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(Journal of Leukocyte Biology. 2003;73:407-416.)
© 2003 by Society for Leukocyte Biology

Human immunodeficiency virus type 1 (HIV-1) induces activation of multiple STATs in CD4+ cells of lymphocyte or monocyte/macrophage lineages

James J. Kohler*, Daniel L. Tuttle*, Carter R. Coberley*, John W. Sleasman*,{dagger} and Maureen M. Goodenow*,{dagger}

Departments of
* Pathology, Immunology, and Laboratory Medicine, and
{dagger} Pediatrics, Division of Immunology and Infectious Diseases, College of Medicine, University of Florida, Gainesville


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Human immunodeficiency virus type 1 (HIV-1) impacts the activation state of multiple lineages of hematopoietic cells. Chronic HIV-1 infection among individuals with progressive disease can be associated with increased levels of activated signal transducers and activators of transcription (STATs) in peripheral blood mononuclear cells. To investigate interactions between HIV-1 and CD4+ cells, activated, phosphorylated STAT proteins in nuclear extracts from lymphocytic and promonocytic cell lines as well as primary monocyte-derived macrophages were measured. Levels of activated STATs increased six- to tenfold in HUT78 and U937 cells within 2 h following exposure to virions. The response to virus was dose-dependent, but kinetics of activation was delayed relative to interleukin-2 or interferon-{gamma}. Activation of STAT1, STAT3, and STAT5 occurred with diverse viral envelope proteins, independent of coreceptor use or viral replication. Envelope-deficient virions had no effect on STAT activation. Monoclonal antibody engagement of CD4 identified a novel role for CD4 as a mediator in the activation of multiple STATs. Results provide a model for HIV-1 pathogenesis in infected and noninfected hematopoietic cells.

Key Words: EMSA • pathogenesis • CCR5 • CXCR4


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Infection of T lymphocytes or macrophages by human immunodeficiency virus type 1 (HIV-1) leads to acquired immune deficiency syndrome (AIDS), which is characterized by immune dysfunction, opportunistic infections, and/or associated cancers [1 , 2 ]. Immune dysfunction is not restricted to infected CD4+ T cells but reflects a global impact on cellular and humoral immunity [3 4 5 ]. HIV-1 infection results in increased T cell activation and clonal expansion within CD4 and CD8 T lymphocytes [6 ]. Chronic viral antigenemia skews post-thymic T cell differentiation, alters normal cytokine production, increases the production of inflammatory proteins, and ultimately leads to T cell anergy and clonal exhaustion [7 , 8 ]. HIV-1 infection also impacts macrophages, which display decreased chemotaxis and reduced antigen-presenting activity [9 , 10 ]. These diverse immune abnormalities develop early in the course of infection by HIV-1 before severe depletion of CD4+ cells occurs [11 ], indicating that HIV-1 impacts the biological state of multiple lineages of hematopoietic cells by direct as well as indirect mechanisms.

Signal transducer and activator of transcription (STAT) proteins are effectors of cytokine signaling in multiple hematopoietic cell lineages, some of which are targets for HIV-1 infection [12 13 14 15 ]. Cytokine engagement of cell-surface receptors results in autophosphorylation of a specific receptor-associated tyrosine kinase of the Janus kinase (JAK) family, which in turn recruits a specific, constitutively expressed, inactivated STAT from the cytoplasm [15 ]. Each cytokine/surface receptor associates with one of four JAK proteins (JAK1–3 or TYK2), which activates one of seven STATs (STAT1–4, -5a, -5b, or -6). JAK proteins interact with an src-homology 2 domain in STAT proteins to activate STATs by phosphorylation [13 , 16 ]. Activated STATs dissociate from the JAK/receptor complex, dimerize [13 ], translocate to the nucleus, and bind to specific DNA-response elements. The result is regulation of expression of a variety of genes including JunB, C-reactive protein, BclxL, interferon-{gamma} (IFN-{gamma}), Fc-{gamma}RI, ß-casein, and/or 2,3-dioxygenase [12 , 16 , 17 ].

Although STATs were originally discovered in the context of IFN signaling, numerous other cytokines and surface receptors, such as the T cell receptor or glucocorticoid receptor, activate STATs [18 19 20 ]. STAT proteins have a specificity defined by cell type. For example, STAT5 associations with JAK1 or JAK3 play an important role in T lymphocytes by preventing feedback inhibition of cell expansion [21 ]. STAT4 proteins are effectors of T helper cell type 1 (Th1)-mediated inflammation in peripheral blood monocytes, dendritic cells, and macrophages [22 ]. Defects in activation of STAT transcription factors are linked to deficiencies of cellular immunity or oncogenic signaling [23 24 25 ].

