CD28-dependent HIV-1 Transcription Is Associated with Vav, Rac, and NF-κB Activation

Activation of HIV-1-infected T cells through the T cell receptor and costimulatory molecule CD28 induces proviral transcription; however, the mechanism behind this enhanced virus expression is unknown. Jurkat T cells and primary CD4+ T cells expressing a CD8α/CD28 chimeric receptor containing a mutation at tyrosine 200 in the cytoplasmic tail were unable to fully induce HIV-1 proviral transcription in response to CD8α/28 receptor cross-linking in comparison to CD28 costimulation. The loss of transactivation seen with the mutant chimeric receptor correlated with a decrease in Vav tyrosine phosphorylation. CD28-dependent activation of HIV-1 transcription also required the GTPase activity of Rac1, which was not activated during costimulation with the mutated receptor. Furthermore, the mutated receptor was unable to induce NF-κB DNA binding or transactivation, as demonstrated by electromobility shift assays and HIV-1 long terminal repeat and NF-κB-dependent reporter constructs. These studies show that signaling events initiated by tyrosine 200 of CD28 are required for efficient expression of HIV-1 transcription in activated T cells.

Engagement of the T cell receptor (TcR) 1 and costimulatory molecule CD28 results in T cell proliferation, differentiation, and induction of cytokine expression, including IL-2. Human immunodeficiency virus type 1 (HIV-1) also utilizes T cell signaling events to ensure efficient virus transcription and regulation. Productive infection of CD4 ϩ T cells by HIV-1 and efficient HIV-1 proviral transcription requires cell activation through the TcR and costimulatory molecules including CD28 (1)(2)(3)(4)(5). However, signaling through CD28 has been shown to inhibit HIV-1 infection by down-regulating chemokine receptor expression (6). This and other data suggests that CD28 serves as both a positive and negative regulator of HIV-1 infection and replication (1,4,5,(7)(8)(9)(10). CD28 signaling is mediated by four tyrosine residues in its cytoplasmic tail, which when phosphorylated, recruit and activate various kinases, phosphatases, and adapter molecules including LCK, ITK, GRID, GRB2, MKP6, and PI3K leading to changes in IL-2 production as well as HIV-1 transcription (11)(12)(13)(14)(15)(16). Deletion of all four tyrosine residues in the CD28 cytoplasmic tail results in a loss of its ability to enhance IL-2 production as well as HIV-1 transcription, demonstrating the necessity of these residues for proper signaling (17,18). Tyrosine residues 188, 191, and 200 have been shown to positively regulate IL-2 production, but their effects on HIV- 1 have not yet been examined (17). Tyrosine 173 has been shown to be involved in T cell activation, but how it influences IL-2 expression is not clear (19 -24). We have shown that Tyr 173 , which recruits PI3K to the CD28 signaling complex, negatively regulates HIV-1 transcription by a Tat-dependent mechanism (18).
Signaling events initiated by the TcR and CD28 lead to increases in intracellular calcium, changes in cytoskeletal organization, and triggering of several kinase cascades. Activation of these signaling pathways targets various transcription factors including NF-B, NFAT, AP-1, Sp1, and Ets-1 (25)(26)(27)(28). NF-B, NFAT, and AP-1, which have binding sites within the HIV-1 long terminal repeat (LTR), are induced by CD28 costimulation (26, 28 -31). However, it is unclear which signals emanating from CD28 are responsible for the induction of these transcription factors and the activation of HIV-1 transcription.
One potential candidate is the guanine nucleotide exchange factor (GEF) Vav which has been shown to be targeted by CD28 signaling in the absence of TcR signaling (11,15,32,33). Vav has multiple binding domains including SH2 and SH3 domains allowing it to serve as an adapter molecule for T cell signaling. It has also been suggested that Vav is essential for properly integrating TcR and CD28 signals (34). Negative regulation of Vav occurs through its interaction with cbl-b, which, upon CD28 ligation, releases Vav and is targeted for autoubiquitination and degradation (35). Previous studies have shown that downstream of CD28, Vav, in cooperation with Rac1, activates NF-B (33,36,37).
Using Jurkat T cell lines and primary CD4 ϩ T lymphocytes expressing chimeric CD8␣/28 receptors with mutations in critical tyrosines located in the cytoplasmic tail, we have shown that CD28 signaling is required for efficient HIV-1 transcription (18). In this study, we specifically examine the role of Tyr 200 in regulating HIV-1 transcription. We show that Tyr 200 is necessary for efficient HIV-1 transcription in Jurkat T cells and primary CD4 ϩ T cells through initiation of signaling events that enhance Vav phosphorylation, Rac1 activation, and NF-B binding activity.

