Hyper-responsiveness to Stimulation of Human ImmunodeficiencyVirus-infected CD4� T Cells Requires Nef and Tat Virus GeneProducts and Results from Higher NFAT, NF-�B, andAP-1 Induction*

Received for publication, July 6, 2004Published, JBC Papers in Press, July 16, 2004, DOI 10.1074/jbc.M407477200

Jean-Francois Fortin‡§, Corinne Barat¶§, Yannick Beausejour¶, Benoit Barbeau¶�,and Michel J. Tremblay¶**

From the ‡Baxter Laboratory for Genetic Pharmacology, Department of Microbiology and Immunology,Stanford University School of Medicine, Stanford, California 94305-5175 and the ¶Research Center inInfectious Diseases, CHUL Research Center, and Faculty of Medicine, Laval University, Quebec G1V 4G2, Canada

A chronic state of immune hyperactivation is a featureof human immunodeficiency virus type-1 (HIV-1) infec-tion. Studies on the molecular mechanisms by whichHIV-1 can modulate the activation state of T cells indi-cate that both Nef and Tat can alter T cell activation.However, the vast majority of data has been obtainedfrom experiments performed with vectors encoding asingle virus protein. We demonstrate that infection ofhuman CD4� T lymphocytes with fully infectious HIV-1leads to a hyper-responsiveness of the interleukin-2 pro-moter. Hypersensitivity in HIV-1-infected T cells wasobserved upon stimulation with various agents that areengaging different signal transduction pathways. Ex-periments performed with recombinant heat stable an-tigen-encoding HIV-1 indicated that the virus-infectedcells are the cells with an enhanced response. Both Nefand Tat are involved in this virus-mediated enhancingeffect on interleukin-2 promoter activity. Interestingly,whereas Nef seems to be acting mainly through hyper-activation of nuclear factor of activated T cells (NFAT),Tat acts in an NFAT-independent manner. Mobility shiftexperiments demonstrated that the HIV-1-associatedpriming of human T cells for stimulation results in agreater induction of transcription factors recognized asessential players in T cell activation, i.e. NFAT, NF-�B,and AP-1. A hyper-responsive state was also establishedupon HIV-1 infection of a more natural cellular reser-voir, i.e. primary CD4� T lymphocytes. Considering thatthe HIV-1 life cycle is tightly regulated by the T cellsignaling machinery, the priming for activation of a ma-jor viral reservoir represents a means by which thisretrovirus can create an ideal cellular microenviron-ment for its propagation and maintenance.

The hallmark of human immunodeficiency virus type-1(HIV-1)1 infection is the establishment of a progressive im-pairment of immune functions resulting primarily from anumeric loss of CD4� T lymphocytes. Possible causes of CD4�

T cell depletion include a direct destruction of infected cellsand an indirect induction of cell death in uninfected cells(reviewed in Ref. 1). Paradoxically, the HIV-1-associated dis-ease is also characterized by a state of chronic T cell activa-tion, driven in part by the persistence of HIV-1-related anti-gens (including whole virions), but also by antigen-independent processes (e.g. cytokine dysregulation). Forexample, the external envelope glycoprotein gp120 acts as apowerful immunogen for both lymphocytes and macrophages,which results in the induction of proinflammatory cytokines(2, 3). During the course of chronic HIV-1 infection, a height-ened state of systemic immune activation is linked to ele-vated numbers of activated CD8� T lymphocytes present inthe periphery. Such activated cells express on their surfaceseveral activation markers such as HLA-DR, CD38, CD57,and CD71 (4–6). Although there is a progressive loss of CD4�

T cells, a significant number of cells from this cellular subsetsimilarly expresses activation markers such as HLA-DR andCD25 (6). Increased immune activation can also be detectedby measuring soluble immune markers. These include neop-terin, soluble CD8, soluble CD14, soluble CD25, tumor necro-sis factor-�, and �2-microglobulin (4, 7–15).

Previous studies have shed light on the possible mechanismsthrough which HIV-1 infection itself can result in immunehyperactivation. Experiments performed in established T celllines and peripheral blood mononuclear cells (PBMCs) indi-cated that HIV-1 gene products Tat and Nef are both involvedin the modulation of T cell function (16–21). Results from thesestudies demonstrate that these HIV-1-encoded proteins primecells for activation and makes them more responsive to T cellactivation signals, a process that could favor higher virus pro-duction upon stimuli mediated via the T cell receptor (TCR) orother cell surface receptors. On the other hand, other studieshave brought evidence suggesting that both Tat and Nef mightbe negatively affecting T cell activation (22, 23).

Throughout these contradictory studies, conclusions were

* This work was supported in part by Canadian Institutes of HealthResearch HIV/AIDS Research Program Grant HOP-15575 (to M. J. T.).The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked“advertisement” in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.

§ Both authors contributed equally to this work.� Supported by a Scholarship Award (Junior 1 level) from the Fonds

de la Recherche en Sante du Quebec,** Recipient of the Canada Research Chair in Human Immuno-Ret-

rovirology (Tier 1 level). To whom correspondence should be addressed:Laboratory of Human Immuno-Retrovirology, Research Center in In-fectious Diseases, RC709, CHUL Research Center, 2705 Laurier Blvd.,Quebec G1V 4G2, Canada. Tel.: 418-654-2705; Fax: 418-654-2212; E-mail: michel.j.tremblay@crchul.ulaval.ca.