HIV-1 is implicated in constitutive activation of STAT1 and STAT5 in some chronically infected individuals, and treatment of human astrocytes by gp120 leads to tyrosine phosphorylation of STAT1 [26 , 27 ]. HIV-1 infection could directly modulate STAT activation through binding to and/or infection of CD4 target cells. Alternatively, virus/cell interactions could indirectly mediate paracrine stimulation of STATs in bystander cells. We designed a study to investigate direct and immediate interactions between HIV-1 or gp120 and STAT signal-transduction pathways through functional measurement of activated STAT proteins in nuclear extracts from CD4+ lymphocytic and promonocytic cell lines, as well as primary monocyte-derived macrophages.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell cultures
Human cutaneous T cell lymphoma [(HUT)78; Cat. no. TIB-161] and human histiocytic lymphoma (U937; Cat. no. CRL-1593) cell lines were obtained from the American Type Culture Collection (Manassas, VA) [28 , 29 ]. Cell cultures were maintained at 37°C with 5% carbon dioxide in RPMI-1640 media with 10% heat-inactivated fetal bovine serum (FBS), 100 U penicillin per ml, 100 µg streptomycin per ml, and 0.05% sodium bicarbonate. Cell cultures were split every 4 days to maintain cell density in the range of 2.5–15 x 105 cells per ml. Transformed primary embryonal kidney cells (293) were obtained from the NIH AIDS Research and Reference Reagent Program (Rockville, MD) and maintained in Dulbecco’s modified Eagle’s medium with 10% FBS [30 ].

Monocytes from healthy seronegative donors were isolated using RosetteSep (Stem Cell Technologies, Vancouver, BC, Canada), according to the manufacturer’s protocol. This method routinely results in the isolation of cells that are greater than 95% pure monocytes with fewer than 5% B and T cell contamination. Cells were adhered to plastic for 2 h at a concentration of 2 x 106 per ml, washed to remove nonadherent cells, and differentiated into macrophages for 7 days at 37°C with 5% carbon dioxide in RPMI media containing 15% human serum, 2.5 mM Hepes, 100 U penicillin per ml, 100 µg streptomycin per ml, and 10 units granulocyte macrophage-colony stimulating factor (Invitrogen, Carlsbad, CA) per ml [31 ].

Reagents and viral agents
The following reagents were obtained from the NIH AIDS Research and Reference Reagent Program: HIV-1LAI/peripheral blood mononuclear cell (PBMC) [32 , 33 ], HIV-1JR-FL[34 35 36 ], and pNL4–3.Luc.R+E-[37 ], in addition to HIV-1BaL gp120, recombinant (Cat. no. 4961), HIV-1IIIB gp120 (ImmunoDiagnostics, Inc., Woburn, MA; Cat. no. 3926), HIV-1MN gp120 (ImmunoDiagnostics, Inc.; Cat. no. 3927), HIV-1SF2 gp120 [38 ], or HIV-1SF2 gp120 (Env2–3) nonglycosylated [39 40 41 42 43 ]. HIVLAI and HIVJR-FLvirus stocks were prepared by coculture with uninfected PBMC, titrated by serial dilution in phytohemagglutinin-activated PBMC, and tissue-culture infectious dose (TCID)50-calculated [44 ]. Cell cultures (3.0x107 cells) were routinely incubated with replication-competent (125 TCID50 per ml) or single-cycle HIV-1 virions (750 ng p24 per ml) for 2 h at 37°C in 5% CO2. Supernatants from 293 cells transfected with pNL4–3.Luc.R+E- were collected after 48 h and filter-purified (0.45 µm) for use in mock infections. Purified HIV gp120 proteins were added (0.5–5 µg) to cells in serum-free RPMI-1640 media (incomplete) and incubated for 2 h before performing nuclear extraction. Monoclonal anti-human CD4 clone Q4120 (Sigma Chemical Co., St. Louis, MO) was used in CD4-binding studies by 30 min incubation with HUT78 or U937 cells before nuclear extraction.

Single-cycle, envelope-pseudotyped, luciferase-tagged viruses were produced from pNL4–3.Luc.R+E-, as described previously [45 ]. Envelope V1–V5 domains from patient-derived, primary isolates had the following characteristics: P1 (macrophage tropic-CCR5), P2 (dual macrophage and T cell tropic-CXCR4), and P3 (dual tropic-CCR5 and -CXCR4) [46 ].

STAT specificity was determined by incubating nuclear extracts with a panel of anti-STAT antibodies including mouse monoclonal IgG1 anti-STAT1 p84/p91 (C-136), rabbit polyclonal anti-STAT3 (C-20), mouse monoclonal IgG1 anti-STAT5 (G-2), and a nonspecific control antibody, mouse monoclonal IgG1 anti-Ying Yan (YY1; H-10). All antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Four-color flow cytometry
Cells were fixed in 1% paraformaldehyde in phosphate-buffered saline (PBS) for 1 h at 4°C, washed, and resuspended in fluorescein-activated cell sorter (FACS) wash (0.02% azide, 10% FBS, 2% human serum in PBS). Fixed cells were stained for 1 h at 4°C with the following antibodies and isotype controls (Becton Dickinson, San Jose, CA): CD4-allophycocyanin (APC; SK3), IgG1-APC (x40), CXCR4-phycoerythrin (PE; 12G5), IgG1-PE (x40), CCR5-fluorescein isothiocyanate (FITC; 2D7), and IgG1-FITC isotype (x40). Following staining, cells were washed and resuspended in FACS wash. Four-color flow cytometry analysis was performed on a Becton Dickinson FACSCalibur in the University of Florida Interdisciplinary Center for Biotechnology Research Flow Cytometry Core Laboratory (Gainesville). Compensation was set using single-stained cells from the same cultures; channel markers were set based on isotype-control antibodies for each stain. Data analysis was performed using WinMDI version 2.8 (Joseph Trotter, Scripps University, San Diego, CA).

