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Originally published In Press as doi:10.1074/jbc.M407477200 on July 16, 2004

J. Biol. Chem., Vol. 279, Issue 38, 39520-39531, September 17, 2004
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Hyper-responsiveness to Stimulation of Human Immunodeficiency Virus-infected CD4+ T Cells Requires Nef and Tat Virus Gene Products and Results from Higher NFAT, NF-{kappa}B, and AP-1 Induction*

Jean-François Fortin{ddagger}§, Corinne Barat¶§, Yannick Beauséjour¶, Benoit Barbeau¶||, and Michel J. Tremblay¶**

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

Received for publication, July 6, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A chronic state of immune hyperactivation is a feature of human immunodeficiency virus type-1 (HIV-1) infection. Studies on the molecular mechanisms by which HIV-1 can modulate the activation state of T cells indicate that both Nef and Tat can alter T cell activation. However, the vast majority of data has been obtained from experiments performed with vectors encoding a single virus protein. We demonstrate that infection of human CD4+ T lymphocytes with fully infectious HIV-1 leads to a hyper-responsiveness of the interleukin-2 promoter. Hypersensitivity in HIV-1-infected T cells was observed upon stimulation with various agents that are engaging different signal transduction pathways. Experiments performed with recombinant heat stable antigen-encoding HIV-1 indicated that the virus-infected cells are the cells with an enhanced response. Both Nef and Tat are involved in this virus-mediated enhancing effect on interleukin-2 promoter activity. Interestingly, whereas Nef seems to be acting mainly through hyperactivation of nuclear factor of activated T cells (NFAT), Tat acts in an NFAT-independent manner. Mobility shift experiments demonstrated that the HIV-1-associated priming of human T cells for stimulation results in a greater induction of transcription factors recognized as essential players in T cell activation, i.e. NFAT, NF-{kappa}B, and AP-1. A hyper-responsive state was also established upon HIV-1 infection of a more natural cellular reservoir, i.e. primary CD4+ T lymphocytes. Considering that the HIV-1 life cycle is tightly regulated by the T cell signaling machinery, the priming for activation of a major viral reservoir represents a means by which this retrovirus can create an ideal cellular microenvironment for its propagation and maintenance.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The hallmark of human immunodeficiency virus type-1 (HIV-1)1 infection is the establishment of a progressive impairment of immune functions resulting primarily from a numeric loss of CD4+ T lymphocytes. Possible causes of CD4+ T cell depletion include a direct destruction of infected cells and an indirect induction of cell death in uninfected cells (reviewed in Ref. 1). Paradoxically, the HIV-1-associated disease is also characterized by a state of chronic T cell activation, driven in part by the persistence of HIV-1-related antigens (including whole virions), but also by antigen-independent processes (e.g. cytokine dysregulation). For example, the external envelope glycoprotein gp120 acts as a powerful 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 heightened state of systemic immune activation is linked to elevated numbers of activated CD8+ T lymphocytes present in the periphery. Such activated cells express on their surface several 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 subset similarly expresses activation markers such as HLA-DR and CD25 (6). Increased immune activation can also be detected by measuring soluble immune markers. These include neopterin, soluble CD8, soluble CD14, soluble CD25, tumor necrosis factor-{alpha}, and {beta}2-microglobulin (4, 7–15).

Previous studies have shed light on the possible mechanisms through which HIV-1 infection itself can result in immune hyperactivation. Experiments performed in established T cell lines and peripheral blood mononuclear cells (PBMCs) indicated that HIV-1 gene products Tat and Nef are both involved in the modulation of T cell function (16–21). Results from these studies demonstrate that these HIV-1-encoded proteins prime cells for activation and makes them more responsive to T cell activation signals, a process that could favor higher virus production upon stimuli mediated via the T cell receptor (TCR) or other cell surface receptors. On the other hand, other studies have brought evidence suggesting that both Tat and Nef might be negatively affecting T cell activation (22, 23).

Throughout these contradictory studies, conclusions were mainly drawn from transient expression of HIV-1-encoded Tat or Nef in human T cells. These types of experimental design are likely to generate biased results because of overexpression of the tested viral protein. In addition, given that the priming of T cells for activation could be influenced by HIV-1 proteins other than Tat and/or Nef or by a combination of viral proteins, the present study was aimed at assessing the potential impact of complete HIV-1 particles on T cell activation pathways. Cells stably transfected with IL-2- and nuclear factor of activated T cells (NFAT)-dependent reporter gene constructs were inoculated with fully competent 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 potent protein tyrosine phosphatase inhibitor bpV[pic]. Our results indicate that infection with competent viruses renders human CD4+ T cells more prone to activation than uninfected cells through increased NFAT, NF-{kappa}B, and activator protein-1 (AP-1) binding activities. Both Tat and Nef proteins were likely implicated in the noticed modulation of T cell activity.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells Used in the Present Study—The human leukemic T cell line Jurkat (clone E6.1) was obtained from the American Type Culture Collection (ATCC) (Manassas, VA). These cells were maintained in complete culture medium made of RPMI 1640 supplemented with 10% fetal bovine serum (Hyclone Laboratories, Logan, UT), glutamine (2 mM), penicillin G (100 units/ml), and streptomycin (100 µg/ml). Stably transfected Jurkat cells were obtained by electroporation, as previously described (24). Briefly, 10 x 106 cells in mid-log phase were washed once and resuspended in 400 µl of complete RPMI medium containing 20 µg of either pIL-2-LUC or pNFAT-LUC vector (see below). This mixture was transferred to a 0.4-cm gap electroporation cuvette (Bio-Rad). Cells were transfected in a Bio-Rad apparatus using standard voltage and capacitance conditions (250 V and 960 microfarads). After a 10-min incubation period, transfected cells were resuspended at a density of 1 x 106/ml in complete RPMI medium for 24 h. Cells were then diluted to 5 x 104 cells/ml and 1 mg/ml of the selective agent G418 (Invitrogen) was added. After 2 weeks of selection, G418-resistant Jurkat-derived cells were pooled and identified as either J-IL-2-LUC or J-NFAT-LUC (24).

PBMCs from healthy donors were isolated by Ficoll-Hypaque density gradient centrifugation. Human T helper cells (i.e. CD4+) were negatively isolated from fresh PBMCs using the CD4+ T cells negative purification kit according to manufacturer's instructions (Miltenyi Biotec). 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 that depletes the cell population of every cell type, except CD4+ T lymphocytes upon application to a magnetically charged column. Before being infected, PBMCs and CD4+ T cells were first cultured in complete RPMI medium containing 10% fetal bovine serum in the presence of 1 µg/ml PHA-L (Sigma) and 30 units/ml of recombinant human IL-2 for 3 days at 37 °C under a 5% CO2 atmosphere.

