Attachment of Human Immunodeficiency Virus-1 (HIV-1) Particles Bearing Host-encoded B7-2 Proteins Leads to Nuclear Factor-kappa B- and Nuclear Factor of Activated T Cells-dependent Activation of HIV-1 Long Terminal Repeat Transcription*

Salim BounouDagger, Nancy Dumais, and Michel J. Tremblay§

From the Centre de Recherche en Infectiologie, Centre Hospitalier Universitaire de Québec, Pavillon CHUL, and Département de Biologie médicale, Faculté de Médecine, Université Laval, Ste-Foy, Québec G1V 4G2, Canada

Received for publication, March 16, 2000, and in revised form, October 25, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Previous studies have shown that human immunodeficiency virus type-1 (HIV-1) can incorporate several surface proteins of host origin. Recent findings indicate that host-encoded cell surface constituents retain their functionality when found embedded into the viral envelope. The primary objective of the current study was to define whether interaction between some specific virion-bound host proteins with their natural cognate ligands present on target cells could mediate intracellular signaling cascade(s). For this purpose, we have generated a whole series of isogenic virus stocks (NL4-3 backbone) bearing or not bearing on their surface foreign CD28, CD54 (ICAM-1), CD80 (B7-1) or CD86 (B7-2) proteins. Our results indicate that incubation of human T lymphoid cells with virions bearing host-derived B7-2 proteins and anti-CD3 antibody can potently activate HIV-1 long terminal repeat-driven gene expression. This up-regulating effect necessitates the involvement of nuclear factor-kappa B (NF-kappa B) and nuclear factor of activated T cells (NFAT) as revealed by the use of vectors coding for dominant negative versions of both transcription factors (i.e. Ikappa Balpha S32A/36A and dnNFAT) and band shift assays. The increase of NF-kappa B activity was abolished when infection with B7-2-bearing HIV-1 particles was performed in the presence of the fusion protein CTLA-4 Ig suggesting that the interaction between virally embedded B7-2 and CD28 on the target cell is responsible for the observed NF-kappa B induction. The findings presented here provide the first demonstration that host-encoded proteins acquired by HIV-1 can mediate signal transduction events.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The attachment of HIV-11 to target cells is occurring via high affinity binding between the external viral envelope gp120 and the cell surface CD4 glycoprotein. Recently, several studies have identified chemokine receptors as major fusion cofactors for T cell- and macrophage-tropic HIV-1 isolates (reviewed in Ref. 1). It is known that a threshold number of interactions between viral and cellular surface molecules is necessary to achieve an efficient viral infection because the surfaces of both CD4+ T cells and HIV-1 are highly negatively charged (2). McKeating and co-workers (3) have reported that the observed spontaneous shedding of gp120 in vitro was linked to a loss of HIV-1 infectivity. The shedding of gp120 in vitro is also taking place in vivo (4). Thus, one may assume that the infection process will be greatly jeopardized if the number of gp120 molecules is too low to provide the threshold binding energy required to overcome the repulsive electrostatic forces between cellular and viral membranes (2). Changes in infectivity have dramatic consequences in viral output. For instance, a reduction in infectivity of 50% will diminish the viral production to 0.0032% of the expected output after only five replicative cycles (5). The binding of virus-associated gp120 to cellular CD4 is often weak, and most cell types that are permissive for HIV-1 infection express low levels of CD4. Thus, other interactions between the viral entity and the host cell surface could play a dominant role in the attachment process. Others and we have postulated that host-derived proteins present on the viral surface could influence the initial interaction between the virion and its target (6-8).

The incorporation of cellular constituents in newly formed viruses has been demonstrated to occur in retroviruses (9-13). Similar studies were extended to HIV-1, and a vast array of cell membrane proteins was found to be acquired by this retrovirus such as the HLA-DR, -DP, and -DQ determinants of major histocompatibility complex class-II (MHC-II), ICAM-1, LFA-1, beta 2-microglobulin, CD3, CD43, CD44, CD55, CD59, CD63, and the transferrin receptor (CD71) (reviewed in Ref. 14). It should be stated that incorporation of selected host cell molecules was found to be conserved among different HIV-1 subtypes and strains that were expanded on phytohemagglutinin-activated peripheral blood mononuclear cells (15). An initial report has shown that incorporation of MHC-I molecules is not essential for HIV-1 infectivity (16), but data from recent studies clearly indicate that several virion-acquired host proteins have functional effects on the biology of HIV-1 (reviewed in Ref. 14).

Although it is clear now that virion-anchored foreign proteins play an essential role in the attachment process of HIV-1 to its target, there is no information available yet with respect to putative signaling events that could be mediated by interaction between HIV-1-bound host proteins and their physiologic counter-receptors located on the cell surface. This is somewhat surprising, based on previous observations suggesting that intracellular signal transduction events can be mediated upon the virus attachment process. Indeed, interaction between the HIV-1 envelope and CD4/chemokine coreceptors has been shown to mediate several intracellular signaling events, including phosphorylation of phosphatidylinositol 3-kinase (PI 3-kinase), tyrosine phosphorylation of Pyk2, focal adhesion kinase and CCR5, activation of PI 4-kinase, Raf-1, and several mitogen-activated protein kinase pathways (e.g. mitogen-activated protein kinase/extracellular signal-regulated kinase kinase, c-Jun N-terminal kinase, p38) (17-28). Thus, it can be proposed that binding of some virally incorporated host proteins with their normal counter-receptors can also lead to signal transduction into target cells.

Thus, the primary goal of this study was to define whether different intracellular biochemical events can be initiated depending of the foreign host cell surface constituent that is found embedded within the HIV-1 envelope. Our experiments were performed using isogenic virions bearing or not bearing on their surface host-derived B7-1 (CD80), B7-2 (CD86), CD28, and ICAM-1 (CD54) proteins. We demonstrate that the nature of virion-anchored host protein is indeed of utmost importance with respect to HIV-1-mediated signal transduction pathway that is seen upon virus-cell attachment. Furthermore, we provide evidence that such signaling events can positively modulate HIV-1 LTR-driven gene expression via activation of nuclear factor-kappa B (NF-kappa B) and nuclear factor of activated T cells (NFAT), two transcription factors.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Lines and Culture Conditions-- The 1G5 T cell line, a Jurkat E6.1 derivative that harbors two stably integrated constructs constituted of the luciferase gene under the control of the HIV-1SF2 LTR, was obtained from Dr. Estuardo Aguilar-Cordova and Dr. John Belmont through the AIDS Research and Reference Reagent Program (Division of AIDS, NIAID, National Institutes of Health, Bethesda, MD) (29). The human T lymphoid cell line Jurkat clone E6.1 was used in this study because it is considered as a model cell line for the study of T cell signaling machinery (30). We have also used DT30, which are murine Fc receptor-bearing mastocytoma P815 cells stably expressing cell surface human B7-1 (CD80) proteins (31). Both cell lines were obtained from the American Type Culture Collection (Rockville, MD). Cells were grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (Life Technologies, Inc.), 2 mM glutamine, 100 units/ml penicillin G, 100 µg/ml streptomycin, 0.22% NaHCO3, and were maintained at 37 °C under a 5% CO2 humidified atmosphere. DT30 cells were maintained under the pressure of 1 mg/ml selective agent G418 (Life Technologies, Inc.). DT30 cells were fixed briefly in 1% paraformaldehyde, washed extensively with phosphate-buffered saline (PBS), and then stored frozen in aliquots at a density of 2 × 106/ml of PBS. Since such cells are fixed, they do not grow or secrete factors that could mediate signal transduction in studied target cells. For our experiments aimed at stimulating Jurkat cells, 2 × 104 DT30 was added to 105 Jurkat cells.