1 The abbreviations used are: HIV-1, human immunodeficiency virustype-1; PBMC, peripheral blood mononuclear cell; TCR, T cell receptor;IL-2, interleukin-2; PHA, phytohemagglutinin; PMA, phorbol 12-myris-tate 13-acetate; Iono, ionomycin; HSA, heat stable antigen; GFP, greenfluorescent protein; PBS, phosphate-buffered saline; NFAT, nuclearfactor of activated T cells; AP-1, activation protein-1.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 38, Issue of September 17, pp. 39520–39531, 2004© 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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mainly drawn from transient expression of HIV-1-encoded Tat orNef in human T cells. These types of experimental design arelikely to generate biased results because of overexpression of thetested viral protein. In addition, given that the priming of T cellsfor activation could be influenced by HIV-1 proteins other thanTat and/or Nef or by a combination of viral proteins, the presentstudy was aimed at assessing the potential impact of completeHIV-1 particles on T cell activation pathways. Cells stably trans-fected with IL-2- and nuclear factor of activated T cells (NFAT)-dependent reporter gene constructs were inoculated with fullycompetent viruses and were next subjected to various stimuli,either used alone or in combination, namely phytohemagglutinin(PHA), phorbol 12-myristate 13-acetate (PMA), ionomycin (Iono),anti-CD3 (clone OKT3), anti-CD28 (clone 9.3), and the potentprotein tyrosine phosphatase inhibitor bpV[pic]. Our results in-dicate that infection with competent viruses renders humanCD4� T cells more prone to activation than uninfected cellsthrough increased NFAT, NF-�B, and activator protein-1 (AP-1)binding activities. Both Tat and Nef proteins were likely impli-cated in the noticed modulation of T cell activity.

MATERIALS AND METHODS

Cells Used in the Present Study—The human leukemic T cell lineJurkat (clone E6.1) was obtained from the American Type CultureCollection (ATCC) (Manassas, VA). These cells were maintained incomplete culture medium made of RPMI 1640 supplemented with 10%fetal bovine serum (Hyclone Laboratories, Logan, UT), glutamine (2mM), penicillin G (100 units/ml), and streptomycin (100 �g/ml). Stablytransfected Jurkat cells were obtained by electroporation, as previouslydescribed (24). Briefly, 10 � 106 cells in mid-log phase were washedonce and resuspended in 400 �l of complete RPMI medium containing20 �g of either pIL-2-LUC or pNFAT-LUC vector (see below). Thismixture was transferred to a 0.4-cm gap electroporation cuvette (Bio-Rad). Cells were transfected in a Bio-Rad apparatus using standardvoltage and capacitance conditions (250 V and 960 microfarads). Aftera 10-min incubation period, transfected cells were resuspended at adensity of 1 � 106/ml in complete RPMI medium for 24 h. Cells werethen diluted to 5 � 104 cells/ml and 1 mg/ml of the selective agent G418(Invitrogen) was added. After 2 weeks of selection, G418-resistant Ju-rkat-derived cells were pooled and identified as either J-IL-2-LUC orJ-NFAT-LUC (24).

PBMCs from healthy donors were isolated by Ficoll-Hypaque densitygradient centrifugation. Human T helper cells (i.e. CD4�) were nega-tively isolated from fresh PBMCs using the CD4� T cells negativepurification kit according to manufacturer’s instructions (Miltenyi Bio-tec). Negative isolation was used to avoid signal transduction events

during the purification process that might impact on our studies.Briefly, we have used an antibody mixture and a magnetic colloid thatdepletes the cell population of every cell type, except CD4� T lympho-cytes upon application to a magnetically charged column. Before beinginfected, PBMCs and CD4� T cells were first cultured in completeRPMI medium containing 10% fetal bovine serum in the presence of 1�g/ml PHA-L (Sigma) and 30 units/ml of recombinant human IL-2 for 3days at 37 °C under a 5% CO2 atmosphere.

Plasmids and Antibodies—The molecular constructs pIL-2-LUC andpNFAT-LUC contain the complete 320-bp IL-2 promoter and the min-imal IL-2 promoter with three tandem copies of the NFAT1-bindingsite, respectively (kindly provided by Dr. G. Crabtree, Howard HughesMedical Institute, Stanford, CA) (25). pNF-�B-LUC was purchased

FIG. 1. HIV-1 infection of human Tcells enhances IL-2 promoter activityin response to several stimuli. Jurkatcells stably transfected with an IL-2 pro-moter-directed luciferase construct wereeither left uninfected or infected withNL4-3. Eight days post-infection, cellswere either left untreated or were stimu-lated with the listed agents. After 8 h,cells were lysed to monitor luciferase ac-tivity. Results are presented as themean � S.D. of quadruplicate samples.Data shown are representative of threeindependent experiments. The -fold dif-ferences between infected and unin-fected Jurkat cells are shown at the top ofeach bar corresponding to HIV-1-infectedsamples.

FIG. 2. Stimuli-mediated NFAT induction is also increasedupon HIV-1 infection of human T cells. Jurkat cells stably trans-fected with an NFAT-dependent reporter gene vector were either leftuninfected or infected with NL4-3. Eight days post-infection, cells wereeither left untreated or were stimulated with the listed agents. After8 h, cells were lysed to monitor luciferase activity. Results are presentedas the mean � S.D. of quadruplicate samples. Data shown are repre-sentative of three independent experiments. The -fold differences be-tween infected and uninfected Jurkat cells are shown at the top of barscorresponding to HIV-1-infected samples.