EXPERIMENTAL PROCEDURES
Cell Lines and Primary Cells-Jurkat T cell line clone E6 -1 obtained from ATCC were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum, 100 units/ml penicillin, 100 g/ml streptomycin, 0.2 M L-glutamine, and 0.5% Fungizone. 293T human embryonic kidney cells and Chinese hamster ovary (CHO) cells expressing FcR␥II receptors (CHO-Fc) (gift from Dr. I. Mellman, Yale University) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 100 units/ml penicillin, 100 g/ml streptomycin, 0.2 M L-glutamine, and 0.5% Fungizone. Jurkat cell lines overexpressing CD8␣/28 chimeric receptors 8WT and F200 were described previously (17,18,38). Expression of receptors was confirmed by flow cytometry. Primary blood lymphocytes were isolated from whole blood using a Ficoll/Histopaque (Sigma) gradient. Macrophages were separated by adherence to plastic overnight and CD4 ϩ T cells were then positively selected from the non-adherent population using a CD4 ϩ isolation kit (Dynal, Oslo, Norway).
Generation of HIV-1 Infectious Titers and Infections-Vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped HIV-1 was generated by transfecting 293T cells with 15 g of cDNA for either pNL4 -3-Luc(ϩ) Env(Ϫ) Nef(Ϫ) (HIV-luc) (39) or pHXBnPLAP(ϩ)Nef(ϩ) (HIV-PLAP) (40); obtained from NIH AIDS Research and Reference Reagent Program, NIH, 3 g of RSV-Rev, and 3 g LTR VSV-G (41) by CaPO 4 transfection (42). 293T transfection efficiency was assessed by determining luciferase activity using a Promega luciferase kit (Madison, WI). Supernatants were collected and filtered through a 0.45-micron disc prior to infection. Typically, the multiplicity of infection of infectious supernatants was ϳ0.1-0.5. Cells were infected by culturing in the presence of virus stock for 12-24 h before replacing with fresh media. Cells were cultured for an additional 24 h before measuring luciferase activity to assess virus transcription. In some experiments, cells were infected with HIV-PLAP and 72-h post-infection HIV-PLAP positive cells were selected using CELLection Pan Mouse IgG kit (Dynal, Lake Success, NY) along with anti-human PLAP antibodies (Sigma).
Activation of T Cells-Jurkat cells were washed and serum-starved for 4 h prior to activation. CHO-Fc cells were plated at 2 ϫ 10 5 cells/well in a 24-well plate, incubated for 12 h to allow adherence, treated with mitomycin-C (Sigma) at 10 g/ml and incubated in the absence of serum for 2 h before using to activate the Jurkat cell lines. 1 ϫ 10 6 Jurkat cells were activated by coculturing cells with the CHO-Fc and 0.1 g/ml mouse anti-human CD3 and 1.0 g/ml anti-human CD28 or 1.0 g/ml anti-human CD8␣ antibodies (BD Pharmingen, San Diego, CA). Following stimulation, Jurkat cells were harvested and luciferase activity measured.
Transient Transfections-1.2 ϫ 10 7 Jurkat cells were resuspended in 400 l of RPMI 1640 and placed on ice for 15 min along with 15 g HIV-luc, LTR-luc, Ϫ205LTR-luc, Ϫ158LTR-luc, or Ϫ205mB-luc and then electroporated using a T280 square electroporation system (BTX, San Diego, CA) (39). For some experiments, 12 g of LTR-luc and 10 g of RacN17 (generously provided by Dr. M. A. Schwartz, The Scripps Research Institute, La Jolla, CA) or 10 g of pEF empty vector DNA were added to cells. Cells were given 1 pulse for 25 ms at 240 V in a 4-mm cuvette. Cells were then cultured for 24 h and stimulated as described above. Primary CD4 ϩ cells were stimulated for 12 h with 10 ng/ml PMA and 2 g/ml PHA prior to electroporation. Following stimulation, 1.2 ϫ 10 7 primary CD4 ϩ T cells were resuspended in 400 l of 10% fetal calf serum RPMI 1640 and placed on ice for 15 min with 20 g of HIV-luc, and 20 g of pMHneo 8WT or F200 (17). DNA was transfected into primary T cells by electroporation using the following conditions: 4 mm cuvette, 250 volts, 1 pulse, and 40 ms (T280 square electroporation system). Cells were cultured 24 h prior to stimulation by anti-CD3, anti-CD28, or anti-CD8␣. Receptor expression was monitored by flow cytometry.