Plasmids-- We have used pLTR-Luc (HIV-1 LTR from strain HXB2) and pmkappa BLTR-Luc plasmids, which were kindly provided by Dr. K. L. Calame (Columbia University, New York, NY). Such molecular constructs contain the luciferase reporter gene under the control of the wild-type (GGGACTTTCC) or the NF-kappa B-mutated (CTCACTTTCC) HIV-1 LTR domain (-453 to +80) (32). The pkappa B-TATA-LUC plasmid only contains the HIV-1 enhancer region (-105/-70) and a TATA box placed upstream of the luciferase gene (33). The dominant negative Ikappa Balpha -expressing vector pCMV-Ikappa Balpha S32A/36A has been described previously (33). These two latter molecular constructs were generous gifts from Dr. W.C. Greene (J. Gladstone Institutes, San Francisco, CA). The pCDNA3-dnNFAT vector codes for a dominant negative NFAT mutant and was supplied by Dr. R. J. Davis (Howard Hughes Medical Institute, Worcester, MA) (34). pNFAT-Luc, containing the minimal IL-2 promoter with three tandem copies of the NFAT1-binding site, was kindly provided by Dr. G. Crabtree (Howard Hughes Medical Institute, Stanford, CA) (35). The commercial pNFkappa B-Luc molecular construct contains five consensus NF-kappa B-binding sequences placed in front of the luciferase gene along with a minimal promoter (Stratagene). pNL4-3 is a full-length infectious molecular clone of HIV-1 (provided by the AIDS Research and Reference Reagent Program) (36). pCD1.8 is an eukaryotic expression vector containing the entire human ICAM-1 cDNA and was obtained from Dr. T. A. Springer (Center for Blood Research, Boston, MA) (37). pHbeta Apr-1-neo is a human expression vector containing the entire human CD28 cDNA and has been described previously (a generous gift from Dr. D. Olive, INSERM U119, Marseille, France) (38). Mammalian expression vectors coding for human B7-1 (pCDL-SRalpha -B7-1) and B7-2 (pCN-B7-2) were obtained from Dr. A. Truneh (SmithKline Beecham Pharmaceuticals, King of Prussia, PA) (39).

Antibodies and Purified Proteins-- Anti-CD3 hybridoma OKT3 (specific for the epsilon  chain of the CD3 complex) was obtained from the American Type Culture Collection (Rockville, MD). Antibodies from this hybridoma were purified with mAbTrap protein G affinity columns according to manufacturer's instructions (Pharmacia Biotechnology AB, Uppsala, Sweden). The monoclonal antibody BU-63 (IgG2a) is specific for human B7-2 (CD86) (40) and has been supplied by Dr. D. L. Hardie (University of Birmingham, Birmingham, United Kingdom). UCHL-1 (IgG2a) is a monoclonal anti-human CD45RO antibody that was used as a control to semiquantitatively estimate the incorporation rate of foreign B7-2 proteins in virus preparations. CTLA-4 Ig is constituted of the extracellular domain of CTLA-4 fused to the Fc fragment of the immunoglobulin G1 (IgG1). Previous studies have indicated that this fusion protein demonstrates a strong affinity for B7-1 and B7-2 molecules and blocks the interaction between CD28 and B7-1/B7-2 (41). Fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin G (IgG) was purchased from Jackson Immunoresearch Laboratories, Inc. (West Grove, PA).

Production of Virus Stocks-- Isogenic virus preparations bearing or not bearing some specific host-derived proteins were produced by calcium phosphate (CaPO4) transfection of 293T cells as we described previously (6, 42-44). In brief, 293T cells were transfected with pNL4-3 alone (virus stock called NL4-3/Null) or were cotransfected with either pCDL-SRalpha -B7-1 (virus stock called NL4-3/B7-1), pCN-B7-2 (virus stock called NL4-3/B7-2), pHbeta Apr-1-neo (virus stock called NL4-3/CD28), or pCD1.8 (virus stock called NL4-3/ICAM-1). At 16 h after transfection, cells were washed twice with 3 ml of PBS and were incubated for an additional 24 h with 3 ml of Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Virion-containing supernatants were filtered through a 0.45-µm cellulose acetate membrane (Millipore, MA), aliquoted in 200-µl fractions, and were finally frozen at -85 °C until needed. Virus stocks were normalized for virion content using a commercial available enzyme-linked immunosorbent assay for the viral major core protein p24 (Organon Teknika, Durham, NC). The standardization on p24 contents is based on our previous observation indicating that virus preparations harvested from transfected 293T cells contain minimal amounts of p24 that are not associated with infectious virions (44). All virus stocks underwent one freeze-thaw cycle prior to initiation of infection studies. It should be stated that virus preparations were made from 293T cells expressing comparable levels of studied host cell surface constituents (i.e. B7-1, B7-2, CD28, and ICAM-1) and the physical presence of foreign proteins on the exterior of HIV-1 particles was assessed using our previously described immunomagnetic-based virus capture assay (see below) (45). Virus preparations were also made by infecting human tonsil histocultures as described previously (46). Briefly, human tonsillar tissue removed during a routine tonsillectomy and not required for clinical purposes was received within 5 h of excision. The tonsils were washed thoroughly with medium containing antibiotics and then sectioned into 2-3-mm blocks. These tissue blocks were placed on top of collagen sponge gels in the culture medium at the air-liquid interface and infected for 10 days with NL4-3 (2.5 ng of p24) added to the top of each tissue block (3-5 µl of clarified virus). Productive HIV-1 infection was assessed by measuring p24 in the culture medium with a commercial HIV-1 p24 antigen enzyme-linked immunosorbent assay.

DEAE-dextran Transfections-- Transient transfections were done using the DEAE-dextran method. In brief, cells (5 × 106) were first washed once in TS buffer (25 mM Tris-HCl (pH 7.4), 5 mM KCl, 0.6 mM Na2HPO4, 0.5 mM MgCl2, and 0.7 mM CaCl2) and resuspended in 0.5 ml of TS containing 15-30 µg of used plasmid(s) and 500 µg/ml of DEAE-dextran (final concentration). The cells/TS/plasmid/DEAE-dextran mix was incubated for 25 min at room temperature. Thereafter, cells were diluted at a concentration of 1 × 106/ml using complete culture medium supplemented with 100 µM of chloroquine (Sigma) and transferred into six-well plates. After 45 min of incubation at 37 °C, cells were centrifuged, resuspended in complete culture medium, and incubated at 37 °C for 24 h. To minimize variations in plasmid transfection efficiencies, cells were transfected in bulk and were next separated into various treatment groups.