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from Stratagene and contained five consensus NF-�B binding se-quences cloned upstream from the luciferase gene along with a minimalpromoter. pNL4-3 is a full-length infectious molecular clone of HIV-1(26). The pNL4-3.HSA.R�E� vector leads to the production of HIV-1particles encoding for the murine heat stable antigen (HSA) CD24 gene(27, 28). The pHCMV-G molecular construct encodes for the broadhost-range vesicular stomatitis virus envelope glycoprotein G under thecontrol of the human cytomegalovirus promoter (29). The NL4.3-GFPmolecular clone contains the green fluorescent protein gene and aninternal ribosome entry site inserted upstream from the Nef gene in theNL4-3 clone. NL4-3-GPF virions are fully competent and carry allknown virus genes (30). The hybridoma producing the anti-CD3 anti-body (clone OKT3, which is specific for the � chain of the CD3 complex)was obtained from ATCC. Antibodies from this hybridoma were puri-fied with mAbTrap protein G affinity columns according to the manu-facturer’s instructions (Amersham Biosciences). Purified anti-CD28 an-tibodies (clone 9.3) were a generous gift from Dr. J. A. Ledbetter(Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton,NJ). Purified goat anti-mouse IgG antibodies were purchased fromJackson ImmunoResearch Laboratories (West Grove, PA). The R-phy-coerythrin-conjugated rat anti-human IL-2 antibody and the isotype-matched control antibody were purchased from BD Pharmingen.

Preparation of Virus Stocks and Infection—Fully infectious NL4-3viral entities were generated by calcium phosphate transfection of 293Tcells as described previously (31). The infectivity of virus preparationswas monitored by terminal dilution microassay using PHA-stimulatedPBMCs as targets. End-point titration was performed in flat-bottomedmicrotiter wells using four parallel series of 10-fold dilutions. After 7

days of incubation, virus production was assessed by measuring the p24content with an in-house double antibody sandwich enzymatic assay(32). Parental Jurkat, J-IL-2-LUC, and J-NFAT-LUC cells were inocu-lated with NL4-3 at a multiplicity of infection of 0.05. Cells were theneither left untreated or were stimulated for 8 h as described below.Pseudotyped HSA-encoding HIV-1 particles were generated by cotrans-fection of 293T cells with pNL4-3.HSA.R�E� and pHCMV-G. Virusstocks were normalized for virion content using the p24 test. J-IL-2-LUC cells (5� 106) were infected initially with such pseudotyped vi-ruses (200 ng of p24). Forty-eight hours post-infection, virus-infectedcells, which express cell surface mouse CD24, were purified by positiveselection using rat monoclonal anti-mouse CD24 antibodies (CYMBUSBiotechnology) and BioMag goat anti-rat IgG (Fc specific)-coated mag-netic beads (Polysciences). Cells were then either left untreated or werestimulated for 8 h as described below.

Transfections and Reporter Gene Assays—Transient transfections wereperformed using the DEAE-Dextran method (33). To minimize variationsin plasmid transfection efficiencies, cells were transfected in bulk andwere next separated into various treatment groups at a density of 105 cellsper well (100 �l) in 96-well flat-bottom plates. Except for those used ascontrols, cells were treated with PHA-P (3 �g/ml; Sigma), PMA (20 ng/ml;Sigma), Iono (1 �M; Calbiochem), anti-CD3 antibody (3 �g/ml)/anti-CD28antibody (1 �g/ml) along with goat anti-mouse IgG (5 �g/ml), and bpV[pic](10 �M) in a final volume of 200 �l. Next, cells were incubated at 37 °C for8 h unless otherwise specified. Luciferase activity was determined follow-ing a previously described protocol (33). -Fold induction was obtained bycalculating the ratio between measured relative light units of treatedsamples over untreated samples.

FIG. 3. Hypersensitivity of HIV-1-infected CD4� T cells to stimuli-mediated IL-2 gene activity is influenced by the course of HIV-1infection. J-IL-2-LUC cells were either left uninfected or infected with NL4-3. At 3 (panel A), 8 (panel B), and 11 days post-infection (panel C),cells were either left untreated or were stimulated with the listed agents. After 8 h, cells were lysed to monitor luciferase activity. Results areexpressed as -fold increase in luciferase activity in stimulated over untreated samples and represent the mean of quadruplicate samples. Standarddeviations were always less than 10%. Data shown are representative of three independent experiments. Samples were also taken to estimate thenumber of CD4-expressing cells by flow cytometry analysis (panel D).

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Cytofluorometry—Flow cytometry analyses were performed with 106

cells that were incubated with 100 �l of phosphate-buffered saline(PBS, pH 7.4) containing a saturating amount of a monoclonal anti-CD4antibody (i.e. clone SIM.4) for 30 min on ice. After being washed withcold PBS, the cells were labeled for 30 min on ice with 100 �l of asaturating amount of R-phycoerythrin-conjugated goat anti-mouse IgG(Caltag). Finally, cells were washed and analyzed on a cytofluorometer(EPICS XL, Coulter Corp., Miami, FL). Intracellular flow cytometrywas performed as follows. Cells (5 � 105) were washed once in PBS,fixed with 25 �l of reagent A (Fix & Perm cell permeabilization kit fromCALTAG Laboratories), and incubated 15 min at room temperature.Cells were washed in PBS, resuspended with 25 �l of reagent B towhich was added the anti-p24 monoclonal antibody 31-90-25 (ATCC),vortexed gently, and incubated for 15 min at room temperature. Cellswere subsequently washed with PBS supplemented with 1% sodium azideand resuspended with 100 �l of PBS containing a fluorescein isothiocya-nate-labeled goat anti-mouse IgG antibody (1 �g total) and further incu-bated for 15 min at room temperature. Finally, cells were centrifuged andresuspended in 1% paraformaldehyde in PBS before being analyzed byflow cytometry. For detection of intracellular IL-2, cells were initiallyinoculated with GFP-encoding NL4-3 particles and were stimulated 4days later with PMA and ionomycin or with anti-CD3/anti-CD28 antibod-ies for 6 h in the presence of BD GolgiStop (2 �M). Cells were then fixed,permeabilized, and stained with phycoerythrin-conjugated rat anti-human IL-2 antibody or a rat isotype-matched irrelevant antibody(i.e. IgG2a). Cells were immediately analyzed by flow cytometry.