Plasmids and Antibodies—The molecular constructs pIL-2-LUC and pNFAT-LUC contain the complete 320-bp IL-2 promoter and the minimal IL-2 promoter with three tandem copies of the NFAT1-binding site, respectively (kindly provided by Dr. G. Crabtree, Howard Hughes Medical Institute, Stanford, CA) (25). pNF-{kappa}B-LUC was purchased from Stratagene and contained five consensus NF-{kappa}B binding sequences cloned upstream from the luciferase gene along with a minimal promoter. 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-1 particles encoding for the murine heat stable antigen (HSA) CD24 gene (27, 28). The pHCMV-G molecular construct encodes for the broad host-range vesicular stomatitis virus envelope glycoprotein G under the control of the human cytomegalovirus promoter (29). The NL4.3-GFP molecular clone contains the green fluorescent protein gene and an internal ribosome entry site inserted upstream from the Nef gene in the NL4-3 clone. NL4-3-GPF virions are fully competent and carry all known virus genes (30). The hybridoma producing the anti-CD3 antibody (clone OKT3, which is specific for the {epsilon} chain of the CD3 complex) was obtained from ATCC. Antibodies from this hybridoma were purified with mAbTrap protein G affinity columns according to the manufacturer's instructions (Amersham Biosciences). Purified anti-CD28 antibodies (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 from Jackson ImmunoResearch Laboratories (West Grove, PA). The R-phycoerythrin-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 NL4-3 viral entities were generated by calcium phosphate transfection of 293T cells as described previously (31). The infectivity of virus preparations was monitored by terminal dilution microassay using PHA-stimulated PBMCs as targets. End-point titration was performed in flat-bottomed microtiter wells using four parallel series of 10-fold dilutions. After 7 days of incubation, virus production was assessed by measuring the p24 content with an in-house double antibody sandwich enzymatic assay (32). Parental Jurkat, J-IL-2-LUC, and J-NFAT-LUC cells were inoculated with NL4-3 at a multiplicity of infection of 0.05. Cells were then either left untreated or were stimulated for 8 h as described below. Pseudotyped HSA-encoding HIV-1 particles were generated by cotransfection of 293T cells with pNL4-3.HSA.R–E– and pHCMV-G. Virus stocks were normalized for virion content using the p24 test. J-IL-2-LUC cells (5x 106) were infected initially with such pseudotyped viruses (200 ng of p24). Forty-eight hours post-infection, virus-infected cells, which express cell surface mouse CD24, were purified by positive selection using rat monoclonal anti-mouse CD24 antibodies (CYMBUS Biotechnology) and BioMag goat anti-rat IgG (Fc specific)-coated magnetic beads (Polysciences). Cells were then either left untreated or were stimulated for 8 h as described below.

Transfections and Reporter Gene Assays—Transient transfections were performed using the DEAE-Dextran method (33). To minimize variations in plasmid transfection efficiencies, cells were transfected in bulk and were next separated into various treatment groups at a density of 105 cells per well (100 µl) in 96-well flat-bottom plates. Except for those used as controls, cells were treated with PHA-P (3 µg/ml; Sigma), PMA (20 ng/ml; Sigma), Iono (1 µM; Calbiochem), anti-CD3 antibody (3 µg/ml)/anti-CD28 antibody (1 µg/ml) along with goat anti-mouse IgG (5 µg/ml), and bpV[pic] (10 µM) in a final volume of 200 µl. Next, cells were incubated at 37 °C for 8 h unless otherwise specified. Luciferase activity was determined following a previously described protocol (33). -Fold induction was obtained by calculating the ratio between measured relative light units of treated samples over untreated samples.

Cytofluorometry—Flow cytometry analyses were performed with 106 cells that were incubated with 100 µl of phosphate-buffered saline (PBS, pH 7.4) containing a saturating amount of a monoclonal anti-CD4 antibody (i.e. clone SIM.4) for 30 min on ice. After being washed with cold PBS, the cells were labeled for 30 min on ice with 100 µl of a saturating 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 cytometry was performed as follows. Cells (5 x 105) were washed once in PBS, fixed with 25 µl of reagent A (Fix & Perm cell permeabilization kit from CALTAG Laboratories), and incubated 15 min at room temperature. Cells were washed in PBS, resuspended with 25 µl of reagent B to which was added the anti-p24 monoclonal antibody 31-90-25 (ATCC), vortexed gently, and incubated for 15 min at room temperature. Cells were subsequently washed with PBS supplemented with 1% sodium azide and resuspended with 100 µl of PBS containing a fluorescein isothiocyanate-labeled goat anti-mouse IgG antibody (1 µg total) and further incubated for 15 min at room temperature. Finally, cells were centrifuged and resuspended in 1% paraformaldehyde in PBS before being analyzed by flow cytometry. For detection of intracellular IL-2, cells were initially inoculated with GFP-encoding NL4-3 particles and were stimulated 4 days later with PMA and ionomycin or with anti-CD3/anti-CD28 antibodies for 6 h in the presence of BD GolgiStop (2 µM). 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 Shift Assays—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 terminated by the addition of ice-cold PBS, and nuclear extracts were prepared according to the previously described microscale preparation protocol (34). Protein concentrations were determined by the bicinchoninic assay with a commercial protein assay reagent (Pierce). Nuclear extracts (10 µg) were incubated for 20 min at room temperature in 20 µl of 1x binding buffer (10 mM HEPES, pH 7.9, 4% glycerol, 1% Ficoll, 25 mM KCl, 1 mM dithiothreitol, 0.5 mM EDTA, 25 mM NaCl, 2 µg of poly(dI-dC), 10 µg of nuclease-free bovine serum albumin fraction V) containing 0.8 ng of {gamma}-32P-labeled double-stranded DNA oligonucleotide. The following double-stranded DNA oligonucleotides were used as probes and/or competitors: the distal NFAT-binding site from the murine IL-2 promoter (5'-TCGAGCCCAAAGAGGAAAATTTGTTTCATG-3'); the consensus NF-{kappa}B-binding site (5'-ATGTGAGGGGACTTTCCCAGGC-3'); and the consensus binding site for AP-1 (5'-CGCTTGATGACTCAGCCGGAA-3'). DNA-protein complexes were resolved from unbound labeled DNA by electrophoresis in native 4% (w/v) polyacrylamide gels. The gels were subsequently dried and autoradiographed. Cold competition assays were carried out by adding a 100-fold molar excess of unlabeled double-stranded DNA oligonucleotide simultaneously with the labeled probe.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
HIV-1 Infection of Human T Cells Enhances IL-2 Promoter Activity upon Stimulation—Although the effect of the HIV-1 proteins Tat and Nef on T cell activation has been previously documented, there is still no consensus as to whether this effect is positive or negative. Moreover, the possible induction of a hyperactive state to various stimuli has been rarely investigated in cells infected with complete virions. In this study, an experimental setting was designed to provide a more relevant assessment of the changes occurring during T cell activation following HIV-1 infection. Because the hallmark of T cell activation remains the induction of IL-2 gene expression, IL-2 promoter activity was used as a marker for T cell activation. To examine the possible effect of HIV-1 infection on T cell activation, 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 prototypic CXCR4 using virus isolate that encodes all known HIV-1 proteins (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 following stimulation with various agents. Among the stimuli used in the present study are agents that mimic antigen stimulation, namely the plant lectin PHA or antibodies that cross-link the TCR and the costimulatory CD28 molecule (reviewed in Ref. 36). Other agents tested are the tumor promoter phorbol ester PMA, an activator of protein kinase C; the calcium ionophore ionomycin, an inducer of intracellular calcium mobilization; and bpV[pic], a protein-tyrosine phosphatase inhibitor shown to be a potent activator of several transcription factors (24, 33, 37–41). These activators were selected because, when added alone or in various combinations, they have been demonstrated to activate transcription factors important for IL-2 gene activation (i.e. NF-{kappa}B, NFAT, and/or AP-1).

