©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
HIV-1 Envelope Glycoproteins Induce Activation of Activated Protein-1 in CD4 T Cells (*)

(Received for publication, February 2, 1995; and in revised form, June 2, 1995)

Narendra Chirmule (§) Harris Goonewardena Sunil Pahwa (1) Regina Pasieka Vaniambadi S. Kalyanaraman (1) Savita Pahwa (§)

From the Department of Pediatrics, North Shore University Hospital, Cornell University Medical College, Manhasset, New York 11030 and Advanced BioScience Labs Inc., Kensington, Maryland 20872

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Activation of CD4 positive T cells is a primary requirement for human immunodeficiency virus (HIV) entry, efficient HIV replication, and progression to AIDS. Utilizing CD4 positive T cell lines and purified T cells from normal individuals, we have demonstrated that native envelope glycoproteins of HIV, gp160, can induce activation of transcription factor, activated protein-1 (AP-1). The stimulatory effects of gp160 are mediated through the CD4 molecule, since treatment of gp160 with soluble CD4-IgG abrogates its activity, and CD4 negative T cell lines fail to be stimulated with gp160. Immunoprecipitation of the gp160-induced nuclear extracts with polyclonal antibodies to Fos and Jun proteins indicates that AP-1 complex is comprised of members of these family of proteins. The gp160-induced AP-1 complex is dependent upon protein tyrosine phosphorylation and is protein synthesis-independent. This stimulation can also be abolished by inhibitors of protein kinase C, but it is unaffected by calcium channel blocker or cyclosporine A. This gp160 treatment adversely affects the functional capabilities of T cells; pretreatment of CD4+ T cells with gp160 for 4 h at 37 °C inhibited anti-CD3-induced interleukin-2 secretion. Effects similar to gp160 were seen with anti-CD4 mAb. The aberrant activation of AP-1 by gp160 in CD4 positive T cells could result in up-regulation of cytokines containing AP-1 sites, e.g. interleukin-3 and granulocyte macrophage colony-stimulating factor, and concurrently lead to T cell unresponsiveness by inhibiting interleukin-2 secretion.


INTRODUCTION

The CD4 molecule is the binding site of the human immunodeficiency virus via the envelope glycoprotein, gp160/gp120(1) . This interaction occurs at a specific region at the external domain of the CD4 molecule. There have been conflicting reports on the ability of gp160/gp120 to transduce biochemical signals through the CD4 molecule on T cells. While increase in intracellular calcium, hydrolysis of phosphatidyl inositol, and activation of tyrosine kinases have been demonstrated by some(2, 3, 4, 5) , others have failed to observe these events(6, 7) .

The interaction of the CD4 molecule with the nonpolymorphic beta2 domain of the MHC (^1)class II molecule has been demonstrated to play a vital role in activation of mature T cells and in T cell development in the thymus(8) . Several studies have now demonstrated that the CD4-MHC class II interaction is essential for effective signal transduction, at low antigen concentrations, to increase the avidity, in co-receptor-dependent systems(9, 10) . Studies demonstrating the association of the src homologous tyrosine kinase p56 and the putative p32 G-protein with the cytoplasmic tail of the CD4 molecule have demonstrated that biochemical signals can be transduced through the CD4 molecule(11, 12) . In this respect, exposure of CD4+ T cells to anti-CD4 mAb or HIV gp120 has been shown to induce activation of the Raf-1-related 110-kDa polypeptide, and phosphatidylinositol 3- and phosphatidylinositol 4-kinases (13) and activation of NF-kappaB(14) .

Utilizing soluble envelope glycoproteins of HIV-1, gp160, we have previously demonstrated that CD4-mediated signals result in biological effects. These include up-regulation of CD40 ligand in CD4+ T cells, resulting in polyclonal B cell differentiation(15) ; induction of IL-3, IL-6, and granulocyte macrophage colony-stimulating factor mRNA and cytokine secretion, which induce increased myelopoiesis(16) ; and increased expression of Fas antigen on CD4+ T cells, resulting in accelerated apoptosis in peripheral blood mononuclear cells(17) . To delineate the nature of the biochemical signals transduced through the CD4 molecule on T cells, we have investigated the ability of gp160 and anti-CD4 mAbs to induce activation of the transcription factor, activated protein 1 (AP-1).