Detection of Virion-bound Host B7-2 Proteins by a Virus Capture assay-- The presence of virion-bound host B7-2 proteins was semiquantitatively estimated using a modified version of a previously described virus capture assay (45). Briefly, 12.5 × 106 magnetic beads (BioMag, Fc-specific; PerSeptive Diagnostics, Inc., Cambridge, MA), previously coated with the anti-B7-2 antibody (BU-63), were incubated with similar amounts of studied virus preparations standardized in terms of the viral core p24 protein (2500 pg of p24) in a final volume of 1 ml of binding medium (PBS + 0.1% bovine serum albumin). This mixture was incubated for 1 h at 4 °C on a rotating plate. The beads were washed three times in binding medium with a magnetic separation unit and were resuspended in 100 µl of binding medium. The amount of immunocaptured HIV-1 particles was assessed by measuring viral p24 protein content found associated with the immunomagnetic beads by a commercial p24 enzymatic assay. Magnetic beads coated with an isotype-matched antibody (i.e. IgG2a) specific human CD45RO (clone UCHL-1) were used as a negative control because CD45 has been shown to be excluded from HIV-1 envelope (47).

Stimulations and Reporter Gene Assays-- Transiently transfected Jurkat E6.1 cells were seeded at a density of 2 × 105 cells/well (100 µl) in 96-well flat-bottom plates. Cells were next incubated for 1 h on ice with virions bearing or not bearing host B7-1 (NL4-3/B7-1), B7-2 (NL4-3/B7-2), CD28 (NL4-3/CD28), or ICAM-1 (NL4-3/ICAM-1) (10-200 ng of p24). Cells inoculated with HIV-1 particles were next either left untreated or were treated with the anti-CD3 antibody (clone OKT3) at 3 µg/ml. As controls, uninfected cells were either left unstimulated or were treated in a final volume of 200 µl with the following stimuli: anti-CD3 antibody (clone OKT3 at 3 µg/ml), anti-CD28 antibody (clone 9.3 at 1 µg/ml), or the combination of anti-CD3 antibody and DT30 cells (2 × 104 DT30/105 transfected Jurkat cells). Finally, cells were incubated at 37 °C for 7 h unless otherwise specified. For some experiments, before the addition of activators, virions were either left untreated or were pretreated with CTLA-4 Ig or by freeze-thaw cycles. Following the incubation period, 100 µl of cell-free supernatant were drawn from each well and 25 µl of cell culture lysis buffer (25 mM Tris phosphate (pH 7.8), 2 mM dithiothreitol, 1% Triton X-100, and 10% glycerol, final concentrations) were added before incubation at room temperature for 30 min. The extracts (20 µl) were analyzed for luciferase activity in 96-well plates using a Dynex MLX luminometer. Each well was injected with 100 µl of luciferase assay buffer (20 mM Tricine, 1.07 mM (MgCO3)4·Mg(OH)2·5H2O, 2.67 mM MgSO4, 0.1 mM EDTA, 270 µM coenzyme A, 470 µM luciferin, 530 µM ATP, and 33.3 mM dithiothreitol). Light output was measured for 20 s with a 2-s delay. Values are expressed in terms of relative light units as measured by the apparatus. Results shown are expressed as -fold inductions relative to basal luciferase activity in untreated/uninfected control cells.

Virus Infection-- Similar amounts of each recombinant luciferase-encoding virus stocks (130 ng of p24 for NL4-3/Null and NL4-3/B7-2 virions) were used to infect to 2 × 105 1G5 cells in a 96-well flat bottom tissue culture plate in a final volume of 200 µl. After 48 h of infection, cells were lysed and luciferase activity was monitored using a microplate luminometer (MLX; Dynex Technologies, Chantilly, VA).

Electrophoretic Mobility Shift Assay-- Nuclear extracts were prepared according to the microscale preparation protocol. Briefly, Jurkat E6.1 cells were left untreated, treated with stimuli, or incubated with virus stocks (150 ng of p24) in the presence of anti-CD3 for 60 min at 37 °C. The incubation of cells with the stimulating agents and/or viruses was terminated by the addition of ice-cold PBS and nuclear extracts were then prepared as described previously (48, 49). Six micrograms of nuclear extracts were used to perform electrophoretic mobility shift assay. Nuclear extracts were incubated for 30 min at room temperature in 15 µl of binding buffer (100 mM HEPES (pH 7.9), 40% glycerol, 10% Ficoll, 250 mM KCl, 10 mM dithiothreitol, 5 mM EDTA, 250 mM NaCl, 2 µg of poly(dI-dC), 10 µg of nuclease-free bovine serum albumin fraction V) containing 1 ng of gamma -32P-5'-end-labeled, double-stranded (dsDNA) oligonucleotide. This mixture was incubated for 30 min at 37 °C, and the reaction was stopped with 5 µl of 0.2 M EDTA. The labeled oligonucleotide was extracted with phenol/chloroform and passed through a G-50 spin column. The dsDNA oligonucleotide, which was used as a probe, contains the consensus NF-kappa B-binding site corresponding to the sequence 5'-ATGTGAGGGGACTTTCCCAGGC-3' (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). DNA-NF-kappa B complexes were resolved from free labeled DNA by electrophoresis in native 4% (w/v) polyacrylamide gels and 0.5× TBE buffer. The gels were subsequently dried and autoradiographed. Cold competitor assays were carried out by adding a 100-fold molar excess of homologous unlabeled dsDNA NF-kappa B oligonucleotide simultaneously with the labeled probe. Supershift assays were performed by preincubation of nuclear extracts with 1 µl of either specific anti-p50 or anti-p65 polyclonal antibodies (Dr. Nancy Rice, NCI, National Institutes of Health, Frederick, MD) in the presence of all the components of the binding reaction described above for 20 min at room temperature prior to the addition of the probe.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Incubation of Cells with B7-2-bearing Virions and Anti-CD3 Antibody Leads to Induction of HIV-1 LTR-mediated Activity-- In an attempt to define whether attachment of HIV-1 particles bearing some specific host-encoded cell surface proteins can lead to signaling events, the TCR/CD3-, CD4-, CD28-, LFA-1-, and CXCR4-expressing human T lymphoid cell line Jurkat was incubated with isogenic virions bearing or not bearing foreign B7-1, B7-2, CD28, and ICAM-1 proteins. Transfection of such cells with a vector made of the luciferase reporter gene driven by the regulatory element of HIV-1 (pLTR-Luc) prior to incubation with virus preparations allowed us to assess the putative up-regulating effect on virus transcription mediated by the binding step. Our initial set of experiments was performed by incubating transiently transfected Jurkat cells with similar amounts of isogenic virions standardized in terms of p24. As expected, HIV-1 LTR transcription was not activated neither by antibody-mediated cross-linking of cell surface TCR/CD3 complex nor by multivalent occupancy of CD28 by using B7-1-expressing DT30 cells (Fig. 1A). However, the occupancy of both TCR/CD3 and CD28 led to a marked activation of HIV-1 LTR-dependent luciferase expression. These results are in agreement with previous observations indicating that the CD28-mediated signal transduction pathway is considered as one of the dominant costimulatory pathways to achieve the complete activation of the T cell and induction of HIV-1 transcription/expression (50-52). The HIV-1 long terminal repeat was not modulated by attachment of infectious HIV-1 particles not bearing (NL4-3/Null) or bearing host B7-1 (NL4-3/B7-1), B7-2 (NL4-3/B7-2), CD28 (NL4-3/CD28), and ICAM-1 proteins (NL4-3/ICAM-1).