Nuclear protein extraction
Cultured cells (3.0x107 cells) were preincubated in incomplete RPMI-1640 media overnight and treated with viruses for 2 h or with 100 U interleukin (IL)-2 (Boehringer Mannheim, GmbH, Germany) or 100 U IFN-{gamma} (Sigma Chemical Co.) per ml for 15 min for optimal stimulation, as determined by kinetic studies. Nuclear extracts were prepared as described with minor modifications [47 ]. Cells were washed twice in PBS followed by two washes in PBS plus phosphatase inhibitors (1 mM sodium orthovanadate, 5 mM sodium fluoride). The cells were rinsed with hypotonic buffer {40 mM HEPES, 2 mM EDTA, 2 mM EGTA, 20 mM sodium fluoride, 1 mM sodium orthovanadate, 1 mM sodium pyrophosphate, 1 mM dithiothreitol (DTT), and 0.5 mM protease inhibitor cocktail [4-(2-aminoethyl) benzenesulfonyl fluoride, pepstatin A, trans-epoxysuccinyl-L-leucylamido (4-guanidino) butane, bestatin, leupeptin, and aprotinin]}. The cell pellet was resuspended into hypotonic buffer with Nonidet P-40 (0.2%), microcentrifuged for 20 s at 4°C, resuspended in high salt buffer (840 mM sodium chloride, 40 mM HEPES, 2 mM EDTA, 2 mM EGTA, 40% glycerol, 20 mM sodium fluoride, 1 mM sodium orthovanadate, 1 mM sodium pyrophosphate, 1 mM DTT, and 0.5 mM protease inhibitor cocktail), and incubated in a tube rotator for 30 min at 4°C. The lysed cells were centrifuged for 20 min at 4°C, and the supernatant was collected and stored at -80°C. Total protein concentrations in each extract were determined (BioRad DC protein assay kit, BioRad Laboratories, Hercules CA).

Electrophoretic mobility shift assay (EMSA)
Probe to detect activated STATs was derived from the c-fos gene promoter hSIE (AGC TTC ATT TCC CGT AAA TCC CTA, and AGC TTA GGG ATT TAC GGG AAA TGA) [25 ]. Activated STAT1 and STAT3 dimers bind to hSIE sequence with high affinity, and STAT5 binds to hSIE with a lower affinity than STAT1 or STAT3 [13 , 25 ]. Annealed probe (2x10-12 mol) was labeled with {alpha}[32P]deoxycytidine 5'-triphosphate (3000 Ci/mmol) and {alpha}[32P]dTTP (3000 Ci/mmol) using a Klenow fragment of DNA polymerase I (New England Biolabs, Beverly, MA). Specific activity (cpm/µg DNA) of labeled probe was calculated for each reaction. Nuclear extracts containing 10 µg total protein were incubated with an equal volume of labeled probe mix [10 mM HEPES, 10% glycerol, 1 mM DTT, 0.1 µg/µl poly(dI:dC), 0.5 µg per µl bovine serum albumin, radiolabeled probe (4000 cpm/µg DNA)] for 30 min at 37°C in 0.9 M Tris base, 0.9 M boric acid, and 2.5 mM NaEDTA (TBE) buffer. Specific cold competitors were generated using annealed primers hSIE (STAT1- or STAT3-specific) or MGFe (STAT5-specific), a mammary gland factor in the ß-casein gene promoter (AGC TAG ATT TCT AGG AAT TCA A and AGC TTT GAA TTC CTA GAA ATC T) [48 ] without {alpha}[32P]-labeled dNTPs. A nonspecific cold competitor was generated using annealed primers UCR, a YY1-binding site located within the Moloney murine leukemia virus promoter (ACG TTA GCG CCA TTT TAC GG) [49 ]. Cold competitors were included at a concentration of 14:1 over {alpha}[32P]-labeled probe. Specific anti-STAT1 (C-136), STAT3 (C-20), or STAT5 (G-2) antibodies, obtained at a concentration of 2 mg/ml, were added as 2–4 µg per reaction into a total volume of 20 µl. The use of larger amounts of anti-STAT antibodies up to 8 µg was determined to have no additional effect on the signal produced by the STAT/probe complex. Samples and probe mix were resolved on 5% acrylamide gels [50% glycerol, 40% (39:1) acrylamide solution, 30% ammonium persulfate, 0.5 mM N,N,N',N',-tetramethylethylenediamine in TBE buffer]. Gels were fixed (10% acetic acid and 10% methanol), rinsed with deionized water, transferred, and dried onto Whatman paper. Radiographic images were captured over 8–48 h exposures, and phosphorimages were detected using a Storm 860 scanner (Molecular Dynamics, Sunnyvale, CA) after 4-h exposures.

To quantitate levels of STAT activation from independent experiments, we developed a mathematical equation, which accounts for differences between gels. The specific fold increase in STATs for each sample was determined by a formula derived from standard approaches to phosphorimage technology: Fold increase = (average intensity of sample band–average intensity of probe alone)/(average intensity of nonstimulated control–average intensity of probe alone).

Samples analyzed on separate gels were compared by converting to relative equivalents using a ratio determined by the respective IL-2-stimulated (positive control) average intensity and mean of all IL-2-stimulated fold increases from 36 gels: Sample equivalent = sample fold increase gel #1 x (IL-2 fold increase gel #1)/(mean total IL-2 fold increases from all gels).

