a lymphoid cell line, which encodes several mutations on the DNA binding domain of p53 (see Table 2 ). These mutations have been described to abolish transcriptional activation of p53-dependent genes [31 ], so that it is unable to activate its classic, transcriptional targets, such as p21 or Bax. Following a similar strategy than with HeLa P4-R5 MAGI cells, we transduced retroviral vectors expressing previously described shRNAs targeting p53, the HIV-1 Rev protein [23 ], luciferase, and GFP [24 ] into SupT1 cells, and cells expressing the shRNAs were selected with puromycin for 3 weeks and then tested for p53 mRNA expression by qRT-PCR (Fig. 2A ) and protein expression by Western blot (Fig. 2B) . p53 suppression was stable for more than 3 months.


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  		Table 2. Mutation Sites in SupT1 p53


Figure 2
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  		Figure 2. Stable interference of p53 in lymphoid SupT1 cells. (A) Relative p53 mRNA levels determined by real-time qPCR and normalized to GADPH mRNA levels in the different SupT1 types. Mean ± SEM of two independent experiments is shown. (B) p53 levels determined by Western blot analysis. (C) Proliferation of SupT1 cells [{blacksquare}, SupT1 wt; {blacktriangleup}, SupT1shGFP; {diamondsuit}, SupT1sh luciferase (shLuc); {blacktriangledown}, SupT1shRev; and •, SupT1shp53].

Similar to HeLa cells, SupT1 expressing shRNAs were tested for IFN-{alpha}, inflammatory cytokines, and OAS1 expression. Cell growth (Fig. 2C) and CD4/CXCR4 expression (data not shown) were also evaluated.

Inhibition of p53 expression blocked HIV-1 replication and Tat-dependent transactivation of the HIV-1 LTR
HeLashp53 cells were acutely infected with HIV-1 strain NL4-3 and tested for virus replication and Tat-dependent transactivation of the HIV-1 LTR promoter. HeLashp53 cells showed lower replication levels than wild-type or HeLashCD40 control cells (Fig. 3A ). To determine if the observed effect was a result of a transcriptional event, a HIV-1 Tat expression plasmid (pcTat) was transfected into p53-negative cells. Tat-induced activation of HIV-1 promoter was reduced in HeLashp53 clones at low levels of pcTat transfection (Fig. 3B) as compared with wild-type or HeLashCD40 control cells. However, when higher levels of pcTat were transfected, only HeLashp53 c2 showed reduced transcription levels. These results are in agreement with the infection experiments, where both clones showed an inhibition of ß-gal activity, but the reduction in Tat transactivation was higher in Clone 2.


Figure 3
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  		Figure 3. Effects of p53 down-regulation in HIV-1 replication or HIV-1 Tat-induced transcription. (A) ß-gal fold induction after 5-day infection with HIV-1 NL4-3 strain. Mean ± SEM of three independent experiments is shown. (B) ß-gal fold induction in HeLa P4-R5 MAGI cells transfected with 0.5 µg or 2 µg HIV-1 Tat expression plasmid, pcTat, after 24 h. Mean ± SEM of three independent experiments is shown. (C) p53 expression estimation by Western blot in one representative experiment of pcTat transfection.

Also, HIV-1 Tat protein has been described recently to suppress RNAi [32 ]. Thus, we asked if Tat expression could recover the expression of p53 in p53-interfered cells. However, we did not observe any significant recovering of p53 expression in Tat-expressing cells by qRT-PCR (data not shown) or Western blot analysis (Fig. 3C) .

As observed in HeLa cells, interference of p53 expression in the lymphoid SupT1 cells blocked virus replication in acutely infected cells, as measured by HIV-1 p24 antigen production. SupT1shp53 cells showed lower HIV-1 replication levels than wild-type SupT1 cells, SupT1shGFP, or SupT1shLuc cells used as control (Fig. 4A ). The extent of inhibition of HIV-1 replication induced by silencing of p53 was not as potent as that observed when a shRNA targeting HIV-1 Rev was expressed, suggesting that p53 partially controls HIV-1 replication in lymphoid cells. Although SupT1 cells express a p53 protein that cannot transcribe its classical target genes, the results above indicate that mutant p53 has the same function as wild-type p53 in the regulation of HIV replication. This suggests that p53 exerts its effect on HIV-1 transactivation independently on its capacity to transcribe cellular genes.