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Fig. 1.   Activation of HIV-1 LTR-driven gene activity is seen following incubation with anti-CD3 antibody and B7-2-bearing HIV-1NL4-3. A, at 18 h following transient transfection of Jurkat with pLTR-Luc, cells were incubated for 7 h with soluble anti-CD3 antibody, DT30 cells, a combination of anti-CD3 and DT30 cells, or 150 ng of p24 for each isogenic virus stocks tested (i.e. NL4-3/Null, NL4-3/B7-1, NL4-3/B7-2, NL4-3/CD28, and NL4-3/ICAM-1). B, transiently transfected Jurkat cells were also incubated for 7 h with virions bearing or not bearing host-derived B7-1, B7-2, CD28, and ICAM-1 proteins in the presence of anti-CD3 antibody. C, 1G5 cells were incubated for 7 h in the presence of soluble anti-CD3 antibody, DT30 cells, or a combination of anti-CD3 and DT30 cells. 1G5 cells were also incubated for 7 h with anti-CD3 antibody and NL4-3/Null or NL4-3/B7-2 viruses (130 ng of p24). Cells were subsequently lysed, and the lysates were then assayed for luciferase activity with a microplate luminometer. Results shown are the means (± S.D.) of quadruplicate samples and are expressed as -fold induction relative to basal luciferase activity in untreated/uninfected control cells. These results are representative of three independent experiments.

Given that effective activation of T lymphocytes necessitates the coupling of antigen-nonspecific signal with antigen-specific interactions of the TCR/CD3 complex, we next performed similar experiments in the presence of soluble anti-CD3 antibodies. Again, the biochemical signals provided by the ligation of TCR/CD3 complex (alpha -CD3) and CD28 (DT30) resulted in a strong activation of HIV-1 gene expression (Fig. 1B). Interestingly, incubation of transiently transfected Jurkat cells with ligand to CD3 and virions bearing host-derived B7-2 proteins resulted in a greater induction of HIV-1 LTR as compared with isogenic HIV-1NL4-3 particles devoid of this host cell surface constituent (i.e. NL4-3/Null). In an attempt to define whether such a virus-mediated modulation of HIV-1 LTR activity can still be observed in the context of an integrated provirus, 1G5 cells were incubated with anti-CD3 antibodies and B7-2-bearing virus particles. As depicted in Fig. 1C, HIV-1 LTR-driven gene activity was still up-regulated with B7-2-bearing virions and not with isogenic viral entities devoid of foreign B7-2 proteins, hence confirming the results obtained in transient transfection experiments.

The HIV-1 Enhancer Region Is the Target for LTR Activation by B7-2-bearing Virions and Anti-CD3 Antibody-- It has been shown that the HIV-1 enhancer is the main responding region of the LTR and is primarily responsible for the transcriptional increase observed following T cell activation (53). The HIV-1 enhancer is composed of two NF-kappa B-binding sites separated by an AP-2 site that has recently been demonstrated to be important for the binding of another transcription factor, NFAT (54). To determine whether the enhancer domain is the target for the observed increase in LTR activity, pkappa B-TATA-Luc, which only contains the HIV-1 enhancer region (-105/-70) upstream of a TATA box, was transfected into Jurkat cells. The combined action of anti-CD3 and DT30 cells resulted in a significant increase in pkappa B-TATA-Luc gene expression (Fig. 2A). Anti-CD3 antibodies and progeny viruses devoid of foreign B7-2 proteins (i.e. NL4-3/Null) had no detectable effect on HIV-1 promoter-driven gene activity. However, a dose-dependent increase in pkappa B-TATA-Luc gene expression was noticed when cells were incubated with anti-CD3 antibodies along with isogenic B7-2-bearing HIV-1 particles. For example, addition of 150 ng of p24 of NL4-3/B7-2 resulted in a significant 95-fold increase in HIV-1 enhancer-dependent luciferase activity. The direct involvement of the interaction between virion-bound B7-2 and CD28 on the target cell surface in the observed activation of HIV-1 enhancer was assessed by inhibiting interaction between B7-2 and CD28. This goal was achieved by adding a soluble form of CTLA-4 (CTLA-4 Ig) to the mixture composed of NL4-3/B7-2 virions, anti-CD3 antibodies, and Jurkat cells transiently transfected with pkappa B-TATA-Luc vector. Data from Fig. 2B indicate that CTLA-4 can totally abrogate the significant up-regulation of HIV-1 enhancer-driven luciferase activity mediated by anti-CD3 and isogenic B7-2-bearing virions. It should be noted that incubation of transfected Jurkat cells with B7-2-bearing progeny viruses in the absence of anti-CD3 could not induce up-regulation of HIV-1 enhancer region.



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Fig. 2.   Interaction between virion-bound host B7-2 and cell surface CD28 is playing a key role in the observed up-regulation of LTR activity. A, Jurkat cells were transiently transfected with a construct bearing the HIV-1 enhancer placed in front of the luciferase reporter gene (pkappa B-TATA-Luc) and were incubated 18 h later with anti-CD3, DT30 cells, the combination of anti-CD3 antibody and DT30 cells, or anti-CD3 antibody with increasing concentrations of virons bearing or not bearing host-encoded B7-2 proteins. B, in some experiments, the CTLA-4 Ig fusion protein was added before the incubation period. C, 1G5 cells were infected for 48 h with 130 ng of p24 of NL4-3/Null () or NL4-3/B7-2 (black-square) virus preparations that previously underwent three freeze-thaw cycles. D, Jurkat cells transiently transfected with pkappa B-TATA-Luc were treated with anti-CD3 antibody and NL4-3/Null virions or NL4-3/B7-2 (130 ng of p24) that previously underwent three freeze-thaw cycles. E, 1G5 cells were treated with increasing concentrations of the Tat inhibitor Ro 24-7429 for 45 min and cells were then infected with NL4-3/Null (100 ng of p24). Luciferase activity was assayed 48 h after virus inoculation. F, Jurkat cells transiently transfected with pkappa B-TATA-Luc were treated with the Tat inhibitor Ro 24-7429 for 45 min and were next incubated for 7 h in the presence of anti-CD3 antibody and NL4-3/Null or NL4-3/B7-2 virions (100 ng of p24). Next, cells were lysed and the lysates were assayed for luciferase activity with a microplate luminometer. Results shown are the means (± S.D.) of quadruplicate samples and are expressed as -fold induction relative to basal luciferase activity in untreated/uninfected control cells. These results are representative of three independent experiments.