Preparation of Nuclear Extracts and Electrophoretic Mobility ShiftAssays—Uninfected and HIV-1-infected cells were either left untreated

or were incubated for 1 h at 37 °C with PMA/Iono, anti-CD3/anti-CD28,or bpV[pic]. Incubation with the various stimulating agents was termi-nated by the addition of ice-cold PBS, and nuclear extracts were pre-pared according to the previously described microscale preparationprotocol (34). Protein concentrations were determined by the bicincho-ninic assay with a commercial protein assay reagent (Pierce). Nuclearextracts (10 �g) were incubated for 20 min at room temperature in 20 �lof 1� binding buffer (10 mM HEPES, pH 7.9, 4% glycerol, 1% Ficoll, 25mM KCl, 1 mM dithiothreitol, 0.5 mM EDTA, 25 mM NaCl, 2 �g ofpoly(dI-dC), 10 �g of nuclease-free bovine serum albumin fraction V)containing 0.8 ng of �-32P-labeled double-stranded DNA oligonucleo-tide. The following double-stranded DNA oligonucleotides were used asprobes and/or competitors: the distal NFAT-binding site from themurine IL-2 promoter (5�-TCGAGCCCAAAGAGGAAAATTTGTTTCA-TG-3�); the consensus NF-�B-binding site (5�-ATGTGAGGGGACTTT-CCCAGGC-3�); and the consensus binding site for AP-1 (5�-CGCTTG-ATGACTCAGCCGGAA-3�). DNA-protein complexes were resolved fromunbound labeled DNA by electrophoresis in native 4% (w/v) polyacryl-amide gels. The gels were subsequently dried and autoradiographed.Cold competition assays were carried out by adding a 100-fold molarexcess of unlabeled double-stranded DNA oligonucleotide simulta-neously with the labeled probe.

RESULTS

HIV-1 Infection of Human T Cells Enhances IL-2 PromoterActivity upon Stimulation—Although the effect of the HIV-1proteins Tat and Nef on T cell activation has been previously

FIG. 4. Higher sensitivity to NFAT activation also requires an established HIV-1 infection. J-NFAT-LUC cells were either leftuninfected or infected with NL4-3. At 3 (panel A), 8 (panel B), and 11 days post-infection (panel C), cells were either left untreated or werestimulated with the listed agents. After 8 h, cells were lysed to monitor luciferase activity. Results are expressed as -fold increase in luciferaseactivity in stimulated over untreated samples and represent the mean of quadruplicate samples. Standard deviations were always less than 10%.Data shown are representative of three independent experiments. Samples were also taken to estimate the number of CD4-expressing cells by flowcytometry analysis (panel D).

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documented, there is still no consensus as to whether this effectis positive or negative. Moreover, the possible induction of ahyperactive state to various stimuli has been rarely investi-gated in cells infected with complete virions. In this study, anexperimental setting was designed to provide a more relevantassessment of the changes occurring during T cell activationfollowing HIV-1 infection. Because the hallmark of T cell acti-vation remains the induction of IL-2 gene expression, IL-2promoter activity was used as a marker for T cell activation. Toexamine the possible effect of HIV-1 infection on T cell activa-tion, a Jurkat derivative stably expressing an IL-2 promoter-driven luciferase construct was therefore used (i.e. J-IL-2-LUC). This cell line was infected with NL4-3, a prototypicCXCR4 using virus isolate that encodes all known HIV-1 pro-teins (26, 35). Eight days post-infection, the IL-2 promoter-directed luciferase activity was measured 8 h later in the J-IL-2-LUC cells, either without further treatment, or followingstimulation with various agents. Among the stimuli used in thepresent study are agents that mimic antigen stimulation,namely the plant lectin PHA or antibodies that cross-link theTCR and the costimulatory CD28 molecule (reviewed in Ref.36). Other agents tested are the tumor promoter phorbol esterPMA, an activator of protein kinase C; the calcium ionophoreionomycin, an inducer of intracellular calcium mobilization;and bpV[pic], a protein-tyrosine phosphatase inhibitor shownto be a potent activator of several transcription factors (24, 33,37–41). These activators were selected because, when addedalone or in various combinations, they have been demonstratedto activate transcription factors important for IL-2 gene acti-vation (i.e. NF-�B, NFAT, and/or AP-1).

Upon HIV-1 infection and stimulation by the studied activa-tors, J-IL-2-LUC cells responded with a remarkable level ofactivation (Fig. 1). Indeed, a superinduction of IL-2 promoteractivity was observed with most agents tested upon HIV-1infection. For example, the treatment of HIV-1-infected cellswith PHA, PMA/PHA, PMA/Iono, and OKT3/9.3 caused a re-spective 14-, 20-, 87-, and 4-fold increase of reporter gene ac-tivity, in comparison to the activity of uninfected J-IL-2-LUCcells. An even greater increase in luciferase activity was seenfollowing the addition of bpV[pic] to NL4-3-infected Jurkatcells (155-fold). As expected, treatment with PMA alone did notresult in IL-2 activation in either infected or uninfected cells.These results indicate that infection with fully competentHIV-1 particles renders human Jurkat T cells hyper-respon-sive to stimuli affecting the IL-2 gene expression.

NFAT Plays a Role in HIV-1-mediated T Cell Hyperactiva-tion—The transcription factor NFAT is a critical regulator ofthe IL-2 gene transcription during normal T cell activation (42,43). To investigate if NFAT might contribute to the positiveeffect of HIV-1 infection in the stimuli-dependent increase inIL-2 transcriptional activity, a Jurkat-derived cellular clonestably transfected with a reporter plasmid expressing lucifer-ase under the control of three tandem short binding sites forNFAT (�286 to �257 of IL-2 enhancer) (44) was inoculatedwith HIV-1 virions. Such J-NFAT-LUC cells were treated withall stimuli, and NFAT-dependent luciferase activity was mon-itored following an 8-h stimulation period. The process ofHIV-1 infection enhanced NFAT transcriptional activity sever-alfold (Fig. 2), although the magnitude of augmentation inJ-NFAT-LUC cells was lower than in J-IL-2-LUC cells. Again,the addition of PMA alone did not lead to a significant increasein luciferase activity in uninfected or infected cells. These re-sults thus indicate that the NFAT transcription factor can alsobe activated in HIV-1-infected cells and might be involved inthe superinduction of the IL-2 promoter activity observed ininfected cells.