Figure 4
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  		Figure 4. HIV-1 replication was blocked significantly in acutely infected SupT1shp53 cells. (A) Extracellular p24 antigen after 6-day infection of SupT1 cells with HIV-1 NL4-3. Mean ± SEM of three independent experiments is shown. (B) Replication of HIV-1 measured in a single-cycle assay as relative luciferase activity after 3-day infection of SupT1 cells. Mean ± SEM of three independent experiments is shown.

Lymphoid cells may be susceptible to HIV-1 envelope-induced, single-cell death [33 34 35 36 ] or p53 activation during HIV-1-mediated, cell-to-cell fusion [10 , 11 ]. To ensure that these events were not responsible for p24 depletion, we performed single-cycle infection assays in which these events are minimized. Then, HIV-NL4-3, carrying a luciferase reporter gene and pseudotyped with HIV-1 envelope (HIVenv-HIV), was used to infect SupT1 cells. Viral replication was measured as the luciferase production detected in a chemiluminescent assay (Fig. 3B) . Results confirmed that HIV-1 replication was inhibited in SupT1shp53 cells when compared with wild-type SupT1 or SupT1shGFP. Conversely, both shRNAs against HIV-1 Rev and luciferase had an inhibitory effect. As expected, AMD3100, an antagonist of CXCR4, and the HIV RT inhibitor AZT blocked HIVenv-HIV virus.

Death of HIV-1-induced syncytia was reduced in cocultures with persistently infected HIV-1 cells
HIV-1 envelope induces the formation of multinuclear cells, termed syncytia, engaging an apoptotic process in which p53 has been involved, being a model to study envelope-induced pathogenesis [10 , 11 ]. Thus, we asked what would be the role of p53 in syncytia formation and viability in our model. Lymphoid MOLT cells persistently infected with the HIV-1 strain NL4-3 (MOLT-NL4-3) were cocultured with different HeLa- or SupT1-derived cell types (in a ratio of 1:20 or 1:10, respectively). After 24 h of coculture, the size of syncytia was quantified as a measure of viability [37 ] by counting the number of nuclei per syncytium with Hoescht staining, in the case of HeLa cocultures, or measuring syncytial relative area from contrast-phase microscopy analysis in the case of SupT1 cocultures (Fig. 5 ). The pan-caspase inhibitor Z-VAD-fluoromethylketone (Z-VAD-fmk) allowed for the formation of larger syncytia in HeLa and SupT1 cocultures, that is, increased syncytium viability. We also observed that suppression of p53 increased the viability of syncytia as compared with control cells in HeLa cocultures. However, this was not observed when SupT1 were the target cells. Moreover, rapamycin, an inhibitor of the mammalian target of rapamycin-mediated p53 activation, only increased the size of syncytia of cocultures with HeLa cells but not of SupT1 syncytia. Mutations in p53 expressed in SupT1 cells may explain the differences between the two cell types, so SupT1 cells would be unable to respond through p53 to the apoptotic pathway induced by HIV-1 envelope.


Figure 5
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  		Figure 5. Inhibition of p53 increases syncytium viability. (A) The different types of HeLa P4-R5 MAGI cells were cocultured with persistently infected HIV-1 cells (MOLT-NL4-3) in a 20:1 ratio. After 24 h, syncytia were stained with the nuclear dye Hoechst 33342. Two representative conditions are shown. (B) Nuclei per syncytium determined by contrast-phase microscopy. Mean ± SEM of three independent experiments is shown. Z-VAD, Z-Val-Ala-Asp. (C) SupT1 cells were cocultured with MOLT-NL4-3 in a 10:1 ratio. After 24 h, syncytia in coculture were microphotographed. Four representative conditions are shown. (D) Syncytia size was quantified by analysis of contrast-phase photographs of the cocultures. Mean ± SEM of three independent experiments is given.