Physiological activation of T cells through the T cell receptor results in the activation of AP-1(18) . AP-1 is a collection of homodimeric and heterodimeric protein complexes of the c-fos and c-jun proto-oncogene products(19) . These proteins interact with a common DNA binding site, the TPA-responsive element (TGA(C/G)TCA) and activate gene transcription(20) . The binding of AP-1 to the TPA-responsive element has been attributed to post-translational modification of preexisting members of the Fos and Jun family of proteins, involving phosphorylation and dephosphorylation events(21) . Our results have demonstrated that the CD4-induced signals transduced by gp160 or anti-CD4 mAb gp160 in T cells result in activation of AP-1 by a mechanism that involves post-translational activation of Fos and Jun family of proteins.

Depression of antigen-specific T cell responses is a relatively early feature of HIV infection and precedes the quantitative decline of CD4+ T cells(22) . Several investigators have clearly demonstrated the inhibitory effects of gp120 on normal T cell functions (for review, see (23) ). The mechanism of gp120-mediated inhibition of T cell responses involves inhibition of intracellular calcium mobilization, hydrolysis of inositol phosphates, and activation of protein kinase C, and kinase activity of p56(24, 25, 26, 27, 28) . The reduced proliferative responses were attributed to inhibition of decreased IL-2 mRNA expression and IL-2 secretion(25) . In this study, we have suggested that binding of envelope glycoproteins of HIV to CD4+ T cells induces aberrant activation of the transcription factor AP-1 (which plays a critical role in IL-2 gene transcription, (18) ) and results in inhibition anti-CD3 mAb-induced IL-2 secretion.


MATERIALS AND METHODS

Envelope Glycoproteins

gp160 and gp120 were purified from culture supernatants of a clone of HIV-infected Hut-78 cells, 6D4, as described earlier(28) . Briefly, supernatant of cells grown in serum-free HB104 medium was concentrated and passed through a lentil-lectin Sepharose column. Glycoproteins were eluted with 400 mM alpha-methyl mannoside. gp160 was further purified by affinity chromatography over anti-HIV mAb-Sepharose 4B column. The envelope glycoprotein preparations were >95% pure and were not contaminated with endotoxins, as tested by the Limulus amoebocyte lysate assay (E-TOXATE, Sigma).

Antibodies and Reagents

The following reagents and resources were used: mAb to CD4 (Leu3a, IgG1; Becton Dickinson, Mountainview, CA); mAb to CD3 (mAb 454, IgG2a, gift from Dr. N. Chiorazzi, North Shore University Hospital, Manhasset, NY); nonimmune mouse Ig (mIg; Chrompure IgG, Jackson ImmunoResearch, West Grove, PA). Polyclonal antibodies to Fos and Jun proteins used were as follows; rabbit anti-c-Jun/AP1 and anti-c-Fos (Santa Cruz Biotechologies, Santa Crus, CA) recognize all of the members of the Fos/Jun family of proteins. Polyclonal rabbit anti-gp120 was developed by Advanced BioScience Labs Inc., Kensington, MD; soluble CD4-IgG was a gift from Genentech, San Fransisco, CA. Herbimycin A (Life Technologies, Inc.), cyclosporine A (Sandoz, East Hanover, NJ), H-7, cycloheximide, 2-mercaptoethanol, verapamil, and phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma.

Cells

CD4 positive clone of Jurkat T cells, E6-1, obtained from the National Institutes of Health AIDS Reference Reagent Program, Bethesda, MD, (donated by Dr. A. Weiss, (29) ) was maintained in RPMI 1640 medium (Whittaker) supplemented with pennicillin and streptomycin and 10% fetal calf serum. CD4 positive T cells H9, Molt4 were obtained from ATCC, Betheda, MD. CD4 negative Jurkat T cells (JN) were mutant CD4 negative (CD3+) cells by fluorescence-activated cell sorting analysis following staining with fluorescein isothiocyanate-conjugated anti-CD3 mAb and anti-CD4-conjugated with phycoerythrin (Becton Dickinson, Mountainview, CA).

Peripheral blood lymphocytes were purified by Ficoll-Hypaque density gradient centrifugation. T cells were purified from peripheral blood lymphocytes by rosetting 2 times with neuraminidase-treated sheep red blood cells as described earlier(15) .