A Linear Correlation Is Seen Over Time between the Estab-lishment of HIV-1 Infection and the Hyperactivation of HumanT Cells—To confirm our observations and to estimate the timerequired for a hyper-responsiveness state to appear in humanT cells following virus infection, J-IL-2-LUC and J-NFAT-LUCcells were infected with NL4-3 and reporter gene activity wasevaluated after stimulation, at 3, 8, and 11 days post-infection.In parallel, virus infection was estimated by measuring thepercentage of CD4-expressing cells and monitoring p24 produc-tion. No superinduction of IL-2 promoter transcription wasdetectable at the earliest tested time point (i.e. 3 days post-infection) (Fig. 3A) but it was observed at later time points (i.e.8 and 11 days after infection) and increased over time (Fig. 3,B and C). Monitoring the virus-induced surface CD4 down-regulation and p24 production in culture supernatants to as-sess the spread of HIV-1 infection allowed us to conclude thatHIV-1 replication positively correlates with the hyperactivatedstate (Fig. 3D, and data not shown). Indeed, an important dropof CD4-positive T cells and an increase in p24 production oc-curred at day 8 following infection and these changes weremaintained at day 11, which coincided with the onset of theHIV-mediated increase in IL-2 promoter activity. The trendobserved in J-IL-2-LUC cells was also noted in J-NFAT-LUC-infected cells, i.e. a drop in CD4� T cell count and an augmen-tation of p24 production were paralleled with an increase inNFAT activation in the infected cell population (Fig. 4 and datanot shown). However, the order of magnitude of the effect wassmaller in J-NFAT-LUC cells, which is perfectly in line withmeasurements of reporter gene activity. These data are thusindicative of similar time kinetics in stimuli-mediated NFATactivation and IL-2 gene expression upon HIV-1 infection.

Hyperstimulation Is More Important in HIV-1-infectedCells—Next we monitored whether the virus-infected cells arethe ones showing an enhanced response to stimulation. Thisgoal was achieved through infection of J-IL-2-LUC cells withrecombinant HIV-1 particles that encode for the cell surfacemurine HSA CD24. Progeny viruses were pseudotyped with theenvelope glycoprotein from vesicular stomatitis virus to in-crease virus infectivity. Following infection and stimulation,

FIG. 5. Hyper-responsiveness of CD4� T cells to stimuli-medi-ated IL-2 gene activity is more important in HIV-1-infected cells.J-IL-2-LUC cells (5 � 106) were either left uninfected or inoculated withHSA-encoding HIV-1 particles pseudotyped with vesicular stomatitisvirus envelope glycoprotein G (200 ng of p24). After an incubationperiod of 48 h, cells were either left unsorted or were sorted usingmagnetic beads coated with an anti-HSA antibody. Cells were theneither left untreated or were stimulated with the listed agents. After8 h, cells were lysed to monitor luciferase activity. Results are presentedas the mean � S.D. of quadruplicate samples. Data shown are repre-sentative of three independent experiments. RLU, relative light unit.

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reporter gene activity was assessed in both unsorted and sortedcells (i.e. CD24�). As depicted in Fig. 5, a higher response tomost of the agents tested was seen in HIV-1-infected CD24-expressing cells as compared with unsorted cells (a mixture ofuninfected and virus-infected cells). Measurements of lucifer-ase activity in unsorted and sorted (i.e. HSA-expressing) J-IL-2-LUC cells that are infected with recombinant HSA-encodingviruses indicated that the sorting procedure with magneticbeads coated with the anti-HSA antibody has no effect on IL-2promoter-driven reporter gene activity (data not shown), there-fore confirming that the observed hyperstimulation in CD24-positive cells (i.e. HIV-1-infected) is because of virus infection.

Nef Is Directly Implicated in HIV-1-mediated Hyper-respon-siveness of Human T Cells—To identify virus gene product(s)underlying the HIV-1-induced hypersensitivity to stimulation,J-IL-2-LUC and J-NFAT-LUC cells were inoculated with wild-type NL4-3 virus (i.e. WT-Nef/NL4-3) or an isogenic mutantcontaining a deletion in the nef regulatory gene (i.e. �Nef/NL4-3). Following infection and stimulation with the various acti-vators, the luciferase activity was quantified. The data pre-sented in Fig. 6A demonstrate that Nef plays a pivotal role inthe superactivation of the IL-2 promoter seen in human T cellsupon HIV-1 infection, for all the activators tested. Most nota-

bly, the increment observed in NFAT activation in HIV-1-infected cells was totally abolished when Nef-deficient virionswere used to infect the J-NFAT-LUC cells (Fig. 6B). HIV-1replication, as measured by supernatant-associated HIV-1 p24antigen, did not differ between the two virus types in theinfected Jurkat cell clones throughout the entire time lapse ofinfection (data not shown). Given that Nef-deficient HIV-1viruses have been shown to be less infectious than wild typevirions (45), intracellular flow cytometry was also performed 8days post-infection to evaluate the frequency of p24-expressingcells (provides an estimate of cells productively infected withHIV-1). Infection with �Nef/NL4-3 viruses resulted in a higherpercentage of p24-expressing cells in both J-IL-2-LUC andJ-NFAT-LUC cells as compared with infection with WT-Nef/NL4-3 virions (Fig. 7). These data provide evidence that Nefplays a role in the HIV-1-induced hyperactivation of NFAT,resulting in an important induction of IL-2 promoter activity.