Upon HIV-1 infection and stimulation by the studied activators, J-IL-2-LUC cells responded with a remarkable level of activation (Fig. 1). Indeed, a superinduction of IL-2 promoter activity was observed with most agents tested upon HIV-1 infection. For example, the treatment of HIV-1-infected cells with PHA, PMA/PHA, PMA/Iono, and OKT3/9.3 caused a respective 14-, 20-, 87-, and 4-fold increase of reporter gene activity, in comparison to the activity of uninfected J-IL-2-LUC cells. An even greater increase in luciferase activity was seen following the addition of bpV[pic] to NL4-3-infected Jurkat cells (155-fold). As expected, treatment with PMA alone did not result in IL-2 activation in either infected or uninfected cells. These results indicate that infection with fully competent HIV-1 particles renders human Jurkat T cells hyper-responsive to stimuli affecting the IL-2 gene expression.



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  		FIG. 1.
HIV-1 infection of human T cells enhances IL-2 promoter activity in response to several stimuli. Jurkat cells stably transfected with an IL-2 promoter-directed luciferase construct were either left uninfected or infected with NL4-3. Eight days post-infection, cells were either left untreated or were stimulated with the listed agents. After 8 h, cells were lysed to monitor luciferase activity. Results are presented as the mean ± S.D. of quadruplicate samples. Data shown are representative of three independent experiments. The -fold differences between infected and uninfected Jurkat cells are shown at the top of each bar corresponding to HIV-1-infected samples.

 
NFAT Plays a Role in HIV-1-mediated T Cell Hyperactivation—The transcription factor NFAT is a critical regulator of the IL-2 gene transcription during normal T cell activation (42, 43). To investigate if NFAT might contribute to the positive effect of HIV-1 infection in the stimuli-dependent increase in IL-2 transcriptional activity, a Jurkat-derived cellular clone stably transfected with a reporter plasmid expressing luciferase under the control of three tandem short binding sites for NFAT (–286 to –257 of IL-2 enhancer) (44) was inoculated with HIV-1 virions. Such J-NFAT-LUC cells were treated with all stimuli, and NFAT-dependent luciferase activity was monitored following an 8-h stimulation period. The process of HIV-1 infection enhanced NFAT transcriptional activity severalfold (Fig. 2), although the magnitude of augmentation in J-NFAT-LUC cells was lower than in J-IL-2-LUC cells. Again, the addition of PMA alone did not lead to a significant increase in luciferase activity in uninfected or infected cells. These results thus indicate that the NFAT transcription factor can also be activated in HIV-1-infected cells and might be involved in the superinduction of the IL-2 promoter activity observed in infected cells.



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  		FIG. 2.
Stimuli-mediated NFAT induction is also increased upon HIV-1 infection of human T cells. Jurkat cells stably transfected with an NFAT-dependent reporter gene vector were either left uninfected or infected with NL4-3. Eight days post-infection, cells were either left untreated or were stimulated with the listed agents. After 8 h, cells were lysed to monitor luciferase activity. Results are presented as the mean ± S.D. of quadruplicate samples. Data shown are representative of three independent experiments. The -fold differences between infected and uninfected Jurkat cells are shown at the top of bars corresponding to HIV-1-infected samples.

 
A Linear Correlation Is Seen Over Time between the Establishment of HIV-1 Infection and the Hyperactivation of Human T Cells—To confirm our observations and to estimate the time required for a hyper-responsiveness state to appear in human T cells following virus infection, J-IL-2-LUC and J-NFAT-LUC cells were infected with NL4-3 and reporter gene activity was evaluated after stimulation, at 3, 8, and 11 days post-infection. In parallel, virus infection was estimated by measuring the percentage of CD4-expressing cells and monitoring p24 production. No superinduction of IL-2 promoter transcription was detectable 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 assess the spread of HIV-1 infection allowed us to conclude that HIV-1 replication positively correlates with the hyperactivated state (Fig. 3D, and data not shown). Indeed, an important drop of CD4-positive T cells and an increase in p24 production occurred at day 8 following infection and these changes were maintained at day 11, which coincided with the onset of the HIV-mediated increase in IL-2 promoter activity. The trend observed 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 augmentation of p24 production were paralleled with an increase in NFAT activation in the infected cell population (Fig. 4 and data not shown). However, the order of magnitude of the effect was smaller in J-NFAT-LUC cells, which is perfectly in line with measurements of reporter gene activity. These data are thus indicative of similar time kinetics in stimuli-mediated NFAT activation and IL-2 gene expression upon HIV-1 infection.



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  		FIG. 3.
Hypersensitivity of HIV-1-infected CD4+ T cells to stimuli-mediated IL-2 gene activity is influenced by the course of HIV-1 infection. 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 are expressed as -fold increase in luciferase activity 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 flow cytometry analysis (panel D).

 



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  		FIG. 4.
Higher sensitivity to NFAT activation also requires an established HIV-1 infection. J-NFAT-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 are expressed as -fold increase in luciferase activity 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 flow cytometry analysis (panel D).