Immunoprecipitation and Immunoblots-Jurkat cells were either left uninfected or infected as described above with HIV-luc or HIV-PLAP. The infected Jurkat cells were serum-starved 4 h prior to stimulation with 0.1 g/ml mouse anti-human CD3, 1.0 g/ml anti-human CD28, or 1.0 g/ml anti-human CD8␣ antibodies (BD Pharmingen) and cross-linked using 5.0 g/ml goat anti-mouse Ig (New England Biolabs, Beverly, MA) for 5 min. Protein extracts were prepared with lysis buffer (10 mM Tris-CL (pH 7.4), 150 mM NaCl, 1.0 mM EDTA (pH 8.0), 2.0 mM sodium vanadate, 10 mM sodium fluoride, 10 mM sodium pyrophosphate, 1% Nonidet P-40, 1.0 mM phenylmethylsufonyl fluoride, 1.0 mM pepstatin). Protein A/G (Santa Cruz Biotechnologies) was incubated with whole cell extracts for 2 h to preclear the lysates. Protein A/G beads were precoated with 1.0 g of anti-Vav (New England Biolabs), washed twice in lysis buffer before adding to precleared whole cell extracts (5 ϫ 10 6 cells/sample to 20 l of beads) for 15 h at 4°C. Protein-bound beads were washed three times in lysis buffer before adding 1ϫ SDS loading buffer containing dithiothreitol. Samples were then treated at 100°C for 5 min before resolving on 8% SDS-PAGE. Proteins were transferred to nitrocellulose membrane (Schleicher and Schuell, Keene, NH) and associated Vav was detected with primary antibodies against phosphotyrosine or anti-human Vav (New England Biolabs). Blots were developed using the Amersham Biosciences ECL kit.
Transduction of Primary CD4 ϩ -Retroviral expression vectors for CD8␣/28 8WT and CD8␣/28 F200 were generated by blunt-ended ligation of a HindIII/EcoRI fragment containing the chimeric receptors from the original pMHneo vector into blunted EcoRI and XhoI sites of murine stem cell virus vector 2.2 (MSCV 2.2; generously provided by G. Nolan, Stanford University, CA). MSCV-8WT or MSCV-F200 retrovirus were then packaged in 293T cells by transfecting 15 g of the viral DNA with 3 g of VSV-G envelope, 3 g of pECO, and 3 g of Tat by CaPO 4 transfection (42). Supernatants were collected and filtered through a 0.45-micron disc prior to infection. Typically, the multiplicity of infection of infectious supernatants was ϳ0.1-0.5. Cells were infected by culturing in the presence of virus stock and 10 ng/ml PMA and 2 g/ml PHA (Sigma) for 12-24 h before replacing with fresh media. Cells were cultured an additional 3 days prior to sorting with CD8 positive isolation kit (Dynal, Oslow, Norway). Sorted cells were allowed to rest overnight prior to activation.
Rac GTP Exchange Assay-Cells were stimulated as described above for immunoprecipitations. Lysates were made according to Rac1 Activation Assay Kit (Upstate Technologies, Lake Placid, NY). Briefly, cells were lysed in magnesium lysis buffer (MLB) supplemented with 10 g/ml leupeptin, 10 g/ml apoprotin, 1 mM sodium vanadate, and 1 mM sodium flouride. Cell lysates were precleared for 10 min with GST beads. Lysates were then incubated with PAK-1 PBD-agarose for 1 h at 4°C. Beads were washed three times in MLB and samples were prepared for electrophoresis by adding 1ϫ SDS loading dye. Samples were boiled for 5 min and resolved by 12% SDS-PAGE. Proteins were transferred to nitrocellulose membrane and GTP-bound Rac1 was identified by anti-Rac1 antibody.