Tat Also Mediates Hyperactivation of the IL-2 Promoter butIndependently of NFAT—In an attempt to be consistent withour previous experiments, it would have been appropriate touse Tat-deficient viruses to scrutinize the involvement of Tat instimuli-dependent regulation of the IL-2 promoter and NFATactivity. Unfortunately, HIV-1 proviral mutants that lack the

FIG. 6. HIV-1-mediated up-regula-tion of IL-2 promoter activity in-volves Nef and NFAT induction. J-IL-2-LUC (panel A) and J-NFAT-LUC cells(panel B) were infected with wild-typeNL4-3 or Nef-deleted NL4-3 mutant.Eight days post-infection, cells were ei-ther left untreated or were stimulatedwith the listed agents. After 8 h, cellswere lysed to monitor luciferase activity.Results are presented as the mean � S.D.of quadruplicate samples. Data shown arerepresentative of three independent ex-periments. RLU, relative light unit.

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viral regulatory gene tat are unable to replicate in human Tcells. Thus, co-transfection studies were performed to investi-gate if expression of HIV-1 Tat could render human T cellsmore sensitive to the tested stimuli. We transfected Jurkat Tlymphoid cells with either pIL-2-LUC or pNFAT-LUC, andwith or without a Tat expression vector. Twenty-four hoursafter transfection, the reporter gene activity in these cells wasmeasured, either without further treatment, or following a 8-hstimulation with PHA, PMA/PHA, PMA/Iono, OKT3/9.3, andbpV[pic]. The graph shown in Fig. 8A demonstrates that Tatexpression in Jurkat T cells induces a much stronger IL-2promoter activity upon stimulation with any of the tested ac-tivating agents. On the other hand, when co-transfections wereconducted with the pNFAT-LUC vector, no such overinductionof luciferase reporter gene expression could be attributed to Tatexpression (Fig. 8B). These results thus indicate that Tat alsocontributes to the superinduction of IL-2 promoter activityupon HIV-1 infection. However, as opposed to Nef, transcrip-tion factor(s) other than NFAT are likely to be targeted by Tat.

Stimuli-mediated Nuclear Translocation of NFAT, NF-�B,and AP-1 Is Augmented upon HIV-1 Infection—In addition toNFAT, the activation of the IL-2 promoter involves the induc-tion of other transcription factors such as NF-�B and AP-1.Thus to confirm the participation of NFAT and to examinewhether the induction of NF-�B and AP-1 is also augmentedupon stimulation of HIV-1-infected cells, mobility shift assayswere performed using the appropriate �-32P-labeled probes.Nuclear extracts from uninfected and HIV-1-infected Jurkatcells were derived from cells, either untreated, or treated withthe listed agents. Electrophoretic mobility shift assay analysisrevealed that complexes specific for NFAT, NF-�B, and AP-1were more intense in nuclear extracts from HIV-1-infected cellsthan from uninfected cells for the majority of the tested acti-vators (Fig. 9, panels A–C). Interestingly, in unstimulated cellsthe nuclear levels of these transcription factors were higherupon HIV-1 infection especially for NFAT and AP-1. For eachsignal, the specificity of the signal was confirmed throughcompetition experiments. These results suggest that HIV-1infection leads to higher levels of NFAT, NF-�B, and AP-1nuclear translocation following stimulation of Jurkat T cells.

Hyperstimulation Is Also Observed upon HIV-1 Infection of aMore Natural Cellular Reservoir, i.e. CD4� T Lymphocytes—Toverify if the HIV-1-induced hyperstimulation observed in Jur-kat cells can also take place in primary human cells, purifiedCD4-expressing T lymphocytes were infected with GFP-encod-ing HIV-1 particles, an experimental strategy allowing an easydiscrimination of infected versus uninfected cells by flow cy-tometry. Four days post-infection, about 10% of CD4� T cellswere GFP-positive, i.e. infected. Following stimulation, intra-cellular IL-2 measurement indicated that a greater proportionof IL-2-expressing cells was present in the HIV-1-infected pop-ulation (i.e. GFP-positive) when compared with the uninfectedpopulation (i.e. GFP-negative) (Fig. 10).

DISCUSSION

This study describes a strong positive effect on T cell sig-naling, and more precisely on IL-2 promoter transcription,upon infection of human T cells with complete HIV-1 progenyvirus. This effect is suggested to implicate at least two HIV-1regulatory proteins, i.e. Nef and Tat, and to be mediated viainduction of three transcription factors recognized as impor-tant players in the regulation of IL-2, i.e. NFAT, NF-�B, andAP-1. Although some previous studies have also reportedpositive effects on T cell activation by these two viral pro-teins, in these cases, this phenomenon was observed when asingle virus protein (i.e. Nef or Tat) was present via eithertransient or stable expression in target cells (16–21). Thepresent work thus more closely parallels in vivo situationsbecause we used fully competent viruses instead of singlyexpressed HIV-1 gene products. Our results also suggest thatthe effect on IL-2 and NFAT hyperactivation is dependent onthe development over time of HIV-1 infection and intracellu-lar expression of viral proteins (as opposed to extracellularfactors). A selective isolation of virus-infected cells with theuse of recombinant virus coding for surface murine CD24allowed us to demonstrate that the hyper-responsivenessstate is mainly present in cells harboring HIV-1.