Immunomagnetic Separation of CD4+ and CD8+ T Cells

Purified T cells stimulated with medium alone or with gp160 for 4 h were incubated with anti-CD8 mAb conjugated immunomagnetic beads (Dynal, Great Neck, NY) for 30 min at 4 °C on a rotating shaker, as recommended by the manufacturer. The cells were subjected to a magnetic field, and the unbound cells (designated CD8 negative, CD8- T cells) were carefully aspirated. The cells bound to the beads were designated CD8 positive (CD8+) and were >90% CD4+ as determined by flow cytometry. Nuclear proteins were extracted as described below.

Nuclear Extracts

Small scale nuclear extracts were made from 2 10^7 unactivated or activated E6-1 cells as described previously(18) . Unless otherwise stated, cells were stimulated with medium alone or various stimuli for 4 h at 37 °C. Cells were washed and resuspended in 10 mM Tris, pH 7.4, 10 mM NaCl, 3 mM MgCl(2), 0.5 mM dithiothreitol and 0.5 mM phenylmethylsulfonyl fluoride and lysed by the addition of Nonidet P-40 to a final concentration of 0.5%. Nuclei were pelleted and washed in the same buffer without Nonidet P-40, and nuclear proteins were extracted in buffer C (20 mM HEPES, pH 7.4, mM 0.42 NaCl, 1.5 mM MgCl(2), 0.2 mM EDTA, 25% (v/v) glycerol, 0.01% NaN(3))(30) . After pelleting neclear debris, the supernatant was removed and diluted with an equal volume of buffer D (20 mM HEPES, pH 7.4, 50 mM KCl, 0.2 mM EDTA, 20% (v/v) glycerol, 0.01% NaN(3)). This extract was used directly in the electrophoretic mobility shift assay (EMSA). The equivalence of the extracts was verified by protein estimation using the BCA protein kit (Pierce).

EMSA

Double-stranded oligonucleotides corresponding to the consensus AP-1 binding sequence (5`-CGC TTG ATG AGT CAG CGC GAA-3` (31) ) were obtained commercially (Promega, Madison, WI) and end-labeled with [P]ATP and polynucleotide kinase. In some experiments, the binding site of AP-1 to the human IL-2 promoter (5`-AATTCCAAAGAGTCATCAGA-3`) was used as a competitor(18) . For each binding reaction, 10,000 cpm (0.2-0.5 ng) of end-labeled oligonucleotide was incubated for 30 min at room temperature with 5-8 µg of nuclear extract in the presence of 3 mg of sheared poly(dI-dC) (Pharmacia Biotech Inc.). The resulting DNA-protein complexes were analyzed by electrophoresis at 4 °C of 4% polyacrylamide gels. Unlabeled oligonucleotides used for competitions were added to nuclear extracts and poly(dI-dC) prior to the addition of labeled probe. Gels were dried on a gel drier (Bio-Rad) and visualized by autoradiography. In some experiments, nuclear extracts were incubated with antibodies to c-Jun, c-Fos, or normal rabbit serum for 1 h on ice, followed by incubation with Protein A-conjugated Sepharose (Pharmacia) for 30 min at 4 °C. After centrifugation at 5000 g for 10 min, supernatants were analyzed for AP-1 binding in EMSA, as described above.

IL-2 Secretion

CD4+ E6-1 cells, CD4- JN cells, or purified CD4+ T cells were pretreated with medium, various concentrations of gp120, or anti-CD4 mAb (mAb Leu3a) followed by stimulation with anti-CD3 mAb (mAb 454) plus 10 ng/ml PMA for 24 h. Culture supernatants were collected and analyzed for the presence of IL-2 by commercial ELISA kit (R & D Systems, Minneapolis, MN) according to the manufacturer's protocol.