Electrophoretic Mobility Shift Assays-Nuclear extracts from Jurkat and primary cells were prepared as previously described (43). Electrophoretic mobility shift assays (EMSAs) were performed by incubating 4 g of protein from nuclear extracts with 4 g of poly dIdC (Amersham Biosciences), 0.25 mM HEPES (pH 7.5), 0.6 M KCl, 9.0% glycerol, 1.0 mM EDTA, 7.5 mM dithiothreitol, 50 mM MgCl 2 . Reaction mixtures were preincubated with 50-fold excess specific or nonspecific competitors, or 0.5 g of polyclonal antibodies against NF-B subunits or C/EBP for 20 min at room temperature. Samples were loaded onto a 6% polyacrylamide gel and electrophoresed at 120 V in 0.5ϫ Tris borate-EDTA. Probes for EMSA were generated by annealing oligonucleotides representing the HIV-1 NF-B sites 5Ј-AGCTCCTGGAAAGTCCCCAGCGG-AAAGTCCCTT-3Ј and 5Ј-AGCTAAGGGACTTTCCGCTGGGGACTTT-CCAGG-3Ј and C/EBP site 5Ј-GATCGCCTAGCATTTCATCACACGT-3Ј and 5Ј-GATCACGTGTGATGAAATGCTAGGC-3Ј. Probes were generated by end filling with the Klenow fragment of Escherichia coli polymerase in the presence of [␣-P 32 ]dCTP (44).

Tyr 200 Is Necessary for CD28-induced HIV-1 Transcription-
We have previously shown that Tyr 173 within the cytoplasmic tail of CD28 initiates a negative signal in CD28-mediated costimulation of HIV-1 transcription (18). However, CD28 signaling overall increases HIV-1 transcription suggesting that other residues must be required for this activity. We tested the role of Tyr 200 in CD28-dependent HIV-1 transcription using CD8␣/28 chimeric receptors in which the extracellular and transmembrane domains derived from CD8␣ were fused to the CD28 cytoplasmic domain, which is sufficient for T cell costimulation (17). Jurkat cell lines were generated that express CD8␣/28 chimeric receptors, in which all four tyrosine residues are maintained (8WT), or the Tyr 200 has been mutated to phenylalanine (F200) (17). CD8␣/28 receptor expression was confirmed using antibodies directed against CD8␣ and quantified by flow cytometry. Expression of the 8WT or F200 chimeric receptor was comparable between both cell lines and did not affect CD3 or CD28 expression (Ref. 18, Fig. 1A). In order to determine if Tyr 200 was necessary for provirus expression, 8WT and F200 cell lines were infected with an HIV-1 clone that included a luciferase reporter (HIV-luc) in place of the viral Nef gene (39). These cells were stimulated with different combinations of antibodies against CD3, CD8␣, and CD28 and crosslinked with CHO-Fc as described under "Experimental Procedures." Luciferase activity was assessed as a measure of proviral transcription. Cells stimulated through the 8WT chimeric receptor plus CD3 showed induction of HIV-1 transcription comparable to that observed when they were stimulated through the endogenous CD28 plus CD3 (Ref. 18, Fig. 1B). However, stimulating cells through CD3 and the F200 receptor did not result in induction of proviral transcription over the levels of CD3 stimulation alone (Fig. 1B). Engagement of CD3, CD8␣, or CD28 alone did not efficiently activate viral transcription. These results suggest that tyrosine residue 200 in CD28 is necessary for efficient induction of HIV-1 transcription following CD3 and CD28 activation.
To determine whether these chimeric receptors were functional in primary T cells and mimic normal signals, 8WT and F200 receptors were transiently transfected with the HIV-luc cDNA into CD4 ϩ T cells. Expression of the chimeric receptors was confirmed by flow cytometry with antibodies directed against CD8␣ (Fig. 1C). Receptors were stimulated with antibodies against CD3 plus CD28 or CD8␣ and cocultured with CHO-Fc for cross-linking. Activation through the wild type chimeric receptor resulted in induction of transcription that was comparable to that observed when endogenous CD28 was used as the costimulatory signal (Fig. 1D). However, cells transfected with the F200 mutant chimeric receptor and stimulated with anti-CD3 plus anti-CD8␣ were unable to induce HIV-1 transcription over background levels (Fig. 1D). These data show that the CD8␣/28 chimeric receptors are functional in primary cells, as well as Jurkat cells, and suggest that Jurkat cells are a suitable model system for investigating the mechanisms of CD28 signaling. More importantly, results from both the primary CD4 ϩ T cells and the Jurkat T cell lines demonstrate that Tyr 200 is necessary for CD28-dependent HIV-1 activation.