An exciting feature of this series of investigations consists inthe demonstration that Nef also exerts its modulatory role withrespect to T cell signaling pathways even in the context of the

FIG. 7. Higher levels of p24-expressing cells are achieved upon infection with Nef-deficient NL4-3 viruses. J-IL-2-LUC (panels A andB) and J-NFAT-LUC cells (panels C and D) were infected with wild-type (WT) (panels B and D) or the Nef-deleted NL4-3 mutant (panels A andC). Eight days post-infection, the percentage of p24-expressing cells was monitored by intracellular flow cytometry.

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complete viral genome. Previous work has shown that Nef hasthe potential to alter signal transduction events most likelythrough interactions with several signaling molecules (re-viewed in Ref. 46). For example, Nef associates with numerouscellular partners such as the Nef-associated kinase identifiedas a member of the p21-activated kinase family (47–50), aserine kinase (51), mitogen-activated protein kinase (52), c-Raf-1 (53), p53 (54), protein kinase C� (55), and members of theSrc family of tyrosine kinases (e.g. Lck, Hck, Lyn, and Fyn) (51,52, 56–60). The molecular mechanism of action of Nef is linkedwith its ability to induce transcription factors such as NFAT,NF-�B, and AP-1 (20, 21). The findings that Nef interacts withendogenous inositol triphosphate receptor (19) and increasesthe levels of signaling molecules within rafts (61) represent

mechanisms through which Nef can promote T cell activation.Our observations are perfectly in line with such findings, con-sidering that we observed that HIV-1 infection increases stim-uli-mediated induction of NFAT, NF-�B, and AP-1. More im-portantly, we have confirmed the importance of Nef in NFATsuperactivation in the context of HIV-1 infection. Our resultstherefore agree with the previously suggested increase in acti-vation of NFAT by Nef (20).

Although previous works have demonstrated that PMAalone could become an activator of NFAT in cells transfectedwith a Nef expressing vector (21, 62), our results suggest noinduction of NFAT or IL-2 gene expression upon PMA stimu-lation of HIV-1-infected cells. We cannot provide a clear expla-nation for these discrepancies at this point but one mightspeculate that Nef levels might affect the extent to which signaltransduction is altered and/or the presence of viral proteinsother than Nef might exert a varying effect on signaling path-ways normally affected by Nef alone. This reinforces the valid-ity of our data as more physiologic levels of Nef were present inthe tested Jurkat cells in combination with other HIV-1 pro-teins through the natural HIV-1 infection process.

The HIV-1-encoded transactivating Tat protein is essentialfor viral replication and gene expression (reviewed in Ref. 63).This protein of viral origin can also modulate T cell activation.Indeed, HIV-1 Tat has been reported to enhance IL-2 promoteractivity upon treatment with phorbol ester and calcium iono-phore through the NFAT motif (18). Moreover, the expressionof Tat in human T cells is associated with an augmentation ofIL-2 production in response to engagement of CD3 and CD28surface receptors (17). The effect of Tat has been shown to bemediated by the CD28-responsive element (CD28RE) in theIL-2 promoter (17). The unique CD28RE motif was identified asan enhancer element located at positions �164 to �154 of theminimal IL-2 promoter (64). Full responsiveness to CD28-me-diated signals requires the CD28RE/AP-1 composite element,which comprises the CD28RE and the contiguous AP-1 site(65). The CD28RE/AP-1 element contains binding sequencesfor both NF-�B and AP-1 transcription factors (65–69). Datafrom co-transfection experiments revealed that NFAT is notplaying a role in Tat-dependent hyperactivation of the IL-2promoter, thus suggesting that the effect of Tat on the overin-duction of IL-2 promoter activity might be exclusively relatedto a stronger induction of NF-�B and/or AP-1. Although the useof Tat-deficient viruses would have been more appropriate inthe present study, the inability of such viruses to productivelyinfect human T cells would have complicated the interpretationof the data. Small interfering RNA duplexes could have beenused to block Tat expression. This technical strategy seemslaudable at first sight but targeting the tat gene through thisapproach would have also led to degradation of other unsplicedor singly spliced mRNAs. This is exemplified by a previouswork showing a marked reduction in the level of expression ofall three classes of HIV-1 mRNAs in HIV-1-infected cells (i.e.9.1, 4.3, and 1.8-kbp) treated with Tat-specific small interferingRNAs (70).

Our electrophoretic mobility shift assay data also indicatesthat the overactivation of all three tested transcription factorsalso occurs in unstimulated infected cells. However, this con-trasts with the luciferase reporter gene expression data, inwhich no differences could be measured between HIV-1-in-fected and uninfected Jurkat cells at basal level in terms ofNFAT and IL-2 promoter activation. This suggests that HIV-1infection itself can alter the cascade of events regulating theactivation of these transcription factors, but that either a weakactivation level (for example, NF-�B and AP-1) or the lack ofother post-translational modifications does not allow achieving

FIG. 8. Tat is also involved in the HIV-1-induced hypersensi-tivity of Jurkat cells to stimulation but in an NFAT-independentmanner. Jurkat cells were co-transfected with pIL-2-LUC (panel A) orpNFAT-LUC (panel B) in combination with either a Tat-encoding vectoror an appropriate empty control vector. Twenty-four hours followingtransfection, cells were either left untreated or were stimulated withthe listed agents. After 8 h, cells were lysed to monitor luciferaseactivity. Results are presented as the mean � S.D. of quadruplicatesamples. Data shown are representative of three independentexperiments.

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a competent transcriptional complex for NFAT-dependent andIL-2 promoter-mediated transcription. Experiments are pres-ently underway to shed light on this matter.