To demonstrate that the major positive effect on HIV-1 LTR element by B7-2-bearing viruses is due to signaling from the cell surface as opposed to an increased efficiency of virus entry and infection (e.g. Tat-mediated effect), Jurkat cells transfected with pkappa B-TATA-Luc were incubated with anti-CD3 antibodies and B7-2-bearing virions that initially underwent three freeze-thaw cycles. This treatment results in the production of viruses denuded of their external gp120 envelope proteins and renders such viral entities thus unable to enter target cells. Data from Fig. 2C confirm that such treated virions are no longer infectious for susceptible human T lymphoid cells. Interestingly, B7-2-bearing virions are still able to lead to an increase in HIV-1 LTR-dependent gene activity (30-fold increase) that is not seen with isogenic viruses devoid of foreign B7-2 proteins (Fig. 2D). To further confirm that the observed effect is due to signaling events mediated by virion-anchored B7-2 and not by, for example, virion-associated Tat protein, we carried out a series of experiments with the benzodiazepine Ro 24-7429 Tat inhibitor (55-57). First, 1G5 cells were pretreated with various concentrations of Ro 24-7429 before being inoculated with HIV-1. Data presented in Fig. 2E demonstrate that Ro 24-7429 can potently inhibit the process of HIV-1 infection in a dose-dependent manner. Next, Jurkat cells transiently transfected with pkappa B-TATA-Luc were pretreated with increasing subcytotoxic concentrations of Ro 24-7429 before being incubated with a similar amount of isogenic virions bearing or not on their surface host-encoded B7-2. Interestingly, B7-2-bearing viruses can still up-regulate HIV-1 LTR-driven gene expression (Fig. 2F), therefore eliminating the possibility that the observed effect is due to soluble virion-associated Tat protein. We are now providing conclusive evidence that HIV-1 LTR activation mediated by B7-2-bearing viruses is caused by signaling from the cell surface as opposed to an intracellular Tat-mediated effect.

The Combined Action of Soluble Anti-CD3 Antibody and Binding of NL4-3/B7-2 Virions to Host T Cells Results in Activation of NF-kappa B and NFAT-- To define the cis-acting sequences required for the noticed effects on the HIV-1 enhancer, we transfected a number of plasmids into Jurkat cells, which were then incubated with anti-CD3, DT30, anti-CD30 and DT30, and, finally, anti-CD3 and isogenic virions bearing or not bearing studied host proteins. We initially tested a molecular construct containing the complete regulatory elements of HIV-1 (-453 to +80) mutated at the two NF-kappa B-binding sites (pmkappa BLTR-Luc). When both kappa B-binding sites were mutated, the combined action of anti-CD3 antibody and B7-2-bearing virions was no longer able to up-regulate HIV-1 LTR-driven luciferase activity (Fig. 3A). However, as expected, the engagement of the TCR/CD3 complex in the presence of CD28 costimulatory signal resulted in weak but detectable response of the entire mutated LTR (compare 5.4-fold increase in Fig. 3A and 20-fold increase in Fig. 1A). Data from experiments performed with pmkappa BLTR-Luc permit to conclude that the NF-kappa B transcription factor is an essential factor for the response of the HIV-1 LTR to soluble anti-CD3 antibody and progeny virions bearing host-encoded B7-2 proteins.



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Fig. 3.   Treatment of Jurkat cells with anti-CD3 and B7-2-bearing virions leads to NF-kappa B-dependent activation of HIV-1 enhancer. A, Jurkat cells were transiently transfected with pmkappa BLTR-Luc, a luciferase-encoding vector carrying NF-kappa B-mutated HIV-1 LTR domain, and were incubated 18 h later with soluble anti-CD3 antibody, DT30 cells, a combination of anti-CD3 and DT30 cells, or 150 ng of p24 for each isogenic virus stocks tested (i.e. NL4-3/Null, NL4-3/B7-1, NL4-3/B7-2, NL4-3/CD28, and NL4-3/ICAM-1). B, Jurkat cells were also transfected with a kappa B-driven reporter gene construct (pNFkappa B-Luc) and incubated 18 h later with soluble anti-CD3 antibody, DT30 cells, a combination of anti-CD3 and DT30 cells, or two different concentrations of each isogenic virus stocks tested (100 ng of p24, ; 200 ng of p24, black-square). After 7 h of incubation, cells were lysed and assayed for luciferase activity. Results shown are the means (± S.D.) of quadruplicate samples and are expressed as -fold induction relative to basal luciferase activity in untreated/uninfected control cells. These results are representative of three independent experiments.

To confirm NF-kappa B activation mediated by anti-CD3 antibody and B7-2-bearing virions, we transiently transfected Jurkat cells with a cis-reporter plasmid made of the luciferase reporter gene driven by a basic promoter element (TATA box) joined to five tandem repeats of NF-kappa B binding elements (pNFkappa B-Luc). Exposure of Jurkat cells to anti-CD3 antibody and the highest concentration of NL4-3/B7-2 (i.e. 200 ng of p24) was found to lead to a significant 135-fold increase in NF-kappa B-dependent gene activity (Fig. 3B). Similar levels of NF-kappa B-driven reporter gene expression were seen following treatment with anti-CD3 alone and anti-CD3 antibody in combination with other isogenic virus stocks tested (i.e. NL4-3/Null, NL4-3/B7-1, NL4-3/CD28, and NL4-3/ICAM-1). To directly demonstrate nuclear translocation of NF-kappa B following multivalent occupancy of TCR/CD3 complex and NL4-3/B7-2 attachment to target cells, we performed DNA mobility shift assays. In this series of experiments, the positive control consisted of Jurkat cells treated with TNF-alpha . This proinflammatory cytokine was able to induce the appearance of a specific band corresponding to NF-kappa B as competition assays performed with 100-fold excess of the cold NF-kappa B oligonucleotide led to a complete disappearance of this signal (Fig. 4, compare lanes 4 and 12). Treatment of Jurkat cells with either NL4-3/Null or NL4-3/B7-2 alone did not induce nuclear translocation of NF-kappa B (lanes 5 and 6, respectively). Incubation of Jurkat cells with anti-CD3 antibody in combination with isogenic B7-2-bearing HIV-1 particles resulted in the appearance of a band corresponding to the NF-kappa B factor (lane 8), which was specifically outcompeted by cold excess of the NF-kappa B oligonucleotide (lane 11). The addition of the specific inhibitor of B7-2/CD28 interaction, i.e. CTLA-4 Ig, was found to eliminate the NF-kappa B-specific band (lane 10). We next investigated the identity of the subunit(s) composing the NF-kappa B complex by performing supershift assays with polyclonal anti-p50 and anti-p65 antibodies. Both antibodies led to the complete disappearance of NF-kappa B-specific band (lane 13 for anti-p50 and lane 14 for anti-p65), which was seen following incubation of Jurkat cells with anti-CD3 antibody and B7-2-bearing virions.