 
Hyperstimulation Is More Important in HIV-1-infected Cells—Next we monitored whether the virus-infected cells are the ones showing an enhanced response to stimulation. This goal was achieved through infection of J-IL-2-LUC cells with recombinant HIV-1 particles that encode for the cell surface murine HSA CD24. Progeny viruses were pseudotyped with the envelope glycoprotein from vesicular stomatitis virus to increase virus infectivity. Following infection and stimulation, reporter gene activity was assessed in both unsorted and sorted cells (i.e. CD24+). As depicted in Fig. 5, a higher response to most of the agents tested was seen in HIV-1-infected CD24-expressing cells as compared with unsorted cells (a mixture of uninfected and virus-infected cells). Measurements of luciferase activity in unsorted and sorted (i.e. HSA-expressing) J-IL-2-LUC cells that are infected with recombinant HSA-encoding viruses indicated that the sorting procedure with magnetic beads coated with the anti-HSA antibody has no effect on IL-2 promoter-driven reporter gene activity (data not shown), therefore confirming that the observed hyperstimulation in CD24-positive cells (i.e. HIV-1-infected) is because of virus infection.



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  		FIG. 5.
Hyper-responsiveness of CD4+ T cells to stimuli-mediated IL-2 gene activity is more important in HIV-1-infected cells. J-IL-2-LUC cells (5 x 106) were either left uninfected or inoculated with HSA-encoding HIV-1 particles pseudotyped with vesicular stomatitis virus envelope glycoprotein G (200 ng of p24). After an incubation period of 48 h, cells were either left unsorted or were sorted using magnetic beads coated with an anti-HSA antibody. Cells were then either left untreated or were stimulated with the listed agents. After 8 h, cells were lysed to monitor luciferase activity. Results are presented as the mean ± S.D. of quadruplicate samples. Data shown are representative of three independent experiments. RLU, relative light unit.

 
Nef Is Directly Implicated in HIV-1-mediated Hyper-responsiveness 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 mutant containing a deletion in the nef regulatory gene (i.e. {Delta}Nef/NL4-3). Following infection and stimulation with the various activators, the luciferase activity was quantified. The data presented in Fig. 6A demonstrate that Nef plays a pivotal role in the superactivation of the IL-2 promoter seen in human T cells upon HIV-1 infection, for all the activators tested. Most notably, the increment observed in NFAT activation in HIV-1-infected cells was totally abolished when Nef-deficient virions were used to infect the J-NFAT-LUC cells (Fig. 6B). HIV-1 replication, as measured by supernatant-associated HIV-1 p24 antigen, did not differ between the two virus types in the infected Jurkat cell clones throughout the entire time lapse of infection (data not shown). Given that Nef-deficient HIV-1 viruses have been shown to be less infectious than wild type virions (45), intracellular flow cytometry was also performed 8 days post-infection to evaluate the frequency of p24-expressing cells (provides an estimate of cells productively infected with HIV-1). Infection with {Delta}Nef/NL4-3 viruses resulted in a higher percentage of p24-expressing cells in both J-IL-2-LUC and J-NFAT-LUC cells as compared with infection with WT-Nef/NL4-3 virions (Fig. 7). These data provide evidence that Nef plays a role in the HIV-1-induced hyperactivation of NFAT, resulting in an important induction of IL-2 promoter activity.



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  		FIG. 6.
HIV-1-mediated up-regulation of IL-2 promoter activity involves Nef and NFAT induction. J-IL-2-LUC (panel A) and J-NFAT-LUC cells (panel B) were infected with wild-type NL4-3 or Nef-deleted NL4-3 mutant. Eight days post-infection, cells were either left untreated or were stimulated with the listed agents. After 8 h, cells were lysed to monitor luciferase activity. Results are presented as the mean ± S.D. of quadruplicate samples. Data shown are representative of three independent experiments. RLU, relative light unit.

 



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  		FIG. 7.
Higher levels of p24-expressing cells are achieved upon infection with Nef-deficient NL4-3 viruses. J-IL-2-LUC (panels A and B) 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 and C). Eight days post-infection, the percentage of p24-expressing cells was monitored by intracellular flow cytometry.

 
Tat Also Mediates Hyperactivation of the IL-2 Promoter but Independently of NFAT—In an attempt to be consistent with our previous experiments, it would have been appropriate to use Tat-deficient viruses to scrutinize the involvement of Tat in stimuli-dependent regulation of the IL-2 promoter and NFAT activity. Unfortunately, HIV-1 proviral mutants that lack the viral regulatory gene tat are unable to replicate in human T cells. Thus, co-transfection studies were performed to investigate if expression of HIV-1 Tat could render human T cells more sensitive to the tested stimuli. We transfected Jurkat T lymphoid cells with either pIL-2-LUC or pNFAT-LUC, and with or without a Tat expression vector. Twenty-four hours after transfection, the reporter gene activity in these cells was measured, either without further treatment, or following a 8-h stimulation with PHA, PMA/PHA, PMA/Iono, OKT3/9.3, and bpV[pic]. The graph shown in Fig. 8A demonstrates that Tat expression in Jurkat T cells induces a much stronger IL-2 promoter activity upon stimulation with any of the tested activating agents. On the other hand, when co-transfections were conducted with the pNFAT-LUC vector, no such overinduction of luciferase reporter gene expression could be attributed to Tat expression (Fig. 8B). These results thus indicate that Tat also contributes to the superinduction of IL-2 promoter activity upon HIV-1 infection. However, as opposed to Nef, transcription factor(s) other than NFAT are likely to be targeted by Tat.



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  		FIG. 8.
Tat is also involved in the HIV-1-induced hypersensitivity of Jurkat cells to stimulation but in an NFAT-independent manner. Jurkat cells were co-transfected with pIL-2-LUC (panel A) or pNFAT-LUC (panel B) in combination with either a Tat-encoding vector or an appropriate empty control vector. Twenty-four hours following transfection, cells were either left untreated or were stimulated with the listed agents. After 8 h, cells were lysed to monitor luciferase activity. Results are presented as the mean ± S.D. of quadruplicate samples. Data shown are representative of three independent experiments.

 
Stimuli-mediated Nuclear Translocation of NFAT, NF-{kappa}B, and AP-1 Is Augmented upon HIV-1 Infection—In addition to NFAT, the activation of the IL-2 promoter involves the induction of other transcription factors such as NF-{kappa}B and AP-1. Thus to confirm the participation of NFAT and to examine whether the induction of NF-{kappa}B and AP-1 is also augmented upon stimulation of HIV-1-infected cells, mobility shift assays were performed using the appropriate {gamma}-32P-labeled probes. Nuclear extracts from uninfected and HIV-1-infected Jurkat cells were derived from cells, either untreated, or treated with the listed agents. Electrophoretic mobility shift assay analysis revealed that complexes specific for NFAT, NF-{kappa}B, and AP-1 were more intense in nuclear extracts from HIV-1-infected cells than from uninfected cells for the majority of the tested activators (Fig. 9, panels A–C). Interestingly, in unstimulated cells the nuclear levels of these transcription factors were higher upon HIV-1 infection especially for NFAT and AP-1. For each signal, the specificity of the signal was confirmed through competition experiments. These results suggest that HIV-1 infection leads to higher levels of NFAT, NF-{kappa}B, and AP-1 nuclear translocation following stimulation of Jurkat T cells.