RESULTS

gp160-induced AP-1 Activation in CD4+ T Cells

Fig. 1shows that the basal level of AP-1 activation in the CD4+ E6-1 cells could be enhanced by stimulation with gp160 at a concentration as low as 0.01 mg/ml. This concentration of envelope proteins has been previously reported in the serum of HIV-infected individuals(32) . Soluble gp120, anti-CD4 mAb (Leu3a) and PMA alone could also induce activation of AP-1 in the CD4+ E6-1 cells. Specific binding was demonstrated by abrogation of the AP-1 binding in the presence of excess unlabeled oligonucleotides corresponding to the AP-1 site in the IL-2 promoter (competitor, lane10). The kinetics of the induction of the proteins was examined by using nuclear extracts from E6-1 cells stimulated for increasing amounts of time. The gp160-induced AP-1 binding was observed within 30 min, peaked at 4 h, persisted for 24 h of stimulation (Fig. 2). The gp160-induced AP-1 binding was specific, since it could be abrogated by pretreatment of gp160 with goat anti-gp120 polyclonal antibody (Fig. 3, compare lane2 with lanes6 and 7); normal goat serum had no significant effect on the stimulatory effect of gp160 (lane8). That the constituents of the gp160-induced AP-1 complex comprises Jun and Fos components was confirmed by abrogation of AP-1 binding by immunoprecipitation of the gp160-stimulated nuclear extracts with polyclonal antibodies to c-Jun and c-Fos (Fig. 4); even the basal level binding of AP-1 was inhibited by the addition of these antibodies. Normal rabbit serum had no significant effect on the gp160-induced AP-1 binding. These results indicate that gp160 can induce activation of AP-1 in CD4+ T cells and that the AP-1 complex consists of Fos/Jun family of proteins.


Figure 1: Stimulation of CD4 positive T cells with gp160 induces AP-1 activation. Stimulation of E6-1 cells was carried out by the addition of medium alone (lane1, unsti) or various concentrations of gp160 (lanes2, 3, and 4) and gp120 (lanes5, 6, and 7), or 1 µg/ml of anti-CD4 mAb (Leu3a, lane8) or 50 ng/ml PMA (lane9), for 4 h at 37 °C. Lane10 comprised of competition of AP-1 binding by 10 cold AP-1 oligonucleotides corresponding to the IL-2 AP-1 site. The hollowarrow indicates mobility of free probe, and the solidarrow indicates the position of specific AP-1 binding. The result is a representative of at least five separate experiments.




Figure 2: Kinetics of the gp160-induced AP-1 activation. E6-1 cells were stimulated with 1 mg/ml gp160 for various time intervals indicated. AP-1 binding was analyzed by EMSA. The lowerband shows nonspecific binding (ns).




Figure 3: Pretreatment of gp160 with soluble CD4-IgG or anti-gp160 antibodies abrogates its activity. E6-1 cells were stimulated with medium alone (lane1, unsti) or 1 µg/ml gp160 (lanes2-8) in the presence of 10 or 1 µg/ml soluble CD4-IgG (Genentech, CA) (lanes3 and 4) or 1 µg/ml bovine serum albumin, (lane5), 1:1000, 1:3000 dilution of polyclonal goat anti-gp160 antibodies (lanes6 and 7), or 1:1000 dilution of normal goat serum (lane8). EMSA were performed as described under ``Materials and Methods.''




Figure 4: The gp160-induced AP-1 complex contains Fos and Jun family of proteins. Nuclear extracts were generated from E6-1 cells either unstimulated (lane1) or stimulated with 1 µg/ml gp160 for 4 h at 37 °C (lanes2-8). The gp160-induced nuclear extracts were incubated with medium (lanes1 and 2), 1 µg (in 1 µl), or 0.1 µg (in 0.1 µl) of antibodies to Fos and Jun proteins (which recognize all of the members of the Fos/Jun family of proteins) or normal rabbit serum for 1 h at 4 °C. Immune complexes were immunoprecipitated with Protein A-Sepharose beads (Pharmacia), and supernatants were analyzed for AP-1 binding. Results are representative of five separate experiments.



The gp160-induced Activation of AP-1 Was Mediated through the CD4 Molecule

In order to demonstrate that the cell surface molecule involved in the gp160-mediated stimulatory effects was CD4, gp160 was first preincubated with soluble CD4- IgG for 30 min at 4 °C, prior to addition to the cell cultures. Fig. 3shows that the stimulatory activity of gp160 on E6-1 cells could be abrogated by pretreatment of gp160 with soluble CD4-IgG (compare lane2 with lanes3 and 4). An irrelevant protein, bovine serum albumin, did not have any effect on the gp160-induced activation of AP-1 (lane5).