Fold increases in STAT activation are presented as means with SEM.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
HIVLAIor HIVJR-FLinduced activation of STAT proteins in a T lymphocytic cell line
HIVLAI was selected for its characteristic T cell tropism and specificity for the CXCR4 coreceptor, which is expressed on HUT78 T cells. In contrast, HIVJR-FLis macrophage tropic with specificity for the CCR5 coreceptor. HUT78 T cells expressed undetectable CCR5 by four-color flow cytometry and were refractory to infection by HIVJR-FL (data not shown). As a positive control, IL-2 stimulation of HUT78 cells induced activation of STATs (Fig. 1A , lane 2) by at least tenfold above constitutive levels in nonstimulated cells (Fig. 1A , lane 1) with a specificity that could be abrogated (95%) by cold competitor (Fig. 1A , lane 3). HIVLAI induced an increase in activated STATs that was approximately tenfold greater than levels in nonstimulated cells (Fig. 1A , lanes 4 and 1, respectively). Competition with 14-fold excess cold hSIE probe could abrogate the activated STAT signaling (Fig. 1A , lane 5). Surprisingly, HIVJR-FLalso induced STAT activation (Fig. 1A , lane 6) with a ninefold increase over nonstimulated cells and induced activation of STATs that could be abrogated by competition with 14-fold excess cold hSIE probe (Fig. 1A , lane 7). Activation appeared dependent on envelope, as treatment by HIV-1 envelope-deficient virions (pNL4–3.Luc.R+E-) produced a level of STAT activation (Fig. 1A , lane 8) that was comparable with the level detected in nontreated cells (Fig. 1A , lane 1). Results raised the possibility that the activation of STATs by HIV-1 was coreceptor independent.



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  		Figure 1. HIV-1 induces STAT activation in HUT78 and U937 cells. (A) Radiographic image of EMSA gels demonstrating competition of activated STATs (STAT1, STAT3, and/or STAT5) in HUT78 T lymphocytic cell line. Results shown for HUT78 cells are representative of 10 separate experiments. (B) STAT activation in U937 promonocytic cell line. Results shown for U937 cells are representative of six separate experiments. (C) Effect of nonspecific competitor UCR on detection of STAT activation in U937 cells. In all representative EMSAs, untreated (NoTx), envelope-deficient virion (Env-), STAT dimers (*) and quatramers (**), nonspecific binding (NS), and excess probe (P) are indicated. As a result of the specificity of probes used, detection of STAT complexes is indicative of homodimers or heterodimers of STAT1, STAT3, and/or STAT5. Concentration of competitors was in tenfold excess over {alpha}[32P]-labeled probe.

 
HIVLAI or HIVJR-FLinduced activation of STAT proteins in a promonocytic cell line
To investigate cell-type specificity of STAT activation by HIV-1 strains, cells from a different CD4 lineage were incubated with HIVLAI or HIVJR-FL. U937 promonocytic cell line, similar to the HUT78 T lymphocytic cell line, expressed CD4 and CXCR4 but not CCR5 by four-color flow cytometry (data not shown). IL-2 stimulation had no effect on STAT activation in U937 cells (data not shown), as monocytic cells fail to express the IL-2 receptor. Instead, IFN-{gamma} stimulation activated two types of STAT/probe complexes (Fig. 1B , lane 2), which were abrogated by hSIE or MGFe cold competitors (Fig. 1B , lanes 3 and 4). The two STAT/probe complexes induced by IFN-{gamma} in U937 cells were similar to complexes designated as dimers and quatramers in other studies [50 ]. Treatment by HIVLAI or HIVJR-FL produced increases of activated STATs in promonocytes (Fig. 1B , lanes 5 and 8). In contrast to IFN-{gamma}, virus activated STAT dimers but failed to produce quaternary STAT complexes. The dimeric complexes were specific based on competition with 14-fold excess concentrations of hSIE or MGFe cold probes (Fig. 1B , lanes 6 and 7 and lanes 9 and 10). Envelope-deficient virions had minimal if any effect on STAT activation in promonocytes (Fig. 1B , lane 11). Viral activation of STAT proteins in the U937 promonocytic cell line required envelope but was not specific for CCR5 or CXCR4, similar to results in the HUT78 T lymphocytic cell line.

The specificity of hSIE and MGFe as cold competitors was further defined by comparing a nonspecific cold competitor, UCR, for the ability to abrogate STAT complex formation with {alpha}[32P]-labeled probe. UCR oligonucleotide encodes the binding site that is the target for the ubiquitously expressed YY1 nuclear transcription factor. Although 14-fold excess concentration of hSIE cold competitor effectively abrogated STAT complex signaling induced by IFN-{gamma} in promonocytes (Fig. 1C , lanes 1–3), 14-fold excess concentration of UCR cold competitor had no effect (Fig. 1C , lanes 4 and 5).

STAT activation by HIVLAIand HIVJR-FLis dose-dependent
Although IL-2 or virus activated STAT proteins to similar levels, the kinetics differed (Fig. 2A ). Tenfold activation in T lymphocytic cells by IL-2 appeared rapidly within 15 min (Fig. 2A) but declined by 90 min to less than fivefold over nonstimulated cells (data not shown), demonstrating immediate and transient kinetics. In contrast, increases in STAT activation by HIVLAIwere detectable only after 30 min. Maximum levels were achieved by 2 h and sustained for 2 h before declining to levels that were threefold (±1.0) greater than found in nontreated cells. HIVJR-FLinduced STAT activation with kinetics similar to HIVLAI (data not shown). The impact by virus within 2 h suggested that initial interactions between virus and cells, rather than productive infection, were sufficient for STAT activation. Based on the kinetics of response, 2-h activation by virus was used for all studies.