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  		FIG. 9.
Activation-induced nuclear translocation of NFAT, NF-{kappa}B, and AP-1 is augmented upon HIV-1 infection of CD4+ T cells. Parental Jurkat cells were either left uninfected or were infected with NL4-3. Eight days post-infection, cells were either left untreated or were stimulated for 1 h with the listed agents. Nuclear extracts were next incubated with an NFAT- (panel A), NF-{kappa}B- (panel B), or AP-1-labeled probe (panel C) to be finally analyzed on a 4% native polyacrylamide gel. Competitions were also performed with the appropriate cold probe to verify the specificity of the shifted complexes. The arrows on the left indicate the specific complexes.

 
Hyperstimulation Is Also Observed upon HIV-1 Infection of a More Natural Cellular Reservoir, i.e. CD4+ T Lymphocytes—To verify if the HIV-1-induced hyperstimulation observed in Jurkat cells can also take place in primary human cells, purified CD4-expressing T lymphocytes were infected with GFP-encoding HIV-1 particles, an experimental strategy allowing an easy discrimination of infected versus uninfected cells by flow cytometry. Four days post-infection, about 10% of CD4+ T cells were GFP-positive, i.e. infected. Following stimulation, intracellular IL-2 measurement indicated that a greater proportion of IL-2-expressing cells was present in the HIV-1-infected population (i.e. GFP-positive) when compared with the uninfected population (i.e. GFP-negative) (Fig. 10).



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

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study describes a strong positive effect on T cell signaling, and more precisely on IL-2 promoter transcription, upon infection of human T cells with complete HIV-1 progeny virus. This effect is suggested to implicate at least two HIV-1 regulatory proteins, i.e. Nef and Tat, and to be mediated via induction of three transcription factors recognized as important players in the regulation of IL-2, i.e. NFAT, NF-{kappa}B, and AP-1. Although some previous studies have also reported positive effects on T cell activation by these two viral proteins, in these cases, this phenomenon was observed when a single virus protein (i.e. Nef or Tat) was present via either transient or stable expression in target cells (16–21). The present work thus more closely parallels in vivo situations because we used fully competent viruses instead of singly expressed HIV-1 gene products. Our results also suggest that the effect on IL-2 and NFAT hyperactivation is dependent on the development over time of HIV-1 infection and intracellular expression of viral proteins (as opposed to extracellular factors). A selective isolation of virus-infected cells with the use of recombinant virus coding for surface murine CD24 allowed us to demonstrate that the hyper-responsiveness state is mainly present in cells harboring HIV-1.

An exciting feature of this series of investigations consists in the demonstration that Nef also exerts its modulatory role with respect to T cell signaling pathways even in the context of the complete viral genome. Previous work has shown that Nef has the potential to alter signal transduction events most likely through interactions with several signaling molecules (reviewed in Ref. 46). For example, Nef associates with numerous cellular partners such as the Nef-associated kinase identified as a member of the p21-activated kinase family (47–50), a serine kinase (51), mitogen-activated protein kinase (52), c-Raf-1 (53), p53 (54), protein kinase C{theta} (55), and members of the Src family of tyrosine kinases (e.g. Lck, Hck, Lyn, and Fyn) (51, 52, 56–60). The molecular mechanism of action of Nef is linked with its ability to induce transcription factors such as NFAT, NF-{kappa}B, and AP-1 (20, 21). The findings that Nef interacts with endogenous inositol triphosphate receptor (19) and increases the 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, considering that we observed that HIV-1 infection increases stimuli-mediated induction of NFAT, NF-{kappa}B, and AP-1. More importantly, we have confirmed the importance of Nef in NFAT superactivation in the context of HIV-1 infection. Our results therefore agree with the previously suggested increase in activation of NFAT by Nef (20).

Although previous works have demonstrated that PMA alone could become an activator of NFAT in cells transfected with a Nef expressing vector (21, 62), our results suggest no induction of NFAT or IL-2 gene expression upon PMA stimulation of HIV-1-infected cells. We cannot provide a clear explanation for these discrepancies at this point but one might speculate that Nef levels might affect the extent to which signal transduction is altered and/or the presence of viral proteins other than Nef might exert a varying effect on signaling pathways normally affected by Nef alone. This reinforces the validity of our data as more physiologic levels of Nef were present in the tested Jurkat cells in combination with other HIV-1 proteins through the natural HIV-1 infection process.

The HIV-1-encoded transactivating Tat protein is essential for 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 promoter activity upon treatment with phorbol ester and calcium ionophore through the NFAT motif (18). Moreover, the expression of Tat in human T cells is associated with an augmentation of IL-2 production in response to engagement of CD3 and CD28 surface receptors (17). The effect of Tat has been shown to be mediated by the CD28-responsive element (CD28RE) in the IL-2 promoter (17). The unique CD28RE motif was identified as an enhancer element located at positions –164 to –154 of the minimal IL-2 promoter (64). Full responsiveness to CD28-mediated 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 sequences for both NF-{kappa}B and AP-1 transcription factors (65–69). Data from co-transfection experiments revealed that NFAT is not playing a role in Tat-dependent hyperactivation of the IL-2 promoter, thus suggesting that the effect of Tat on the overinduction of IL-2 promoter activity might be exclusively related to a stronger induction of NF-{kappa}B and/or AP-1. Although the use of Tat-deficient viruses would have been more appropriate in the present study, the inability of such viruses to productively infect human T cells would have complicated the interpretation of the data. Small interfering RNA duplexes could have been used to block Tat expression. This technical strategy seems laudable at first sight but targeting the tat gene through this approach would have also led to degradation of other unspliced or singly spliced mRNAs. This is exemplified by a previous work showing a marked reduction in the level of expression of all 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 interfering RNAs (70).

Our electrophoretic mobility shift assay data also indicates that the overactivation of all three tested transcription factors also occurs in unstimulated infected cells. However, this contrasts with the luciferase reporter gene expression data, in which no differences could be measured between HIV-1-infected and uninfected Jurkat cells at basal level in terms of NFAT and IL-2 promoter activation. This suggests that HIV-1 infection itself can alter the cascade of events regulating the activation of these transcription factors, but that either a weak activation level (for example, NF-{kappa}B and AP-1) or the lack of other post-translational modifications does not allow achieving a competent transcriptional complex for NFAT-dependent and IL-2 promoter-mediated transcription. Experiments are presently underway to shed light on this matter.