In order to further demonstrate that gp160-induced activation of AP-1 was mediated through the CD4 molecule, CD4+ and CD4 negative T cell lines were analyzed. Fig. 5shows that gp160 could stimulate AP-1 activation in CD4+ H9 cells, Molt4 cells, but not in CD4 negative mutant Jurkat T cells (JN). Here again, pretreatment of gp160 with soluble CD4 abrogated AP-1 activation in CD4+ T cells. All of these cells could be effectively induce AP-1 activation upon stimulation with PMA. These results demonstrate that the stimulatory activity of gp160 on AP-1 activation is mediated through the CD4 molecule.


Figure 5: CD4 positive T cell lines, but not CD4 negative T cell lines could be induced by gp160 to increase AP-1 activation. H9 cells were stimulated with medium alone (lane1, unsti), or with PMA, gp160, or gp120 (lanes2, 3, and 4) in the presence of soluble CD4 IgG (lanes5, 6, and 7); E6-1 cells were stimulated with medium alone (lane8), PMA and gp160 (lanes9 and 10) as positive controls. CD4 positive Molt4 and CD4 negative JN (mutant CD4 negative Jurkat cells) were stimulated with medium alone (lanes1 and 7, unsti), gp160, gp120 (lanes2 and 3 and lanes8 and 9) in the presence of soluble CD4-IgG (lanes4 and 5 and lanes10 and 11) or PMA alone (lanes6 and 12).



Gp160 Induced AP-1 Activation in Peripheral Blood CD4+ T Cells

In order to determine the ability of gp160 to activate physiological cells and peripheral blood T cells, purified T cells were first stimulated with gp160 for 4 h at 37 °C. CD4+ T cells were separated from CD8+ T cells by negative selection using anti-CD8 mAb-conjugated magnetic beads; cells bound to the beads were designated CD8+, and unbound T cells designated CD8-. Fig. 6shows that stimulation of CD8- T cells (>90% CD4+ by flow cytometry) with gp160 induced AP-1 activation (upperrightpanel) no activation of AP-1 occurred in the CD8+ T cells (upperleftpanel). The stimulatory effects of gp160 on CD8- T cells could be abrogated by pretreatment of gp160 with soluble CD4- IgG (lowerpanel). Soluble CD4- IgG itself did not induce activation of AP-1 (data not shown).


Figure 6: gp160 can induce CD8- but not CD8+ peripheral blood T cells to induce AP-1 binding. Upper panel, purified T cells were stimulated with medium alone (lane1, unsti), 1 µg/ml gp160 (lanes2 and 3), or gp120 (lanes4 and 5). CD4 and CD8 positive T cells were separated by anti-CD8 mAb-conjugated magnetic beads (Dynal, Great Neck, NY). Adherent cells were denoted CD8 positive and nonadherent cells as CD4 positive. Nuclear extracts were assessed for AP-1 binding by EMSA. The arrow indicates specific binding for AP-1, and the lower band indicates nonspecific binding (ns). Lower panel, gp160 induced AP-1 binding in CD4+ peripheral blood T cells can be abrogated by soluble CD4- IgG. Purified T cells were stimulated with medium alone (lane1, unsti) or 1 µg/ml gp160 (lanes2 and 3) or gp120 (lanes4 and 5) in the presence of soluble CD4-IgG (lanes3 and 5); 50 ng/ml PMA (lane6). Nuclear extracts of CD4+ T cells were analyzed for AP-1 binding by EMSA.



Signal Requirements for the gp160-induced AP-1 Activation

In order to investigate the nature of the signals involved in the activation of AP-1 by gp160, several pharmacological inhibitors were utilized. Fig. 7shows that addition of herbimycin A (HA, inhibitor of tyrosine phosphorylation), and H7 (inhibitor of protein kinase C) abrogated gp160-induced AP-1 activation. A more specific inhibitor of protein kinase C activity, calphostin (33) also inhibited the gp160-induced AP-1 binding (data not shown). Cyclosporine A (CsA) and the protein synthesis inhibitor, cycloheximide (CHX) had no significant effect on activation of AP-1, as did the calcium channel blocker, verapamil (ver). These studies indicate that tyrosine phosphorylation and activation of protein kinase C were involved in the mechanism of the gp160-induced activation of AP-1 binding.