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  		Figure 2. Kinetics and dose-dependent effects of HIV-1 induction of STAT activation. (A) Kinetics of IL-2 or HIVLAI activation of the JAK/STAT signal-transduction pathway in HUT78 cells expressed as fold increase of activated STAT proteins over nonstimulated or mock-infected cells by EMSA. (B) Dose-dependent effect of HIVLAI (diamonds) or HIVJR-FL (squares) on level of STAT activation in HUT78 cells expressed as fold increase over nontreated cells (baseline=1). Represented are means from two experiments for each time point with standard deviations.

 
HUT78 cells were treated with variable doses of HIVLAI or HIVJR-FLbefore nuclear extracts were prepared and analyzed for levels of STAT activation (Fig. 2B) . Twofold increases were detected following exposure to a low dose of HIVLAI (16 TCID50/ml), and a higher viral dose (139 TCID50/ml) resulted in a fivefold (±1.0) activation of STATs. HIVJR-FLalso induced STAT activation in a dose-dependent manner (Fig. 2B) . Increased doses of either virus failed to produce additional STAT activation, indicating that a level of saturation was reached. Differences in mean levels of STAT activation between HIVJR-FL and HIVLAI were not statistically significant. HIVJR-FL virions were as effective as HIVLAI virions in inducing STAT activation, despite the absence of CCR5 expression by HUT78 or U937 cells, indicating that activation was independent of CCR5 or CXCR4.

HIVLAI and HIVJR-FLactivate STAT1, STAT3, and STAT5
Direct competition by 14-fold excess, unlabeled hSIE probe suggested that STAT1 and/or STAT3 were activated, and competition with excess MGFe probe suggested that STAT5 might also be activated [25 ]. To identify the specific STATs induced by HIVLAIor HIVJR-FL, nuclear extracts were treated with a panel of anti-STAT antibodies before interaction with target sequences. Interaction of anti-STAT antibodies with specific STAT proteins can be identified by reduced mobility of the complex (supershift) or by diminished intensity of the specific band, which can occur, as antibody–STAT-binding interactions increase complex size—which limits migration through an acrylamide matrix—or prevent stable complex formation [25 ]. Anti-STAT3 or anti-STAT5 antibodies (1:10) reduced the signal of the STAT complexes in HUT78 cells stimulated with IL-2 (Fig. 3A , lanes 2–4) [12 ]. HIV-induced STAT complexes (Fig. 3A , lanes 6 and 10) were reduced dramatically by anti-STAT1, -STAT3, or -STAT5 antibodies (Fig. 3A , lanes 7–9 and 11–13, respectively). A combination of any two anti-STAT antibodies further diminished but did not completely abrogate the signal produced by the STAT/probe complex (data not shown).



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  		Figure 3. Identification of specific STATs induced by HIVLAI or HIVJR-FL. (A) Radiographic image of EMSA resulting from 30 min preincubation of nuclear extracts from HUT78 cells, stimulated by IL-2 or HIV strains, with a panel of antibodies including anti-STAT1, -STAT3, or -STAT5. (B) Comparison EMSA resulting from nuclear extracts collected from U937 cells is represented. In all cases, the abrogation of STAT complex is defined as a positive antibody interaction. In all representative EMSAs, untreated (NoTx), STAT dimers (*) and quatramers (**), nonspecific binding (NS), and excess probe (P) are indicated.

 
IFN-{gamma} stimulation in monocytes preferentially activates STAT1 [12 , 50 ]. In our studies, anti-STAT1 antibodies diminished the STAT dimer and quaternary complexes induced by IFN-{gamma} stimulation in U937 promonocytic cells (Fig. 3B , lanes 2 and 3), and anti-STAT3 or -STAT5 antibodies had no effect on quaternary forms but reduced dimeric STAT (Fig. 3B , lanes 4 and 5, respectively). STAT dimer complexes induced by HIV-1 viruses were diminished by anti-STAT1 antibodies (Fig. 3B , lanes 7 and 11) as well as by antibodies specific for STAT3 or STAT5 (Fig. 3B , lanes 8 and 9, and 12 and 13).

Envelope glycoproteins induce STAT activation
To determine if STAT activation could occur independent of productive virus replication, single-cycle recombinant viruses were pseudotyped with envelopes containing V1–V5 sequences from HIVLAI, HIVJR-FL, or three different patient isolates (P1, P2, and P3). Envelopes from each patient isolate used CD4 in combination with different coreceptors. Envelope P3 used CCR5 and CXCR4, envelope P1 used CCR5, and envelope P2 used CXCR4 [45 , 46 ].

HIVJR-FLor HIVLAI envelope-pseudotyped viruses resulted in STAT activation in HUT78 cells (Fig. 4A , lanes 3 and 4, respectively). All three recombinant viruses that were pseudotyped by envelopes from patient isolates also induced activation of STAT proteins (Fig. 4A , lanes 5–7). In contrast, envelope-deficient virions produced no effect on the level of STAT activation (Fig. 4A , lane 8). In U937 promonocytes, each of the five envelope-pseudotyped viruses produced increased levels of STAT activation, and envelope-deficient virions still had no effect (data not shown). Competition in the presence of unlabeled hSIE occurred, confirming specificity of STAT/probe complex formation (data not shown). Activation of STATs (STAT1, STAT3, and/or STAT5) was independent of productive viral replication but was dependent on envelope glycoproteins.