Although HIV-1 can gain entry inside resting CD4� T lym-

phocytes that express appropriate entry receptors, the viralreplicative cycle is blocked at the preintegration step unlessactivation signals are provided through the TCR (71–78). Syn-thesis of full-length viral DNA is prevented in quiescent T cells

FIG. 9. Activation-induced nucleartranslocation of NFAT, NF-�B, andAP-1 is augmented upon HIV-1 infec-tion of CD4� T cells. Parental Jurkatcells were either left uninfected or wereinfected with NL4-3. Eight days post-in-fection, cells were either left untreated orwere stimulated for 1 h with the listedagents. Nuclear extracts were next incu-bated with an NFAT- (panel A), NF-�B-(panel B), or AP-1-labeled probe (panel C)to be finally analyzed on a 4% native poly-acrylamide gel. Competitions were alsoperformed with the appropriate coldprobe to verify the specificity of theshifted complexes. The arrows on the leftindicate the specific complexes.

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because of a premature termination of reverse transcription(72). It has also been demonstrated that import of the preinte-gration complex into the nucleus is not efficient in resting

CD4� T cells (79, 80). As the blockade at reverse transcriptioncan be overcome by NFAT (81) and Nef has been shown to beincorporated in mature HIV-1 particles (82, 83), it can be pro-

FIG. 9—continued

FIG. 10. Stimuli-mediated up-regulation of IL-2 production is seen in primary human CD4� T cells upon HIV-1 infection. PurifiedCD4� T lymphocytes were infected with GPF-encoding NL4-3 virions. Four days post-infection, cells were either left untreated or were stimulatedwith the indicated stimuli for 6 h in the presence of BD GolgiStop. Intracellular IL-2 in the GFP-negative and -positive fractions was measuredby two-color flow cytometry. Data shown are representative of two independent experiments.

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posed that Nef proteins released within the cell upon virusentry might favor reverse transcription by diminishing therequirements for NFAT activation. Late events in the HIV-1life cycle, such as viral gene transcription and the assembly ofmature virions, are also highly dependent on T cell activation.It is thus clear that HIV-1 has evolved several mechanisms toexploit the cellular signaling machinery to facilitate its repli-cation and propagation through the infected host. The virusitself, via some of its products such as Nef and Tat, may thuscontribute to the T cell activation process that is required for anefficient virus spread by priming the CD4� T lymphocyte foractivation. In fact, it has been suggested that the process ofHIV-1 infection results in a lowered threshold for T cell acti-vation achieved by priming the TCR signaling complex (20, 84).This may help to explain the high proportion of activated CD4�

T cells in secondary lymphoid organs such as lymph nodesearly after HIV-1 infection (85).

At each end of its genome, HIV-1 carries regulatory domainsknown as long terminal repeat. Various studies have shownthat the HIV-1 long terminal repeat region is composed ofvarious binding motifs that are also present in the regulatoryregions of genes induced after T cell activation such as the IL-2gene. A direct result of the similarity between the architectureof the HIV-1 LTR and the IL-2 promoter is an intimate linkbetween T cell activation and HIV-1 transcription. Becauseinteractions between some HIV-1 viral gene products and thecell signaling machinery can alter the activation state of hostcell, it is legitimate to postulate that the pool of lymphocytesthat can be infected by the virus will be amplified. Indeed, itcan be proposed that upon stimulation of HIV-1-infected cells,the concomitant up-regulation of IL-2 secretion will lead to therecruitment of bystander resting CD4� T lymphocytes and willrender such cells more susceptible to productive virus infection.

At this point, it is important to emphasize that a Tat-de-pendent enhancement of response to T cell activation via theTCR and CD28 receptors has already been reported in pri-mary human T cells (17). Moreover, a Nef-mediated primingupon stimulation with anti-CD3/CD28 antibodies has alsobeen demonstrated in primary CD4� T lymphocytes (20). Ourfindings that a hyperactive state is observed also withinpurified CD4� T cells infected with fully competent GFP-encoding HIV-1 viruses is perfectly in line with these twoprevious reports and provide physiological significance to thepresent work. Our results further reinforce the notion thatHIV-1 infection alters signal transduction in T cells. Suchchanges are likely to influence the onset of infection andmight further help in the spreading of infection in vivo. It isthereby important to clarify the overall HIV-1-associateddysregulation of signal transduction events in the host cellbecause it could provide a novel means for interfering withthe pathologies linked with HIV-1 infection.

Acknowledgments—We thank Dr. M. Dufour for flow cytometricanalyses and Sylvie Methot for editorial assistance.

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TremblayJean-François Fortin, Corinne Barat, Yannick Beauséjour, Benoit Barbeau and Michel J.

B, and AP-1 InductionκNFAT, NF- T Cells Requires Nef and Tat Virus Gene Products and Results from Higher+CD4

Hyper-responsiveness to Stimulation of Human Immunodeficiency Virus-infected

doi: 10.1074/jbc.M407477200 originally published online July 16, 20042004, 279:39520-39531.J. Biol. Chem. 

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Additions and Corrections

Vol. 279 (2004) 39520–39531

Hyper-responsiveness to stimulation of human immunodeficiency virus-infected CD4� T cells requires Nef and Tatvirus gene products and results from higher NFAT, NF-�B, and AP-1 induction.

Jean-François Fortin, Corinne Barat, Yannick Beausejour, Benoit Barbeau, and Michel J. Tremblay

Page 39522, under “Materials and Methods”: Under the section dealing with “Plasmids and Antibodies,” the molecular cloneNL4-3-GFP was misnamed and its source was not fully acknowledged. The following sentence “The NL4-3-GFP molecular clonecontains the green fluorescent protein gene and an internal ribosome entry site inserted upstream from the Nef gene in the NL4-3clone” should be replaced by “The NLENG1-IRES molecular clone, a generous gift from Dr. D. N. Levy (University of Alabamaat Birmingham), contains the enhanced green fluorescent protein gene and an internal ribosome entry site (IRES) insertedupstream from the Nef gene in the NL4-3 clone without removing any viral sequences (30).”

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 10, Issue of March 11, p. 9752, 2005© 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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