Tyr 200 Mediates Vav Phosphorylation and Rac1 Activity-To identify molecules downstream of Tyr 200 , changes in tyrosine phosphorylation were examined following stimulation with anti-CD3 plus anti-CD28 or anti-CD8␣. A decrease in the phosphorylation of an ϳ98 kDa protein was consistently observed in cells activated through CD3 and F200 compared with those in which endogenous CD28 provided the costimulatory signal (data not shown). Vav is guanine nucleotide exchange factor (GEF) with a molecular weight of ϳ95 kDa that has been shown to be phosphorylated post-CD28 ligation (15,36,45). To determine if phosphorylation of Vav was altered in F200 cells, immunoprecipitations were performed using 8WT and F200 cells either untreated or receiving costimulation through the endogenous CD28 or chimeric receptor. Cells activated with anti-CD3 plus anti-CD28 resulted in an increase in Vav phosphorylation over unstimulated cells. Costimulation through the 8WT chimeric receptor resulted in an equivalent induction of Vav phosphorylation; whereas, cells receiving costimulation through the F200 chimeric receptor had significantly less phosphorylated Vav (Fig. 2). These data suggest that Vav is a critical downstream target of CD28 Tyr 200 and plays a role in the activation of HIV-1 transcription.
Vav mediates guanine nucleotide exchange of Rac1 downstream of CD28 signals (36,45,46). To determine whether Rac1 was a critical signaling intermediate for CD28-dependent HIV-1 transcription, a Rac1 dominant negative mutant (RacN17) was transiently transfected along with the HIV-1 LTR linked to a luciferase reporter (LTR-luc) in the F200 cell line. Ectopic expression of RacN17 resulted in the inhibition of the endogenous CD3 plus CD28 signal to the level of induction of cells receiving costimulation through CD3 plus the F200 receptor (Fig. 3A). These data suggest that Vav and Rac1 are important in mediating responses downstream of CD28 Tyr 200 .
To confirm that Rac1, a downstream target of Vav, is a target of signaling through Tyr 200 we directly measured levels of activated GTP-bound Rac1. Cell lysates were prepared from 8WT and F200 cells following costimulation through CD28 or CD8␣ and a PAK PBD GST pull-down was performed to detect GTP-bound Rac1. Activation of the 8WT cells through CD3 and either CD28 or CD8␣ resulted in equivalent induction of GTP

CD28 Activation of HIV-1 Transcription
bound Rac1 over unstimulated cells (Fig. 3B). F200 cells stimulated through CD3 and CD28 resulted in an increase in GTPbound Rac1, but costimulation through the F200 chimeric receptor did not result in any increase in GTP-bound Rac1 (Fig.  3B). These data support the conclusion that Tyr 200 , through the activation of Vav guanine nucleotide exchange activity, targets Rac1 to influence HIV-1 transcription.

CD28-dependent Activation of HIV-1 Transcription Requires NF-B Induction-
The inability of the mutant F200 receptor to induce HIV-1 transcription may reflect inappropriate activation of cellular transcription factors. The role of host transcription factors was examined by transient transfection with HIV-1 LTR luciferase reporter constructs (LTR-luc), which lack viral proteins that may potentially influence virus transcription (18). LTR transcriptional activity was increased 3.5-fold in response to anti-CD3 plus anti-CD28 stimulation compared with the unstimulated cells, whereas stimulation through CD3 and the F200 receptor did not result in significant induction of LTR activity (Fig. 4B), indicating that the Tyr 200 residue of CD28 affects HIV-1 transcription by targeting cellular transcription factors.
To further explore the transcription factors that are downstream of Tyr 200 signaling, a series of HIV-1 LTR-luc constructs harboring deletions and mutations were assayed in the F200 cell line for their ability to respond to CD28 signaling (Fig. 4, A and B) (39). Deleting ϳ342-bp upstream of the transcriptional start site did not alter LTR activity in response to CD3 and endogenous CD28 stimulation (Fig. 4B, Ϫ205 and  Ϫ158). However, activation of F200 cells with anti-CD3 plus anti-CD8␣ did not induce transcription in any of the LTR-dependent reporters. These deletion constructs still contained the NF-B and SP1 binding sites. Mutation of the NF-B sites in the Ϫ205 LTR-luc abolished the ability of CD28 costimulation to induce HIV-1 transcription suggesting that NF-B is required for full induction of HIV-LTR activity. The 8WT cell line was able to induce the LTR-, Ϫ205-, and Ϫ158-dependent reporters with both CD8␣ and CD28 stimulation but was not able to induce the mutant NF-B reporter (data not shown).