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  		Figure 4. Induction of STAT activation by single-cycle envelope recombinant viruses or various gp120 proteins. (A) Nuclear extracts from HUT78 T lymphocytic cells induced by single-cycle recombinant viruses pseudotyped with V1–V5 envelopes from HIVLAI, HIVJR-FL, or one of three different patient isolates (Pt1–Pt3; described in Materials and Methods). Coreceptor use and tropism of recombinant viruses are indicated by CXCR4 (X4) or CCR5 (R5) use and ability to infect T cell lines (T), macrophages (M), or both (dual, D). (B) STAT activation in U937 promonocytic cells following 2 h incubation with gp120-purified proteins derived from HIVBaL, HIVIIIB, HIVMN, HIVSF2, or nonglycosylated HIVSF2 (Env2–3) was compared with IFN-{gamma}, replication-competent HIVJR-FL, or a virion deficient in envelope (Env-). (C) STAT activation in primary monocyte-derived macrophages (MDM) following treatment with IFN-{gamma} or HIV-1 gp120 proteins. A competition assay with 14-fold cold hSIE probe verified the specificity of activated STAT signaling following treatment with IFN-{gamma} or gp120 proteins (1.0 µg) from HIVIIIB or HIVSF2. Results are representative of three independent donors. (D) Kinetics of STAT activation following treatment with gp 120 was determined in U937 promonocytic cells treated with gp120IIIB (1.0 µg) for time indicated (0.5–4 h) by EMSA. (E) Dose effect of gp120 was compared with IL-2 stimulation in T lymphocytic cells. Average levels of STAT activation in 10 µg cell extracts at each treatment were calculated as fold increase over nonstimulated cells. In all representative EMSAs, untreated (NoTx), STAT dimers (*) and quatramers (**), nonspecific binding (NS), and excess probe (P) are indicated.

 
To demonstrate a direct role for envelope gp120 in STAT activation, T lymphocytes or promonocytes were treated by purified gp120s from different viral strains, HIVBaL, HIVIIIB, HIVMN, and HIVSF2 (coreceptor specificity of CCR5, CXCR4, CXCR4, and CCR5/CXCR4, respectively). EMSA analysis of promonocytic nuclear extracts demonstrated an induction of STAT activation by each of the envelope gp120 proteins within 2 h of treatment (Fig. 4B , lanes 4–8). To determine if carbohydrate modification contributed to gp120 activation of STATs, nonglycosylated envelope protein from HIVSF2 (Env2–3) was tested and found sufficient to induce STAT activation (Fig. 4B , lane 9). The impact of glycosylated or nonglycosylated gp120s on STAT activation in T lymphocytes was similar (data not shown).

To evaluate the relationship of STAT activation between promonocytic cells and primary macrophages, differentiated MDM were treated with IFN-{gamma} or gp120 proteins from HIV-1IIIB or HIV-1SF2. As in the cell lines, IFN-{gamma} treatment resulted in activation of STATs with the characteristic quatrameric form that could be competed by a cold hSIE probe (Fig. 4C , lanes 3 and 4). In addition, treatment of MDM with gp120 from either HIV-1 strain resulted in activation of STATs that could be competed by a cold hSIE probe (Fig. 4C , lanes 5–8). MDM results are representative of experimental data obtained from three independent donors.

STAT activation following treatment with gp120 appeared as early as 30 min with a duration of at least 4 h (Fig. 4D , lanes 3–6). The gp120 effect was dose-dependent. Although only a twofold increase in activation was detected with 0.5 µg gp120, a maximum ninefold (±2.0) increase in STAT activation was measured with 1–5 µg gp120 (Fig. 4E) . Together, results from experiments with soluble gp120 and with envelope-deficient virions implicate gp120 as necessary and sufficient for STAT activation.

Specificity of STAT proteins activated by treatment with gp120 was determined using anti-STAT antibodies. Soluble gp120 alone induced activation of STAT1, STAT3, and STAT5 in T lymphocytic cells (Fig. 5A , lanes 4–6) and in promonocytes (Fig. 5B , lanes 6–8). To establish the specificity of antibody/STAT interactions, a nonspecific mouse monoclonal IgG1 anti-YY1 antibody was tested with nuclear extracts from cells treated with IFN-{gamma} or gp120 (Fig. 5B , lanes 4 and 9, respectively). YYI proteins specifically bind to a site in the Moloney murine leukemia virus promoter region. As expected, the anti-YY1 antibody had no effect on the signal produced by STAT/probe complexes.



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  		Figure 5. Identification of specific STATs induced by gp120 or anti-CD4 antibody. (A) EMSA produced by 30 min preincubation with anti-STAT1, -STAT3, or -STAT5 antibodies and nuclear extracts from T lymphocytic cells treated with 1.0 µg gp120 (2 h). (B) Comparison EMSA resulting from nuclear extracts collected from promonocytic cells is represented. A nonspecific antibody, mouse monoclonal IgG1 anti-YY1, was tested as a negative control. (C) EMSA resulting from 30 min preincubation with anti-STAT1, -STAT3, or -STAT5 antibodies and nuclear extracts from MDM that were stimulated by IFN-{gamma} or HIVSF2 gp120 (1.0 µg). (D) EMSA resulting from preincubation of specific anti-STAT antibodies and nuclear extracts treated with 0.62 µg Q4120 antibody (30 min). In all cases, abrogation of signal is defined as a positive antibody interaction. Untreated (NoTx), STAT dimers (*) and quatramers (**), nonspecific binding (NS), and excess probe (P) are indicated.