Although HIV-1 can gain entry inside resting CD4+ T lymphocytes that express appropriate entry receptors, the viral replicative cycle is blocked at the preintegration step unless activation signals are provided through the TCR (71–78). Synthesis of full-length viral DNA is prevented in quiescent T cells because of a premature termination of reverse transcription (72). It has also been demonstrated that import of the preintegration complex into the nucleus is not efficient in resting CD4+ T cells (79, 80). As the blockade at reverse transcription can be overcome by NFAT (81) and Nef has been shown to be incorporated in mature HIV-1 particles (82, 83), it can be proposed that Nef proteins released within the cell upon virus entry might favor reverse transcription by diminishing the requirements for NFAT activation. Late events in the HIV-1 life cycle, such as viral gene transcription and the assembly of mature virions, are also highly dependent on T cell activation. It is thus clear that HIV-1 has evolved several mechanisms to exploit the cellular signaling machinery to facilitate its replication and propagation through the infected host. The virus itself, via some of its products such as Nef and Tat, may thus contribute to the T cell activation process that is required for an efficient virus spread by priming the CD4+ T lymphocyte for activation. In fact, it has been suggested that the process of HIV-1 infection results in a lowered threshold for T cell activation 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 nodes early after HIV-1 infection (85).

At each end of its genome, HIV-1 carries regulatory domains known as long terminal repeat. Various studies have shown that the HIV-1 long terminal repeat region is composed of various binding motifs that are also present in the regulatory regions of genes induced after T cell activation such as the IL-2 gene. A direct result of the similarity between the architecture of the HIV-1 LTR and the IL-2 promoter is an intimate link between T cell activation and HIV-1 transcription. Because interactions between some HIV-1 viral gene products and the cell signaling machinery can alter the activation state of host cell, it is legitimate to postulate that the pool of lymphocytes that can be infected by the virus will be amplified. Indeed, it can be proposed that upon stimulation of HIV-1-infected cells, the concomitant up-regulation of IL-2 secretion will lead to the recruitment of bystander resting CD4+ T lymphocytes and will render such cells more susceptible to productive virus infection.

At this point, it is important to emphasize that a Tat-dependent enhancement of response to T cell activation via the TCR and CD28 receptors has already been reported in primary human T cells (17). Moreover, a Nef-mediated priming upon stimulation with anti-CD3/CD28 antibodies has also been demonstrated in primary CD4+ T lymphocytes (20). Our findings that a hyperactive state is observed also within purified CD4+ T cells infected with fully competent GFP-encoding HIV-1 viruses is perfectly in line with these two previous reports and provide physiological significance to the present work. Our results further reinforce the notion that HIV-1 infection alters signal transduction in T cells. Such changes are likely to influence the onset of infection and might further help in the spreading of infection in vivo. It is thereby important to clarify the overall HIV-1-associated dysregulation of signal transduction events in the host cell because it could provide a novel means for interfering with the pathologies linked with HIV-1 infection.


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

§ Both authors contributed equally to this work. Back

|| Supported by a Scholarship Award (Junior 1 level) from the Fonds de la Recherche en Santé du Québec, Back

** Recipient of the Canada Research Chair in Human Immuno-Retrovirology (Tier 1 level). To whom correspondence should be addressed: Laboratory of Human Immuno-Retrovirology, Research Center in Infectious 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{at}crchul.ulaval.ca.

1 The abbreviations used are: HIV-1, human immunodeficiency virus type-1; PBMC, peripheral blood mononuclear cell; TCR, T cell receptor; IL-2, interleukin-2; PHA, phytohemagglutinin; PMA, phorbol 12-myristate 13-acetate; Iono, ionomycin; HSA, heat stable antigen; GFP, green fluorescent protein; PBS, phosphate-buffered saline; NFAT, nuclear factor of activated T cells; AP-1, activation protein-1. Back