To confirm that NF-B transcriptional activity was deficient in the F200 cells, NF-B, and AP-1 reporters linked to luciferase were transiently transfected into the parental, 8WT, and F200 Jurkat cell lines prior to T cell activation. As expected AP-1 and NF-B-dependent reporters were induced by CD3 plus CD28 stimulation in the parental Jurkat cell line (Fig.   5A). The 8WT cells were able to activate transcription of both NF-B-and AP-1-dependent promoters when stimulated through CD3 plus CD28 or CD8␣. The F200 cell line was unable to efficiently activate NF-B-and AP-1-dependent reporters following CD3 plus CD8␣ stimulation, consistent with Tyr 200 being upstream of AP-1 and NF-B (Fig. 5, B and C). The deletion of AP-1 sites did not decrease the ability of the Ϫ158 LTR reporter to respond to CD28 costimulation suggesting a minimal role for this factor in HIV-1 transcription. Therefore, we focused on the requirement of NF-B in response to CD28 signaling.
Further characterization of NF-B activation was assessed by electrophoretic mobility shift assays (EMSA) performed with nuclear extracts from 8WT or F200 cells stimulated through CD3 and either endogenous CD28 or the chimeric CD8␣ receptor. In some experiments, cells were infected with HIV-PLAP, a recombinant HIV-1 clone that includes a placental alkaline phosphatase marker to positively select infected cells. This virus also encodes the viral protein Nef, which was replaced by the luciferase gene in the previous HIV-luc experiments. Nuclear extracts from HIV-1-infected 8WT and F200 cells stimulated with antibodies against CD3 plus CD28 resulted in an increase in complex formation compared with unstimulated cells, which showed little to no binding activity. FIG. 3. Rac1 is required for CD28 activation for HIV-1 transcription. A, F200 cells were transiently transfected with 12 g of LTR-luc and 10 g of dominant negative Rac1. Cells were allowed to recover for 18 h and then left unstimulated or stimulated with 0.1 g/ml anti-CD3 plus 1.0 g/ml anti-CD28 or anti-CD8␣ on CHO-Fc cells. These data are from a single transfection performed in triplicate that represents three independent experiments. B, 8WT or F200 cells were stimulated for 10 min with 0.1 g/ml anti-CD3 plus 1.0 g of anti-CD28 or anti-CD8␣ and cross-linked with 5 g/ml goat anti-mouse antibody. Cell lysates were immunoprecipitated with PAK-1 PBD-agarose. Associated GTP-bound Rac1 was determined by immunoblotting with anti-Rac1 antibody. The blots shown are from two separate experiments, and the band intensities do not reflect differences in Rac1 levels in the individual cell lines. 8WT cells stimulated through CD3 and CD8␣ resulted in similar levels of NF-B binding when compared with endogenous CD28 costimulation (Fig. 6A). In F200 cells stimulated with CD3 plus CD8␣, no significant increase in complex formation was observed (Fig. 6A). Complexes formed in response to CD3 plus CD28 stimulation were competed away upon the addition of excess unlabeled NF-B probe, but not with an irrelevant unlabeled competitor. Furthermore, these complexes were supershifted with antibodies specific to NF-B (data not shown). Identical EMSA patterns were observed with extracts from F200 cells that were not infected, indicating that viral proteins are not altering NF-B binding activity post CD3 plus CD28 stimulation (data not shown).
We also determined whether Tyr 200 was necessary for NF-B activation in primary CD4 ϩ T cells by EMSA. CD4 ϩ T cells isolated from whole blood were transduced with retroviral vectors carrying the 8WT and F200 chimeric receptors. Cells were sorted for CD8␣ expression and confirmed to be positive for chimeric receptors by flow cytometry. Populations from the sorted cells were ϳ90% positive for CD8␣ expression (data not shown). Sorted cells were stimulated with antibodies against CD3 plus CD28 or CD8␣ in the presence of CHO-Fc cells. 8WT cells induced similar NF-B binding complexes following activation with anti-CD3 plus anti-CD28 or anti-CD8␣ (Fig. 6B). The F200 cells were able to induce NF-B binding in response to CD3 plus CD28 signaling but no significant change in NF-B binding activity was observed when costimulation was provided by the F200 chimeric receptor. The NF-B binding observed in these cells was specific since unlabeled NF-B probe but not unlabeled nonspecific probe competed for binding (Fig.  6B). These data confirm that NF-B is downstream of Tyr 200 and further implicates this pathway in regulating CD28-dependent HIV-1 transcription. DISCUSSION In this report, we demonstrate that residue Tyr 200 of the CD28 receptor is required for the activation of HIV-1 transcrip-tion in both Jurkat and primary CD4 ϩ T cells. We also show that a dominant negative Rac1 inhibits LTR-luc responses and that mutation of Tyr 200 decreases Vav tyrosine phosphorylation and Rac1 activation. These two signaling molecules have been shown to regulate NF-B activation, which is in agreement with a loss of NF-B reporter and binding activity in the F200 cell line. The decrease in NF-B activity severely impairs HIV-1 transcription, consistent with previous studies indicating that this is a critical transcription factor for HIV-1 LTR activity and virus replication (26,31,47).