 
To determine the specific STATs activated by gp120 in primary macrophages, nuclear extracts from MDM were preincubated with anti-STAT1, -STAT3, or -STAT5 antibodies. IFN-{gamma} treatment resulted in the activation of STAT1 (Fig. 5C , lanes 2 and 3), and treatment with gp120 resulted in the activation of STAT1, STAT3, and STAT5 (Fig. 5C , lanes 4–7). The impact of gp120 on specific STAT activation on promonocytic cells or primary macrophages is indistinguishable.

Interaction with CD4 induces STAT activation
Envelope gp120-mediated activation of STATs was CCR5 or CXCR4 coreceptor nonspecific and glycosylation independent, raising the possibility that CD4 could be a cellular component involved in mediating STAT activation. To demonstrate directly that STAT activation can occur through CD4, lymphocytic or promonocytic cells were treated with a mouse monoclonal antibody (mAb), Q4120, which activates T cells by binding to CD4 [51 ]. Q4120 is specific for the CDR2-like loop on the amino-terminal domain 1 of CD4, which is the same site to which gp120 binds with high affinity [29 ].

Treatment of lymphocytic or promonocytic cells by Q4120 antibody increased levels of STAT activation within 30 min and was comparable to the levels achieved following IL-2 or IFN-{gamma} stimulation (data not shown). The specificity of STAT proteins activated by treatment with Q4120 antibodies was compared with HIV-1 gp120 activation. Q4120 binding to CD4 activated STAT1 and STAT3 (Fig. 5D , lanes 3–5), and interaction of STAT complex with anti-STAT5 antibody was less apparent (Fig. 5D , lane 6).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
HIV-1 induces activation of multiple STATs in CD4-expressing T lymphocytic and promonocytic cells, as well as in primary monocyte-derived macrophages. STAT activation at the cellular level occurred rapidly, independent of productive infection or cytokine treatment, and involved viral envelope gp120 glycoproteins. Our studies identified activation of STAT1, STAT3, and STAT5, which extends the repertoire of HIV-1-related activation of STAT1 and STAT5 in primary T cells and PBMC from infected individuals or STAT1 in gp120-treated human astrocytes [26 , 27 ].

A number of viruses alter the activation state of STAT signal-transduction pathways, although with variable consequences. For example, proteins expressed by hepatitis C, simian virus 5, or human cytomegalovirus inhibit the JAK/STAT pathways [52 53 54 ]. In contrast, the latent membrane protein 1 of Epstein-Barr virus activates a JAK/STAT pathway in B lymphocytes [55 ]. Retroviruses, such as Friend leukemia virus or human T cell leukemia/lymphotropic virus type 1, induce constitutive activation of JAK/STAT proteins [56 57 58 ], and various transcription factors are activated in lymphocytes, myeloid cells, or MDM by HIV-1 Nef [26 , 59 , 60 ]. Activation of various STAT-mediated signaling pathways could be a general characteristic of the family of retroviruses, reflecting a relationship that has evolved between many retroviruses and cells of hematopoietic lineages.

Several HIV-1 proteins, including Tat and Nef, can have an effect on JAK/STAT pathways [60 ]. For example, through infection studies with env- and nef-deleted HIV-1 strains, Nef or Env proteins were shown to activate STAT1 in MDM [60 ]. In contrast, our studies demonstrated that initial interactions of gp120 and CD4-expressing cells, independent of productive infection, were sufficient to induce activation, not only of STAT1 but also STAT3 and STAT5. Multiple mechanisms have evolved by which HIV-1 can induce STAT activation, thereby implicating the important role of STATs in the HIV-1 lifecycle.

Virus- or gp120-induced activation of STATs displayed no apparent requirement for CCR5 or CXCR4 coreceptors. Interactions between envelope and other cell-surface proteins, such as minor chemokine receptors or adhesion molecules [27 ], might contribute to activation of STATs, independent of CD4. Conversely, interactions with glycosylation moieties are not likely to impact STAT activation, as the impact of nonglycosylated gp120 was identical to the glycosylated form. In addition, our studies identified a novel, virus-independent role for CD4 as a mediator of STAT1 and STAT3 activation in lymphocytic and promonocytic cell lines. CD4-mediated activation of T lymphocytes induces protein tyrosine phosphorylation, cytoplasmic inositol triphosphate and Ca2+, IL-2 secretion, and cell proliferation [61 , 62 ]. At least some of these downstream targets of activation could be mediated through CD4-dependent activation of STATs. Replication-competent HIV and gp120 proteins can also induce T cell proliferation and IL-2 production [61 , 63 ]. One model is that gp120 binding to CD4 triggers activation of some STATs [64 ]. Direct evidence that the gp120–CD4 interaction mediates STAT activation might be accomplished by including neutralizing CD4 antibodies during treatment with gp120. However, CD4 mAb that abrogate gp120–CD4 interactions such as Q4120 also induce STAT activation [61 ].