Figure 7: The gp160-mediated AP-1 binding is dependent on tyrosine phosphorylation, activation of protein kinase C, but not on protein synthesis or increase in intracellular calcium or CsA. E6- 1 cells were pretreated with various concentrations of cyclosporine A (CsA), herbimycin A (HA), verapamil (ver), cycloheximide (CHX), or H7 and stimulated with gp160 for 4 h at 37 °C. Electromobility shift assays were performed as described under ``Materials and Methods.''



gp120 Inhibits IL-2 Secretion by CD4+ T Cells

E6-1 cells or purified peripheral blood CD4+ T cells were pretreated with 1 µg/ml gp120 or anti-CD4 mAb for 4 h at 37 °C (which induced AP-1 binding) and stimulated with anti-CD3 mAb plus PMA for an additional 24 h at 37 °C, and culture supernatants were analyzed for IL-2 secretion. Table I shows that gp120- or anti-CD4 mAb-treated E6-1 cells and CD4+ peripheral blood T cells were markedly inhibited in their ability to secrete IL-2 in response to anti-CD3 mAb. Pretreatment of the CD4 negative T cells (JN) cells with gp120 or anti-CD4 mAb, however, had no effect on anti-CD3 mAb plus PMA-induced IL-2 secretion.


DISCUSSION

We have demonstrated that the addition of gp160 to CD4+ T cells induces activation of transcription factor AP-1 by signals transduced directly through the CD4 molecule.

Signals transduced through the CD4 molecule on T cells has been shown to play an important role in regulating T cell functional responses mediated through the T cell receptor(8) . Earlier studies had implicated that the binding (adhesion) of the CD4 molecule with its natural ligand, MHC class II molecule, participated in T cell activation by stabilizing the T cell receptor (TCR)-MHC interactions (34) . In addition, inhibition of T cell activation by anti-CD4 mAbs in MHC class II independent systems suggested that inhibitory signals were transduced through the CD4 molecule(35) . In contrast, recent experiments have indicated that positive signals may be induced via the CD4 molecule, either by anti-CD4 mAbs (36) or by HIV envelope glycoproteins, gp160/gp120(2, 3, 4, 5) . Although the CD4-induced signals have been shown to synergize with anti-CD3 or anti-TCR mAb (37) the aberrant persistent activation through this molecule on T cells, may contribute to the pathogenesis of disease, e.g. in HIV infection(38) .

Cellular activation plays a central role in HIV infection(22) . Virus internalization, syncitium formation, and proviral replication have been shown to require cellular activation(39, 40) . Several investigators have demonstrated that binding of gp160/gp120 to CD4 molecules on the cell surface results in activation of biochemical signals(2, 3, 4, 5) . We have demonstrated that binding of gp160, (at concentrations found in vivo,(32) ) to CD4 molecules on T cells can induce biological events, e.g. up-regulation of CD40 ligand(15) , secretion of IL-6, IL-3, granulocyte macrophage colony-stimulating factor, interferon , tumor necrosis factor alpha (16, 17) , and up-regulation of Fas antigen (17) on CD4 T cells. These observations unequivocally demonstrate that gp160 can transduce signals through the CD4 molecule in T cells culminating into biological events.

In the present study, we have demonstrated that gp160 can induce activation of AP-1 in CD4+ T cells. The presence of Fos/Jun family of proteins in the gp160-induced AP-1 complex was confirmed by abrogation of AP-1 binding in immunoprecipitation experiments. Further studies are needed to determine the involvement and functional role of individual Fos/Jun components in the gp160-induced AP-1 complex. The stimulatory effects of gp160 are mediated through the CD4 molecule, since pretreatment of gp160 with soluble CD4- IgG abrogates its activity. Furthermore, cell lines expressing the CD4 molecule (H9, Molt4), but not CD4 negative cell line (JN), can be induced by gp160 to activate AP-1. gp160 can also stimulate peripheral blood CD4 positive cells, but not CD8 positive T cells to activate AP-1. Finally, the stimulatory effects of gp160 can be mimicked by anti-CD4 mAb. These results clearly demonstrate AP-1 activation by direct stimulation through the CD4 molecule.