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Fig. 4.   Nuclear translocation and activation of NF-kappa B is confirmed by mobility shift assays. Jurkat cells were either left untreated (lane 1) or were incubated for 1 h with anti-CD3 (lane 2), 100 µM forskolin (lane 3, used as a negative control), 10 ng/ml TNF-alpha (lane 4, used as a positive control), NL4-3/Null (lane 5, 150 ng of p24), NL4-3/B7-2 (lane 6, 150 ng of p24), anti-CD3 and NL4-3/Null viruses (lane 7, 150 ng of p24), anti-CD3 and NL4-3/B7-2 viruses (lane 8, 150 ng of p24), anti-CD3 + NL4-3/Null viruses (150 ng of p24) + CTLA-4 Ig (lane 9), and anti-CD3 + NL4-3/B7-2 viruses (150 ng of p24) + CTLA-4 Ig (lane 10). Lanes 11 and 12 represent a 100× competition with the unlabeled probe for NF-kappa B for transiently transfected Jurkat cells incubated with anti-CD3 + NL4-3/B7-2 viruses (150 ng of p24) and TNF-alpha , respectively. Lanes 13 and 14 represent a supershift with anti-p50 and anti-p65 antibody, respectively. The nuclear extracts were next incubated with a 32P-end-labeled synthetic double-stranded NF-kappa B probe. The position of the specific complex bound by the kappa B site probe is indicated by an arrow on the left side.

Recent findings indicate that NFAT can synergize with NF-kappa B in transcriptional activation of HIV-1 through its action on the virus enhancer region (54). We therefore evaluated whether NFAT activation could be achieved by the combined action of anti-CD3 antibody and HIV-1 particles having incorporated host B7-2 proteins in their envelope. This task was accomplished by using a vector composed of the luciferase reporter gene placed under the control of the minimal IL-2 promoter containing three tandem copies of the NFAT-binding site (Fig. 5). Treatment of transiently transfected Jurkat cells with anti-CD3 antibody alone led to a 2.9-fold increase in NFAT-dependent gene activity, a value comparable to the one seen when cells are incubated with anti-CD3 and virions devoid of host-encoded B7-2 proteins (i.e. NL4-3/Null). Interestingly, a higher increase in reporter gene activity was seen when cells are incubated with anti-CD3 and isogenic B7-2-bearing viral entities (7-fold activation). Thus, the combined action of TCR/CD3-mediated biochemical events and binding of viruses bearing foreign B7-2 proteins in their envelope enhance activation of the transcription factor NFAT.



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Fig. 5.   Enhancement of NFAT activation by the combined action of anti-CD3 and B7-2-bearing HIV-1 particles. Jurkat cells were transfected with a NFAT-driven vector (pNFAT-Luc) and were incubated for 7 h with soluble anti-CD3 antibody, DT30 cells, a combination of anti-CD3 and DT30 cells, anti-CD3 and NL4-3/Null viruses (150 ng of p24), or anti-CD3 and NL4-3/B7-2 viruses (150 ng of p24). Next, cell lysates were assayed for luciferase activity with a microplate luminometer. Results shown are the means (± S.D.) of quadruplicate samples and are expressed as -fold induction relative to basal luciferase activity in untreated/uninfected control cells. These results are representative of three independent experiments.

Trans-dominant Negative Mutants of Ikappa Balpha and NFAT Block Activation of HIV-1 Enhancer by Anti-CD3 Antibody and B7-2-bearing HIV-1-- Previous studies have demonstrated that nuclear translocation and activation of NF-kappa B is mainly mediated by the degradation of the repressor Ikappa Balpha , which sequesters the NF-kappa B complex in the cytoplasm (58, 59). This degradation is known to be highly dependent on the phosphorylation of the two serine residues 32 and 36 (60, 61). To determine the contribution of both NF-kappa B and NFAT in the noticed increase in HIV-1 enhancer-mediated activity, we used trans-dominant negative mutants of each transcription factor. Initially, we transfected Jurkat cells with a vector coding for a modified version of Ikappa Balpha carrying alanine on both serine 32 and 36 residues. This protein is unable to be serine-phosphorylated but retains its ability to bind to NF-kappa B and can thus act as a dominant negative mutant of wild-type NF-kappa B. Cotransfection of Jurkat cells with pkappa B-TATA-Luc and pCMV-Ikappa Balpha S32A/36A resulted in an almost complete inhibition of the activation of HIV-1 enhancer-driven gene activity mediated by anti-CD3 antibody and B7-2-bearing viruses (Fig. 6A). The positive control made of anti-CD3 antibody and DT30 cells was also dramatically affected by the introduction of Ikappa Balpha S32A/36A. The involvement of NFAT and a possible synergistic effect with NF-kappa B was next investigated with dnNFAT, a dominant negative NFAT mutant that suppresses activation-induced nuclear translocation of all NFAT members (34). The increase in HIV-1 enhancer-dependent luciferase activity mediated by the combination of anti-CD3 and B7-2-bearing virions was diminished by dnNFAT but to a lesser extent than by pCMV-Ikappa Balpha S32A/36A (Fig. 6B). It is of interest to note that such a diminution was further augmented when both negative mutants were introduced into Jurkat cells. Taken together, these results suggest that NF-kappa B and NFAT are both active players in the up-regulation of HIV-1 enhancer activity, which is seen following treatment with anti-CD3 antibody and HIV-1 particles bearing host-derived B7-2 proteins on their surface.



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Fig. 6.   Trans-dominant negative mutant of Ikappa Balpha and NFAT inhibit HIV-1 LTR activity mediated by anti-CD3 and B7-2-bearing virions. A, Jurkat cells were cotransfected with pkappa B-TATA-Luc (15 µg) and an empty vector control (empty bars, 30 µg) or pCMV-Ikappa Balpha S32A/36A (filled bars, 15 µg; striped bars, 30 µg). B, Jurkat cells were also cotransfected with with pkappa B-TATA-Luc (15 µg)/empty vector control (empty bars, 30 µg), pkappa B-TATA-Luc (15 µg)/pCMV-Ikappa Balpha S32A/36A (filled bars, 15 µg), pkappa B-TATA-Luc (15 µg)/pCDNA3-dnNFAT (striped bars, 7.5 µg) or pkappa B-TATA-Luc (15 µg)/pCMV-Ikappa Balpha S32A/36A (15 µg)/pCDNA3-dnNFAT (dotted bars, 7.5 µg). After 18 h, cells were next incubated for 7 h with soluble anti-CD3 antibody, DT30 cells, a combination of anti-CD3 and DT30 cells, anti-CD3 and NL4-3/Null viruses (150 ng of p24), or anti-CD3 and NL4-3/B7-2 viruses (150 ng of p24). The lysates were next assayed for luciferase activity with a microplate luminometer. Results shown are the means (± S.D.) of quadruplicate samples and are expressed as -fold induction relative to basal luciferase activity in untreated/uninfected control cells. These results are representative of three independent experiments.