   ACKNOWLEDGMENTS
 
We thank Dr. M. Dufour for flow cytometric analyses and Sylvie Méthot for editorial assistance.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. McCune, M. (2001) Nature 410, 974–979[CrossRef][Medline] [Order article via Infotrieve]
  2. Khanna, K. V., Yu, X. F., Ford, D. H., Ratner, L., Hildreth, J. K., and Markham, R. B. (2000) J. Immunol. 164, 1408–1415[Abstract/Free Full Text]
  3. Rieckmann, P., Poli, G., Fox, C. H., Kehrl, J. H., and Fauci, A. S. (1991) J. Immunol. 147, 2922–2927[Abstract]
  4. Bass, H. Z., Nishanian, P., Hardy, W. D., Mitsuyasu, R. T., Esmail, E., Cumberland, W., and Fahey, J. L. (1992) Clin. Immunol. Immunopathol. 64, 63–70[CrossRef][Medline] [Order article via Infotrieve]
  5. Liu, Z., Cumberland, W. G., Hultin, L. E., Kaplan, A. H., Detels, R., and Giorgi, J. V. (1998) J. Acquir. Immune Defic. Syndr. Hum. Retrovirol. 18, 332–340[Medline] [Order article via Infotrieve]
  6. Mahalingam, M., Peakman, M., Davies, E. T., Pozniak, A., McManus, T. J., and Vergani, D. (1993) Clin. Exp. Immunol. 93, 337–343[Medline] [Order article via Infotrieve]
  7. Baier-Bitterlich, G., Wachter, H., and Fuchs, D. (1996) J. Acquir. Immune Defic. Syndr. Hum. Retrovirol. 13, 184–193[Medline] [Order article via Infotrieve]
  8. Fahey, J. L., Taylor, J. M., Detels, R., Hofmann, B., Melmed, R., Nishanian, P., and Giorgi, J. V. (1990) N. Engl. J. Med. 322, 166–172[Abstract]
  9. Fahey, J. L., Taylor, J. M., Manna, B., Nishanian, P., Aziz, N., Giorgi, J. V., and Detels, R. (1998) AIDS 12, 1581–1590[CrossRef][Medline] [Order article via Infotrieve]
  10. Aukrust, P., Liabakk, N. B., Muller, F., Espevik, T., and Froland, S. S. (1995) Infection 23, 9–15[CrossRef][Medline] [Order article via Infotrieve]
  11. Aziz, N., Nishanian, P., Taylor, J. M., Mitsuyasu, R. T., Jacobson, J. M., Dezube, B. J., Lederman, M. M., Detels, R., and Fahey, J. L. (1999) J. Infect. Dis. 179, 843–848[CrossRef][Medline] [Order article via Infotrieve]
  12. Lederman, M. M., Kalish, L. A., Asmuth, D., Fiebig, E., Mileno, M., and Busch, M. P. (2000) AIDS 14, 951–958[CrossRef][Medline] [Order article via Infotrieve]
  13. Lien, E., Aukrust, P., Sundan, A., Muller, F., Froland, S. S., and Espevik, T. (1998) Blood 92, 2084–2092[Abstract/Free Full Text]
  14. Nishanian, P., Hofmann, B., Wang, Y., Jackson, A. L., Detels, R., and Fahey, J. L. (1991) AIDS 5, 805–812[Medline] [Order article via Infotrieve]
  15. Zangerle, R., Gallati, H., Sarcletti, M., Weiss, G., Denz, H., Wachter, H., and Fuchs, D. (1994) J. Acquir. Immune Defic. Syndr. 7, 79–85[Medline] [Order article via Infotrieve]
  16. Westendorp, M. O., Weber-Li, M., Frank, R. W., and Krammer, P. H. (1994) J. Virol. 68, 4177–4185[Abstract/Free Full Text]
  17. Ott, M., Emiliani, S., Van Lint, C., Herbein, G., Lovett, J., Chirmule, N., McCloskey, T., Pahwa, S., and Verdin, E. (1997) Science 275, 1481–1485[Abstract/Free Full Text]
  18. Vacca, A., Farina, M., Maroder, M., Alesse, E., Screpanti, I., Frati, L., and Gulino, A. (1994) Biochem. Biophys. Res. Commun. 205, 467–474[CrossRef][Medline] [Order article via Infotrieve]
  19. Manninen, A., and Saksela, K. (2002) J. Exp. Med. 195, 1023–1032[Abstract/Free Full Text]
  20. Wang, J. K., Kiyokawa, E., Verdin, E., and Trono, D. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 394–399[Abstract/Free Full Text]
  21. Manninen, A., Renkema, G. H., and Saksela, K. (2000) J. Biol. Chem. 275, 16513–16517[Abstract/Free Full Text]
  22. Baur, A. S., Sawai, E. T., Dazin, P., Fantl, W. J., Cheng-Mayer, C., and Peterlin, B. M. (1994) Immunity 1, 373–384[CrossRef][Medline] [Order article via Infotrieve]
  23. Gonzalez, E., Punzon, C., Gonzalez, M., and Fresno, M. (2001) J. Immunol. 166, 4560–4569[Abstract/Free Full Text]
  24. Fortin, J. F., Barbeau, B., Robichaud, G. A., Pare, M.-È., Lemieux, A. M., and Tremblay, M. J. (2001) Blood 97, 2390–2400[Abstract/Free Full Text]
  25. Timmerman, L. A., Clipstone, N. A., Ho, S. N., Northrop, J. P., and Crabtree, G. R. (1996) Nature 383, 837–840[CrossRef][Medline] [Order article via Infotrieve]
  26. Adachi, A., Gendelman, H. E., Koenig, S., Folks, T., Willey, R., Rabson, A., and Martin, M. A. (1986) J. Virol. 59, 284–291[Abstract/Free Full Text]
  27. He, J., Choe, S., Walker, R., Di Marzio, P., Morgan, D. O., and Landau, N. R. (1995) J. Virol. 69, 6705–6711[Abstract]
  28. Connor, R. I., Chen, B. K., Choe, S., and Landau, N. R. (1995) Virology 206, 935–944[CrossRef][Medline] [Order article via Infotrieve]
  29. Yee, J. K., Miyanohara, A., Laporte, P., Bouic, K., Burns, J. C., and Friedmann, T. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 9564–9568[Abstract/Free Full Text]
  30. Levy, D. N., Aldrovandi, G. M., Kutsch, O., and Shaw, G. M. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 4204–4209[Abstract/Free Full Text]
  31. Fortin, J. F., Cantin, R., Lamontagne, G., and Tremblay, M. (1997) J. Virol. 71, 3588–3596[Abstract]
  32. Bounou, S., Leclerc, J. E., and Tremblay, M. J. (2002) J. Virol. 76, 1004–1014[Abstract/Free Full Text]
  33. Barbeau, B., Bernier, R., Dumais, N., Briand, G., Olivier, M., Faure, R., Posner, B. I., and Tremblay, M. J. (1997) J. Biol. Chem. 272, 12968–12977[Abstract/Free Full Text]
  34. Schreiber, E., Matthias, P., Müller, M., and Schaffner, W. (1989) Nucleic Acids Res. 17, 6419[Free Full Text]
  35. Mustafa, F., and Robinson, H. L. (1993) J. Virol. 67, 6909–6915[Abstract/Free Full Text]
  36. Linsley, P. S., and Ledbetter, J. A. (1993) Annu. Rev. Immunol. 11, 191–212[Medline] [Order article via Infotrieve]
  37. Roy, J., Audette, M., and Tremblay, M. J. (2001) J. Biol. Chem. 276, 14553–14561[Abstract/Free Full Text]
  38. Ouellet, M., Barbeau, B., and Tremblay, M. J. (1999) J. Biol. Chem. 274, 35029–35036[Abstract/Free Full Text]
  39. Barat, C., and Tremblay, M. J. (2003) J. Biol. Chem. 278, 6992–7000[Abstract/Free Full Text]
  40. Ouellet, M., Barbeau, B., and Tremblay, M. J. (2003) Prog. Nucleic Acids Res. Mol. Biol. 73, 69–105[Medline] [Order article via Infotrieve]