STAT activation by HIV-1 virions was similar to activation by gp120, independent of glycosylated or oligomeric forms of the protein. In contrast, treatment with gp120 differed in the activation of STAT5 from treatment with anti-CD4 antibody. mAb involves binding to a single CD4 epitope, whereas gp120 binding involves more complex interactions with CD4, as well as interactions with other cell-surface molecules. STAT activation induced by gp120 also differed from signaling through cytokine/receptor interactions alone. For example, kinetics of viral-induced STAT activation was significantly delayed in comparison with IL-2- or IFN-{gamma}-induced activation. In addition, HIV-1 gp120 activation involved at least three different STATs, in contrast to limited STAT activation by IL-2 or IFN-{gamma}. The implications are that multiple cytokine-dependent and/or -independent pathways could be induced by viral envelope engagement of CD4. In CD4+ T lymphocytes, IL-2/IL-2 receptor signaling via JAK2 activates STAT3 and STAT5 [61 , 63 , 65 ], which could mediate activation of STAT3 and STAT5 via CD4. Similarly, STAT1 activation in promonocytes could be mediated via IFN-{gamma} signaling. Activation of additional STATs—STAT1 in T cells or STAT3 and STAT5 in promonocytes—may occur through other cytokine signaling or through cytokine-independent means. For example, STAT activation can be initiated by tyrosine kinases, such as p56lck, which associates with CD4 in T lymphocytes [28 , 61 ]. In contrast, the absence of p56lck expression in monocytes would necessitate that STAT activation occurs through different, cell type-specific mechanisms. More importantly, the activation of multiple STATs induced by HIV-1 gp120 represents the potential for a more global alteration of a broad spectrum of gene products downstream.

The effects of HIV-1 on CD4+ cells are complex and can be mediated through multiple signal-transduction cascades [66 , 67 ]. Downstream events, including up-regulation of cytokine, c-myc, and phosphodiesterase-4 protein expression could favor efficient completion of the viral lifecycle and result in high viral production [50 , 62 , 68 69 70 ]. Activation of STAT proteins by gp120 provides a molecular explanation for a bystander effect in multiple lineages of nonproductively infected CD4+ cells, leading to generalized immune dysfunction that is a clinical manifestation of acute HIV-1 infection. Chronically infected HIV individuals can display a down-regulation of activation-induced transcriptional regulation through STAT1 and STAT5 leading to significant inhibition of antigen- and mitogen-induced T cell proliferation [71 ]. In contrast, we demonstrated that early interaction between gp120 and CD4+ cells results in activation of STAT1, STAT3, and STAT5. Activation of the JAK/STAT signaling pathway leads to altered gene expression of specific receptors and/or cytokines, which inevitably affect the activation state of adjacent cells in the microenvironment and their susceptibility to HIV infection. The impact of T cell activation on IL-2 production, for example, could direct the Th1 shift observed in patients during acute and chronic infection [72 , 73 ]. This initial activation may persist and account for the constitutive activation of STAT1 and/or STAT5 detected in patients [26 ]. Further studies to delineate the mechanism of gp120-induced activation of STAT1, STAT3, and STAT5, as well as the downstream effects, will be crucial to understanding one aspect of HIV-1 pathogenesis and designing treatment strategies to maintain the integrity of susceptible hematopoietic cell lineages.


   ACKNOWLEDGEMENTS
 
Studies were supported by PHS awards T32 CA09126, T32 AR07603, R01 HL58005, R01 HD32259, and R01 AI47734; Elizabeth Glaser Pediatric AIDS Foundation PG51154; American Lung Association Florida Seed and Young Investigator awards; and American Foundation for AIDS Research fellowship 70542–20-RFV. We acknowledge the support of UF Shands Cancer Center, Interdisciplinary Center for Biotechnology Research, the Center for Mammalian Genetics, and Molecular Genetics and Microbiology core facilities. The following reagents were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: HIV-1LAI from Drs. Jean-Marie Bechet and Luc Montagnier, courtesy of the Medical Research Council AIDS Directed Program; HIV-1JR-FL from Dr. Irvin Chen (UCLA, Los Angeles); pNL4–3.Luc.R+E- from Dr. Nathaniel Landau (Aaron Diamond AIDS Research Center, The Rockefeller University, New York, NY); HIV-1IIIB gp120 and HIV-1MN gp120 from ImmunoDiagnostics, Inc.; HIV-1BaL gp120, recombinant, from DAIDS, NIAID; and HIV-1SF2 gp120 and nonglycosylated HIV-1SF2 gp120 (Env2–3) from Chiron Corporation (Emeryville, CA). We thank Christina Gavegnano for assistance in collecting nuclear extracts, Dr. Daniel R. Briggs and Steven M. Pomeroy for assistance in generating envelope recombinant viruses from patient isolates of HIV-1, and Dr. Abdolreza Davoodi-Semiromi for providing guidance in interpreting supershift assays.


   FOOTNOTES
 
Correspondence: Maureen M. Goodenow, Ph.D., Department of Pathology, Immunology, and Laboratory Medicine, College of Medicine, University of Florida, P.O. Box 100275, Gainesville, FL 32610. E-mail: goodenow{at}pathology.ufl.edu

Received July 12, 2002; revised November 5, 2002; accepted November 19, 2002.


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