We next compared the degree of virion-anchored B7-2 proteins in viruses originating from our transient-and-expression system (i.e. 293T cells) versus virions expanded in a more physiological cellular milieu. The incorporation of foreign B7-2 into the HIV-1 envelope was assessed using a recently developed virus precipitation assay. The validity of this test was first tested by cotransfecting 293T cells with pNL4-3 and increasing concentrations of the vector coding for human B7-2. Flow cytometry analysis revealed that increasing concentrations of B7-2 expression vector resulted in a concomitant enhancement of the expression of B7-2 on the surface of 293T cells (data not shown). Viruses produced by such transiently transfected 293T cells were next subjected to our virus capture assay. The amount of virus captured was found to be in linear correlation with the level of expression of B7-2 on the surface of 293T cells (Fig. 7A). These results suggest that our virus precipitation assay is not limiting and that the level of B7-2 expression on the producer cells influences the incorporation rate of B7-2 in the HIV-1 envelope. The degree of incorporation of host-encoded B7-2 within virions expanded in a more natural cellular reservoir was next monitored. For this purpose, HIV-1NL4-3 was grown in human tonsil histocultures, a model system that preserves and maintains the mixed cell populations found in secondary lymphoid organs, including T cells, B cells, macrophages, and dendritic cells. This tissue culture system was selected because lymphoid tissue is known as the major site of HIV-1 replication in vivo. Results from a virus capture assay revealed that anti-B7-2 antibodies (clone BU-63) as efficiently captured HIV-1NL4-3 particles produced by 293T cells as viruses produced by histocultures of human tonsils from two different healthy donors (Fig. 7B). These data suggest that comparable levels of host-encoded B7-2 proteins are found embedded on viruses produced either by transiently transfected 293T cells or by human lymphoid tissue following a normal infection with HIV-1.



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Fig. 7.   Comparative analysis of the degree of incorporation of B7-2 in HIV-1NL4-3 produced either in 293T cells or human lymphoid tissue. A, 293T cells were cotransfected with pNL4-3 and increasing concentrations of the pCN-B7-2 expression vector (i.e. 0, 0.1, 0.5, 1, 2.5, and 5 µg). Similar amounts of virus stocks (2.5 ng) were next incubated with magnetic beads coated with antibodies specific for human B7-2 (clone BU-63) and CD45RO (clone UCHL-1). B, viruses (2.5 ng of p24) produced either in transiently transfected 293T cells or histocultures of human lymphoid tissue (i.e. tonsils) were incubated with magnetic beads coated with antibodies specific for human B7-2 (clone BU-63) and CD45RO (clone UCHL-1). The levels of captured virions were quantified by using a p24 enzymatic assay. Magnetic beads coated with the anti-CD45RO antibody served as controls to determine background levels of captured viruses. Data shown represent ratio of captured virions with antibody reactive with human B7-2 and CD45RO, respectively.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

HIV-1 attachment to host cells has been found to profoundly affect the immune system. For example, HIV-1 envelope glycoproteins have been reported to induce secretion of proinflammatory cytokines and mediate enhancement of apoptosis (reviewed in Ref. 62). The underlying basis for the observed biological functions of such virus proteins is thought to involve the interaction of the HIV-1 envelope with the CD4 glycoprotein, the primary cellular receptor for this retrovirus. Upon the discovery of CCR5 as a major coreceptor for HIV-1, many laboratories have demonstrated that HIV-1 envelope can transduce intracellular signals through CCR5 in a manner analogous to that of beta -chemokines (21, 63). The signaling events transduced by the interaction of HIV-1 envelope with CCR5 result in chemotaxis of CD4-expressing T cells, raising the possibility that such response may promote the recruitment of uninfected cells to sites of active viral replication (63). We have previously shown that binding of HIV-1 to its target leads to phosphorylation of phosphatidylinositol 3-kinase (17), as well as to a decrease in HIV-1 transcription and virion production (64, 65). Conflicting data were reported by Deveaux's group (66, 67), who have demonstrated that HIV-1 attachment mediates activation of virus transcription via an NF-kappa B-dependent pathway. Given that numerous functional foreign cell membrane proteins have been found to be acquired by HIV-1, we were interested in defining whether the exact nature of virally embedded host proteins might explain such contrasting findings as we suggested previously (14). This postulate is founded on the observation that the number and type of cell membrane surface proteins acquired by HIV-1 particles can have profound effects on the attachment of virions to target cells, the host cell range, the binding avidity between virus and cell, and the neutralization sensitivity of virions (6, 7, 42, 43, 68-74). It is therefore legitimate to propose that binding of some virally incorporated host proteins with their normal counter-receptors found on the cell surface can lead to signal transduction into target cell. In this work, we report that virions bearing host-derived B7-2 (CD86) proteins can, in association with engagement of the TCR/CD3 complex, activate HIV-1 LTR-driven gene expression through the induction of NF-kappa B and NFAT-dependent second-message pathways.

Our studies were focused on the functionality of some specific virally embedded host proteins, namely CD28, ICAM-1, B7-1, and B7-2. These cells surface constituents were deliberately selected for the following reasons. First, the primary cellular reservoirs of HIV-1 in infected persons, i.e. T helper cells and macrophages, express these surface proteins either in an inducible or constitutive manner. Indeed, CD28 is a homodimeric cell surface glycoprotein that is largely restricted to the T cell lineage. Ninety-five percentage of CD4+ T cells express CD28 and activation of T cells leads to enhanced CD28 expression (75). CD54 (ICAM-1) is widely distributed, and its expression can be induced by a variety of inflammatory cytokines (76). Thus, surface expression of ICAM-1 is high on activated T helper cells and macrophages, two different cell types known to be active producer of virions in infected individuals. B7-1 and B7-2 are expressed on activated macrophages and T cells (75). Enhanced levels of B7-1 were detected upon stimulation in vitro of T cells from HIV-1-infected individuals and also on viral p24-expressing T cells inoculated in vitro with HIV-1 (77, 78). Circulating monocytes from HIV-1-infected patients were also found to express significant amounts of surface B7-1 (79). Second, some of these cell surface markers have been shown to be acquired by field isolates of HIV-1 expanded in mitogen-stimulated peripheral blood mononuclear cells (reviewed in Ref. 14). Moreover, our findings indicate that a laboratory isolate of HIV-1 (NL4-3) expanded in a physiological tissue culture system (i.e. histocultures of human lymphoid tissue (tonsils)) can acquire amounts of foreign B7-2 proteins that are comparable with virions produced by 293T cells. This last observation provides a physiological significance to the current work. Third, the normal counterligands for CD28, ICAM-1, B7-1, and B7-2 proteins are all known to provide signaling cascades upon ligation. For example, antibodies against CD28, B7-1, and B7-2 in T cells elicit different biochemical signals, including phospholipase C-gamma phosphorylation, calcium influx, PI 3-kinase activation, and activation of p21ras, Raf-1, and extracellular signal-regulated kinase/c-Jun N-terminal kinase (80). Although CD28 is primarily seen as a costimulus to the T cell receptor, a recent study indicates that engagement of CD28 alone is sufficient to lead to NF-kappa B activation in human T cells (81). Ligation of CD28 has also been reported to generate tyrosine phosphorylation of the CD28 cytoplasmic tail and the formation of a complex with PI 3-kinase (38). ICAM-1 binding to LFA-1, its cognate ligand, has been demonstrated to up-regulate the activities of PI 3-kinase, sphingomyelinase, and c-Jun N-terminal kinase (83).