The CD28 receptor is an important costimulatory molecule for T cell activation and has four tyrosine residues and a proline-rich domain, which are involved in the recruitment and activation of downstream effector molecules (16). Studies examining the induction of IL-2 and HIV-1 transcription have shown that the activity and interplay of these tyrosine residues is complex and may act competitively or synergistically with one another (11,17,22,23,38,48). Tyr 173 has been identified as recruiting ITK, GRB-2, and PI3K to the receptor (11,14,15). We have previously reported that mutating Tyr 173 or inhibiting PI3K activity increases HIV-1 transcription through a Tat-dependent mechanism (18). However, the contribution of Tyr 200 in activating HIV-1 transcription does not require viral-encoded proteins such as Tat or Nef, but mediates changes in cellular transcription factors (18). We have not yet determined the roles of Tyr 188 and Tyr 191 in HIV-1 transcription. Taken together our results demonstrate that the Tyr 173 and Tyr 200 residues recruit different signaling complexes to activate distinct pathways leading to either negative or positive signals to the HIV-1 LTR.
CD28 activation leads to an increase in Vav phosphorylation, which has been suggested to be mediated by tyrosine Tyr 173 (49); however, we show that Tyr 200 also regulates Vav tyrosine phosphorylation. Vav has been shown to interact with Grb-2, SLP-76, ZAP-70, and Rac1, which contribute to the activation of NFAT and NF-B (33,36,37,45,50). Furthermore, over expression of Vav along with SLP76 circumvented the need for CD3 and CD28 signals to activate NFAT reporters (32,51). Similarly, overexpression of Vav plus Rac1 or MEKK1 were able to circumvent CD3 plus CD28 signaling to induce NF-B reporter activity (36,45,46,53). We confirmed that Rac1 is necessary for induction of the HIV-1 LTR and observe strong activation of the HIV-1 LTR when MEKK1 is constitutively active (data not shown). This would be consistent with reports that MEKK1 can activate NF-B and may be involved downstream of Vav (36,45,46,53). However, additional experiments are required to establish a direct role for MEKK1 in events downstream of CD28 Tyr 200 . Experiments using chemical inhibitors against p38 and MEK1 have suggested that these MAPK pathways are not involved in CD28 Tyr 200 mediated HIV-1 transcription (data not shown).
The viral protein Nef has been shown to influence T cell activation through interactions with several signaling intermediates downstream of the TcR and CD28. Vav is a potential target of Nef, although how this interaction with Vav influences HIV-1 transcription has not been investigated (54). Furthermore, Nef potentially alters NFAT transactivation but, its role in regulating NF-B transactivation is not well characterized (52). In this report, Nef expressing viruses were used to compare NF-B activity downstream of CD3 plus CD28 and CD3 plus CD8␣ by gel shift analysis. Our data indicates that Nef does not compensate for the F200 defect as assayed by NF-B binding, suggesting that Nef has little impact on CD28 signaling through Tyr 200 and minimal affect on NF-B-dependent HIV-1 transcription. Finally, viral proteins do not influence signaling from F200 as suggested by the LTR-reporter constructs, which do not encode the viral proteins.
These data taken together with other reports suggest that CD28 activation of Vav and Rac1 are critical steps in the activation of NF-B (36,37). Furthermore, we demonstrate that this pathway is necessary for the CD28-dependent HIV-1 transcription. Therefore, T cell induction of HIV-1 transcription in response to T cell activation is complex, requiring signals from both the TcR and CD28 to provide efficient transcription of the virus as shown in both the Jurkat T cells and primary CD4 ϩ T lymphocytes.