  41. Ouellet, M., Roy, J., Barbeau, B., Geleziunas, R., and Tremblay, M. J. (2003) Biochemistry 42, 8260–8271[CrossRef][Medline] [Order article via Infotrieve]
  42. Crabtree, G. R. (1999) Cell 96, 611–614[CrossRef][Medline] [Order article via Infotrieve]
  43. Chow, C.-W., Rincón, M., and Davis, R. J. (1999) Mol. Cell. Biol. 19, 2300–2307[Abstract/Free Full Text]
  44. Northrop, J. P., Ullman, K. S., and Crabtree, G. R. (1993) J. Biol. Chem. 268, 2917–2923[Abstract/Free Full Text]
  45. Zheng, Y. H., Plemenitas, A., Linnemann, T., Fackler, O. T., and Peterlin, B. M. (2001) Curr. Biol. 11, 875–879[CrossRef][Medline] [Order article via Infotrieve]
  46. Arendt, C. W., and Littman, D. R. (2001) Genome Biol. 2, Reviews 1030
  47. Lu, X., Wu, X., Plemenitas, A., Yu, H., Sawai, E. T., Abo, A., and Peterlin, B. M. (1996) Curr. Biol. 6, 1677–1684[CrossRef][Medline] [Order article via Infotrieve]
  48. Luo, T., and Garcia, J. V. (1996) J. Virol. 70, 6493–6496[Abstract]
  49. Nunn, M. F., and Marsh, J. W. (1996) J. Virol. 70, 6157–6161[Abstract]
  50. Sawai, E. T., Khan, I. H., Montbriand, P. M., Peterlin, B. M., Cheng-Mayer, C., and Luciw, P. A. (1996) Curr. Biol. 6, 1519–1527[CrossRef][Medline] [Order article via Infotrieve]
  51. Baur, A. S., Sass, G., Laffert, B., Willbold, D., Cheng-Mayer, C., and Peterlin, B. M. (1997) Immunity 6, 283–291[CrossRef][Medline] [Order article via Infotrieve]
  52. Greenway, A., Azad, A., Mills, J., and McPhee, D. (1996) J. Virol. 70, 6701–6708[Abstract/Free Full Text]
  53. Hodge, D. R., Dunn, K. J., Pei, G. K., Chakrabarty, M. K., Heidecker, G., Lautenberger, J. A., and Samuel, K. P. (1998) J. Biol. Chem. 273, 15727–15733[Abstract/Free Full Text]
  54. Greenway, A. L., McPhee, D. A., Allen, K., Johnstone, R., Holloway, G., Mills, J., Azad, A., Sankovich, S., and Lambert, P. (2002) J. Virol. 76, 2692–2702[Abstract/Free Full Text]
  55. Smith, B. L., Krushelnycky, B. W., Mochly-Rosen, D., and Berg, P. (1996) J. Biol. Chem. 271, 16753–16757[Abstract/Free Full Text]
  56. Collette, Y., Dutartre, H., Benziane, A., Ramos, M., Benarous, R., Harris, M., and Olive, D. (1996) J. Biol. Chem. 271, 6333–6341[Abstract/Free Full Text]
  57. Salghetti, S., Mariani, R., and Skowronski, J. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 349–353[Abstract/Free Full Text]
  58. Arold, S., O'Brien, R., Franken, P., Strub, M. P., Hoh, F., Dumas, C., and Ladbury, J. E. (1998) Biochemistry 37, 14683–14691[CrossRef][Medline] [Order article via Infotrieve]
  59. Saksela, K., Cheng, G., and Baltimore, D. (1995) EMBO J. 14, 484–491[Medline] [Order article via Infotrieve]
  60. Cheng, H., Hoxie, J. P., and Parks, W. P. (1999) Virology 264, 5–15[CrossRef][Medline] [Order article via Infotrieve]
  61. Djordjevic, J. T., Schibeci, S. D., Stewart, G. J., and Williamson, P. (2004) AIDS Res. Hum. Retroviruses 20, 547–555[CrossRef][Medline] [Order article via Infotrieve]
  62. Manninen, A., Huotari, P., Hiipakka, M., Renkema, G. H., and Saksela, K. (2001) J. Virol. 75, 3034–3037[Abstract/Free Full Text]
  63. Jones, K. A. (1993) Curr. Opin. Cell Biol. 5, 461–468[CrossRef][Medline] [Order article via Infotrieve]
  64. Fraser, J. D., Irving, B. A., Crabtree, G. R., and Weiss, A. (1991) Science 251, 313–316[Abstract/Free Full Text]
  65. Shapiro, V. S., Truitt, K. E., Imboden, J. B., and Weiss, A. (1997) Mol. Cell. Biol. 17, 4051–4058[Abstract]
  66. Ghosh, P., Tan, T. H., Rice, N. R., Sica, A., and Young, H. A. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 1696–1700[Abstract/Free Full Text]
  67. McGuire, K. L., and Iacobelli, M. (1997) J. Immunol. 159, 1319–1327[Abstract]
  68. Lai, J. H., Horvath, G., Subleski, J., Bruder, J., Ghosh, P., and Tan, T. H. (1995) Mol. Cell. Biol. 15, 4260–4271[Abstract]
  69. Butscher, W. G., Powers, C., Olive, M., Vinson, C., and Gardner, K. (1998) J. Biol. Chem. 273, 552–560[Abstract/Free Full Text]
  70. Coburn, G. A., and Cullen, B. R. (2002) J. Virol. 76, 9225–9231[Abstract/Free Full Text]
  71. Stevenson, M., Stanwick, T. L., Dempsey, M. P., and Lamonica, C. A. (1990) EMBO J. 9, 1551–1560[Medline] [Order article via Infotrieve]
  72. Zack, J. A., Arrigo, S. J., Weitsman, S. R., Go, A. S., Haislip, A., and Chen, I. S. Y. (1990) Cell 61, 213–222[CrossRef][Medline] [Order article via Infotrieve]
  73. Zack, J. A. (1995) Adv. Exp. Med. Biol. 374, 27–31[Medline] [Order article via Infotrieve]
  74. Zack, J. A., Haislip, A. M., Krogstad, P., and Chen, I. S. (1992) J. Virol. 66, 1717–1725[Abstract/Free Full Text]
  75. Spina, C. A., Guatelli, J. C., and Richman, D. D. (1995) J. Virol. 69, 2977–2988[Abstract]
  76. Chun, T. W., Finzi, D., Margolick, J., Chadwick, K., Schwartz, D., and Siliciano, R. F. (1995) Nat. Med. 1, 1284–1290[CrossRef][Medline] [Order article via Infotrieve]
  77. Sonza, S., Maerz, A., Deacon, N., Meanger, J., Mills, J., and Crowe, S. (1996) J. Virol. 70, 3863–3869[Abstract]
  78. Chou, C. S., Ramilo, O., and Vitetta, E. S. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 1361–1365[Abstract/Free Full Text]
  79. Bukrinsky, M. I., Sharova, N., Dempsey, M. P., Stanwick, T. L., Bukrinskaya, A., Haggerty, S., and Stevenson, M. (1992) Proc. Natl. Acad. Sci. U. S. A. 15, 6580–6584
  80. Bukrinsky, M. I., Stanwick, T. L., Dempsey, M. P., and Stevenson, M. (1991) Science 254, 423–427[Abstract/Free Full Text]
  81. Kinoshita, S., Chen, B. K., Kaneshima, H., and Nolan, G. P. (1998) Cell 95, 595–604[CrossRef][Medline] [Order article via Infotrieve]
  82. Pandori, M. W., Fitch, N. J., Craig, H. M., Richman, D. D., Spina, C. A., and Guatelli, J. C. (1996) J. Virol. 70, 4283–4290[Abstract]
  83. Kotov, A., Zhou, J., Flicker, P., and Aiken, C. (1999) J. Virol. 73, 8824–8830[Abstract/Free Full Text]
  84. Schrager, J. A., and Marsh, J. W. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 8167–8172[Abstract/Free Full Text]
  85. Pantaleo, G., Graziosi, C., Demarest, J. F., Butini, L., Montroni, M., Fox, C. H., Orenstein, J. M., Kotler, D. P., and Fauci, A. S. (1993) Nature 362, 355–358[CrossRef][Medline] [Order article via Infotrieve]

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