Although signaling through the TCR/CD3 complex alone is essential for the initial stages of T cell activation, it is not sufficient to induce all events that accompany activation of freshly isolated resting T cells or T cell clones (84). A second costimulatory signal provided by ligand engagement of cell-surface receptor molecules such as CD28 is required (85-87). Interestingly, optimal activation of gene expression directed by the HIV-1 LTR was also found to be exerted by ligands to both TCR/CD3 and CD28 (51, 52). Our results are perfectly in line with such findings because binding of HIV-1 particles bearing B7-2, one of the natural cognate ligands of CD28, is not sufficient per se to up-regulate HIV-1 LTR-driven transcription. Indeed, a concomitant ligation of the TCR/CD3 complex by specific monoclonal antibodies is necessary to achieve activation of virus promoter with B7-2-bearing virions in human T cells carrying an HIV-1 LTR construct either in a transient or stable form. Our results are thus indicating that virally embedded foreign B7-2 proteins are functional even when located within the HIV-1 envelope and that such host-derived proteins can engage with their natural ligand CD28 and mediate CD28-dependent signaling cascades. The observation that NL4-3/Null (i.e. virions devoid of studied host cell surface constituents) and isogenic viruses bearing host-derived B7-1, CD28, or ICAM-1 proteins do not modulate LTR activity even in the presence of anti-CD3 antibodies suggests that the observed phenomenon is really due to the additional interaction between virion-anchored host B7-2 and cell surface CD28 found on the target cell and not to soluble factors present in virus preparations. In addition, a Tat antagonist (i.e. Ro 24-7429) further confirmed that the positive effect on HIV-1 LTR activity is really due to signal transduction pathways engaged upon binding of viron-anchored foreign B7-2 with its CD28 ligand and not to virus-associated Tat.

We were able to demonstrate that transcription factor NF-kappa B is playing a crucial role in the increase of HIV-1 LTR activity, which is seen following ligation of cell surface TCR/CD3 complex and CD28 with specific antibodies and B7-2-bearing virions, respectively. Earlier studies have demonstrated that NFAT is an immediate-early activation factor that plays a crucial role in T cell activation and commitment processes through its control of interleukin-2 gene activation (88). The previously demonstrated synergistic effect between NF-kappa B and NFAT, with respect to activation of HIV-1 transcription (54), prompted us to also investigate the implication of NFAT. We found that NFAT was indeed acting in a synergistic mode with NF-kappa B and that both transcription factors were responsible for the up-regulation of LTR activity mediated by anti-CD3 antibody and B7-2-bearing HIV-1 particles.

Although B7-1 and B7-2 proteins can both interact with CD28 with similar low affinities (89) and provide the costimulatory signal necessary to prevent the induction of T cell anergy and enhance cytokine production (90), only a limited number of studies have compared the signals generated following B7-1 or B7-2 engagement of CD28. Nonetheless, a growing body of evidence suggests that there are different functional outcomes of CD28 engagement by B7-1 and B7-2 (91-93). Our results support this concept since an increase in HIV-1 LTR-driven gene activity was observed only when anti-CD3 antibodies were coupled with B7-2-bearing virions but not with isogenic viruses having incorporated foreign B7-1 proteins.

Based on results from this work and previous studies, we are proposing that intracellular signaling events transduced by HIV-1 envelope and virion-anchored host proteins may directly contribute to the pathogenesis of this retroviral infection. For example, induction of such intracellular biochemical events can modulate the early events in the viral life cycle in uninfected cells and/or, as demonstrated in the present study, affect proviral DNA in already infected cells. In the first scenario, it is known that the replicative cycle of HIV-1 is greatly influenced by the stage of the cell cycle at the time of infection. Successful infection of CD4+ T lymphocytes by HIV-1 requires the activation of target cells whereas infection of quiescent CD4+ T lymphocytes leads to incomplete, labile, reverse transcripts (94-96). Complete HIV-1 reverse transcription was observed only when quiescent peripheral CD4+ T lymphocytes were induced to switch from the G1a phase of the cell cycle to the G1b phase by costimulation through the T cell receptor and CD28 (97). Therefore, depending of the virion-bound cell surface protein, it is quite possible that the initial contact between the viral entity and its target will render the intracellular milieu more favorable for HIV-1 reverse transcription and integration process. The second scenario is based on published work showing that HIV-1 replication is very intimately linked to T cell activation, due to the overlapping of the signal transduction requirement between T cell lymphokine gene expression and HIV-1 LTR transactivation (98). This overlapping is a consequence of the HIV-1 LTR architecture, composed of many different motifs found in regulatory regions of gene induced following T cell activation (53). Our findings clearly suggest that the exact nature of host-encoded protein present on the virion's surface can influence activation of transcription factors recognized as potent inducers of HIV-1 replication (i.e. NF-kappa B and NFAT). Although antibody-mediated engagement of TCR/CD3 complex was found to be necessary to achieve activation of HIV-1 LTR-dependent gene expression by B7-2-bearing progeny virus, it should be kept in mind that substantial amounts of foreign MHC-II proteins are acquired by HIV-1 (45, 82). Therefore, assuming that the nominal antigen occupies the peptide binding groove, interaction with the TCR/CD3 complex can be achieved with virally embedded host MHC-II proteins.

Taken together, our results suggest that attachment of HIV-1 to its target should be considered as an event that, depending of the nature of virion-anchored host proteins, can influence several steps in the virus replicative cycle.


    ACKNOWLEDGEMENTS

We thank Dr. B. Barbeau for critical reading of the manuscript and Dr. M. Dufour for technical assistance in flow cytometry studies.


    FOOTNOTES

* This work was supported in part by Canadian Institutes of Health Research HIV/AIDS Research Program Grant HOP-14438 and by a Fonds de la Recherche en Santé du Québec (Réseau FRSQ SIDA et Maladies Infectieuses) grant (both to M. J. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Recipient of a Ph.D. fellowship from the Fonds de la Recherche en Santé du Québec/Fonds pour la Formation de chercheurs et l'Aide à la Recherche-Program Santé. This work was performed in partial fulfillment of the requirements for a Ph.D. degree at the Faculty of Graduate Studies, Department of Medical Biology, Faculty of Medicine, Laval University.

§ Holder of a Canada Research Chair in Human Immuno Retrovirology. To whom correspondence should be addressed: Laboratoire d'ImmunoRétrovirologie Humaine, Centre de Recherche en Infectiologie, RC709, Centre Hospitalier Universitaire de Québec, Pavillon CHUL, 2705 boul. Laurier, Ste-Foy, Québec G1V 4G2, Canada. Tel.: 418-654-2705; Fax: 418-654-2212; E-mail: michel.j.tremblay@crchul.ulaval.ca.

Published, JBC Papers in Press, November 28, 2000, DOI 10.1074/jbc.M002198200


    ABBREVIATIONS

The abbreviations used are: HIV-1, human immunodeficiency virus type 1; LTR, long terminal repeat; MHC, major histocompatibility complex; PBS, phosphate-buffered saline; NF-kappa B, nuclear factor-kappa B; NFAT, nuclear factor of activated T cells; TNF-alpha , tumor necrosis factor-alpha ; TCR, T cell receptor; dsDNA, double-stranded DNA; PI 3-kinase, phosphatidylinositol 3-kinase; Tricine, N-tris(hydroxymethyl)methylglycine; ICAM-1, intercellular adhesion molecule 1; LFA-1, lymphocyte function-associated antigen-1.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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