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DISCUSSION
 
p53 has been shown to interact with HIV-1 regulatory proteins Tat [2 , 38 ], Nef [39 ], and Vpr [40 ], strongly suggesting that p53 expression modulates HIV-1 infection and virus replication.

Several distinct roles have been proposed for p53 in HIV-1 replication and pathogenesis. Early evidence points to a regulation of HIV-1 transcription in infected cells, alone or in concert with nuclear factor-{kappa}ß or agents that promote LTR-dependent transcription. Most of this evidence comes from gain-of-function studies, in which overexpression of mutant p53 is correlated to increased transcription or viral replication. Nevertheless, wild-type p53 has been described as a cooperator [4 ] but also as a repressor [2 , 38 ] in HIV-1 transcription. We have performed our experiments in two cell lines, which are common references to study HIV infection, HeLa P4-R5 MAGI [42 ] and SupT1 cells [34 ]. Taking into account our results, we suggest that wild-type p53 expressed in HeLa cells cooperates in HIV-1 transcription, similarly to what we observed for mutant p53 in SupT1 cells. Probably, p53 assists the transcriptional machinery required by the LTR promoter [5 , 6 ] or alternatively, could bind directly to the LTR and induce its transcription [43 ].

We have also studied the role of p53 in apoptosis induced by HIV-1 envelope. Although the in vivo relevance of syncytia needs to be elucidated, HIV-1 envelope-induced death of syncytia has been used as a model to study the death of CD4+ lymphocytes after the interaction of HIV-1 envelope with cellular receptors [10 11 12 , 37 ]. Data derived from HeLa cocultures point to a partial requirement of p53 in the apoptosis of the resulting syncytia, as p53-interfered cells showed increased syncytium viability, and p53 appeared to be dispensable for this function in cocultures with SupT1 cells. These differences of behavior between the two cell types may be explained by the mutations in the DNA binding domain of SupT1 p53, which would be unable to exert its classical, transcriptional functions [31 ], including apoptosis induced by a HIV-1 envelope.

It has been shown that human papillomavirus (HPV) 16 E6 protein expression in HeLa cells disrupts the p53-mediated cellular response to DNA damage, suggesting a nonfunctional p53 in this cell type [44 ]. However, evidence that p53 contained in HeLa cells is functional has been reported [45 ]. Our results indicate that p53 expressed in HeLa cells remained functional as to have an effect on HIV replication when compared with cells in which p53 was blocked.

Our results show two different roles for p53 in HIV-1 infection: a cooperative effect with the transcriptional machinery to promote LTR transcription and a proapoptotic function in the death of envelope-induced syncytia. These data support the in vivo observations that p53 RNA levels and p53 activation correlate with HIV-1 progression in patients [1 , 41 ]. Although the relative importance of these seemingly independent mechanisms and how HIV-1 takes advantage of them still remain unknown, the RNAi-based strategy used herein argues in favor of a relevant role of p53 in HIV-1 infection. In view of the pleiotropic effects of modulating p53 expression, a certain degree of controversy has been established about the repressing or cooperative effect of p53 in HIV-1 transcription and replication. Our results help to resolve this controversy by establishing a formal demonstration that down-regulation of p53 hampers HIV-1 infection.

One intriguing question is if HIV-1 is capable of independently regulating the conflicting effects of p53 in HIV-1 infection, i.e., promoting HIV-1 replication but inducing cell death. The interaction of HIV-1 proteins with p53 has been suggested as a way to block apoptosis [39 , 46 ], but if p53 is required for HIV-1 transcription, the virus should develop strategies to block apoptotic functions of p53 without affecting its role in HIV-1 LTR transcription.


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ACKNOWLEDGEMENTS
 
This work was supported in part by the Spanish Ministerio de Educación y Ciencia, Fondo de Investigación Sanitaria (FIS), Fundació Marató de TV3, FIPSE and the European TRIoH Consortium (LSHB-CT-2003-503480). We thank the National Institutes of Health (AIDS Research and Reference Reagent Program) and the National Institute for Biological Standards and Control (AIDS Reagent Program) for reagents. E. P. and J. S. contributed equally to this manuscript.

Received March 13, 2006; revised May 2, 2006; accepted May 11, 2006.


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