Post-translational modifications upon T cell activation involve activation of pre-existing Fos/Jun by intracellular kinases and phosphatases(19) . The observation that protein synthesis inhibitor, CHX failed to abrogate (and in fact augments) the gp160-induced AP-1 binding at 4 h suggests that post-translational modification of preexisting Fos and/or Jun induces AP-1 binding at 4 h. Tyrosine phosphorylation inhibitor, herbimycin A, abrogated the gp160-induced AP-1 binding. In resting cells, the pre-existing Jun is phosphorylated on three sites at the C-terminal domain next to its DNA binding domains (21) ; phosphorylated states of these sites inhibits DNA binding(41) . The activation of Jun requires dephosphorylation of these sites, possibly by a protein kinase C-activated phosphatase(42) . Calcineurin, the phosphatase that modulates NFAT activity(43) , is not involved in c-Jun dephosphorylation, since AP-1 binding is unaffected by treatment of cells with cyclosporine A. While intracellular calcium channel blocker, verapamil, failed to block AP-1 binding, the requirement of protein kinase C activation for the gp160-mediated AP-1 binding was demonstrated by the addition of inhibitors, H7 and calphostin. Understanding the precise nature of the post-transcriptional modification involving activation of the Ha-Ras oncoprotein(44, 45) , mitogen-activated protein kinase pathway(46) , or the newly described JNK and SAPK pathways (47, 48) may give an insight into the regulatory role of CD4-mediated signals in T cell activation.

AP-1 has been demonstrated to be target for T cell clonal anergy, as demonstrated by down-modulation of AP-1 binding and transactivation in anergized T cell clones(49) . We and others have previously demonstrated that pretreatment of CD4+ T cells with envelope proteins of HIV-1 can induce unresponsiveness of T cells upon stimulation through TCRbulletCD3 complex(23, 24, 25, 26) . It is possible that the stimulatory effect of gp160 on AP-1 binding, involving the repressive members of the Jun family, i.e. JunB(50, 51) , which may inhibit IL-2 gene transcription. Our previous studies have demonstrated that pretreatment of T cell clones with envelope glycoproteins or anti-CD4 mAb inhibited IL-2 secretion at the transcriptional level(25, 26) . In this study, we have shown that pretreatment of the CD4+ T cells with gp160 or anti-CD4 mAb for 4 h at 37 °C (which results in activation of AP-1 binding) inhibits anti-CD3 plus PMA-induced IL-2 secretion. Since the inhibition of IL-2 secretion by gp120 in these experiments was independent of the presence of antigen presenting cells, it can be hypothesized that signals mediated through the CD4 molecule in T cells, which results in activation of AP-1, may interfere with signal transduction through the TCRbulletCD3 complex.

Given that the promoters of IL-2, IL-3, IL-6, granulocyte macrophage colony-stimulating factor, TCRbeta, and the HIV long terminal repeat contain AP-1 binding sites(18, 52, 53, 54, 55, 56) , it is possible that the gp160-induced activation of AP-1 in T cells can regulate the expression of these molecules. Since c-Jun and c-Fos have also been implicated in the mechanism of apoptosis(57, 58) , it is possible that gp160-induced AP-1 activation may play a significant role in apoptosis mechanisms in HIV infection. In conclusion, we have demonstrated that soluble envelope glycoproteins of HIV-1, gp160, by binding to the CD4 molecule on T cells, may transduce signals that result in aberrant activation of AP-1. These effects by gp160, or by HIV itself in vivo, may contribute to biological events, e.g. enhanced HIV replication, hypergammaglobulinemia, increased cytokine secretion, hypercellularity in bone marrow, apoptosis, and induction of T cell unresponsiveness.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants AI 28281 (to S. P.) and AI 35414 (to N. C.). The electronic mail services were supported by clinical research Grant MO1 RR 0047. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence may be addressed. Tel.: 516-562-4641; Fax: 516-562-2866.

()
Recipient of a student intern award from the Pediatric AIDS Foundation.

(^1)
The abbreviations used are: MHC, major histocompatibility class; HIV, human immunodeficiency virus; AP-1, activated protein-1; IL, interleukin; PMA, phorbol 12-myristate 13-acetate; EMSA, electrophoretic mobility shift assay; TCR, T cell receptor; mAb, monoclonal antibody.


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