CD28-dependent HIV-1 Transcription Is Associated with Vav, Rac, and NF-κB Activation
2003; Elsevier BV; Volume: 278; Issue: 37 Linguagem: Inglês
10.1074/jbc.m302878200
ISSN1083-351X
AutoresJulie A. Cook, Lee A. Albacker, Avery August, Andrew J. Henderson,
Tópico(s)T-cell and B-cell Immunology
ResumoActivation 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. 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) 1The abbreviations used are: TcR, T cell receptor; HIV-1, human immunodeficiency virus type 1; LTR, long terminal repeat; CHO, Chinese hamster ovary; PLAP, placental alkaline phosphatase; Grb-2, growth receptor-bound protein 2; PI3K, phosphatidylinositol 3-kinase, NFAT, nuclear factor of activated T cells; AP-1, activator protein 1; SH2, Src-homology 2; SH3, Src homology 3; NF-κB, nuclear factor κB; GST, glutathione S-transferase; FITC, fluorescein isothiocyanate; EMSA, electrophoretic mobility shift assay; PBD, P21 binding domain.1The abbreviations used are: TcR, T cell receptor; HIV-1, human immunodeficiency virus type 1; LTR, long terminal repeat; CHO, Chinese hamster ovary; PLAP, placental alkaline phosphatase; Grb-2, growth receptor-bound protein 2; PI3K, phosphatidylinositol 3-kinase, NFAT, nuclear factor of activated T cells; AP-1, activator protein 1; SH2, Src-homology 2; SH3, Src homology 3; NF-κB, nuclear factor κB; GST, glutathione S-transferase; FITC, fluorescein isothiocyanate; EMSA, electrophoretic mobility shift assay; PBD, P21 binding domain. 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+ T cells by HIV-1 and efficient HIV-1 proviral transcription requires cell activation through the TcR and costimulatory molecules including CD28 (1Reiley J.L. Levine B.L. Craighead N. Francomano T. Kim D. Carroll R.G. June C.H. J. Virol. 1998; 72: 8273-8280Crossref PubMed Google Scholar, 2Zack J.A. Arrigo S. Weitsman S.R. Go A.S. Haislip A. Chen I.S. Cell. 1990; 61: 213-222Abstract Full Text PDF PubMed Scopus (1289) Google Scholar, 3Stevenson M. Stanwick T.L. Dempsey M.P. Lamonica C.A. EMBO J. 1990; 9: 1551-1560Crossref PubMed Scopus (644) Google Scholar, 4Levine B. Mosca J. Riley J. Carroll R. Vahey M. Jagodzinski L. Wagner K. Mayers D. Burke D. Weislow O. St. Louis D.C. June C. Science. 1996; 272: 1939-1943Crossref PubMed Scopus (193) Google Scholar, 5Carroll R. Riley J. Levine B. Feng Y. Kaushal S. Ritchey D. 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Using Jurkat T cell lines and primary CD4+ T lymphocytes expressing chimeric CD8α/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 (18Cook J.A. August A. Henderson A.J. J. Immunol. 2002; 169: 254-260Crossref PubMed Scopus (16) Google Scholar). In this study, we specifically examine the role of Tyr200 in regulating HIV-1 transcription. We show that Tyr200 is necessary for efficient HIV-1 transcription in Jurkat T cells and primary CD4+ T cells through initiation of signaling events that enhance Vav phosphorylation, Rac1 activation, and NF-κB binding activity. 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 μg/ml streptomycin, 0.2 m l-glutamine, and 0.5% Fungizone. 293T human embryonic kidney cells and Chinese hamster ovary (CHO) cells expressing FcRγII 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 μg/ml streptomycin, 0.2 m l-glutamine, and 0.5% Fungizone. Jurkat cell lines overexpressing CD8α/28 chimeric receptors 8WT and F200 were described previously (17Teng J.M.C. King P.D. Sadra A. Liu X. Han A. Selvakumar A. August A. Dupont B. Tissue Antigens. 1996; 48: 255-264Crossref PubMed Scopus (22) Google Scholar, 18Cook J.A. August A. Henderson A.J. J. Immunol. 2002; 169: 254-260Crossref PubMed Scopus (16) Google Scholar, 38King P.D. Sadra A. Teng J.M.C. Liu X. Han A. Selvakumar A. August A. Dupont B. J. Immunol. 1997; 158: 580-590PubMed Google Scholar). 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+ T cells were then positively selected from the non-adherent population using a CD4+ 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 μg of cDNA for either pNL4–3-Luc(+) Env(–) Nef(–) (HIV-luc) (39Henderson A.J. Zou X. Calame K.L. J. Virol. 1995; 69: 5337-5344Crossref PubMed Google Scholar) or pHXBnPLAP(+)Nef(+) (HIV-PLAP) (40Chen B.K. Gandhi R. Baltimore D. J. Virol. 1996; 70: 6044-6053Crossref PubMed Google Scholar); obtained from NIH AIDS Research and Reference Reagent Program, NIH, 3 μg of RSV-Rev, and 3 μg LTR VSV-G (41Bartz S.R. Vodicka M.A. Methods Enzymol. 1997; 12: 337-342Crossref Scopus (131) Google Scholar) by CaPO4 transfection (42Pear W.S. Nolan G.P. Scott M.L. Baltimore D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8392-8396Crossref PubMed Scopus (2291) Google Scholar). 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 ∼0.1–0.5. 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 × 105 cells/well in a 24-well plate, incubated for 12 h to allow adherence, treated with mitomycin-C (Sigma) at 10 μg/ml and incubated in the absence of serum for 2 h before using to activate the Jurkat cell lines. 1 × 106 Jurkat cells were activated by coculturing cells with the CHO-Fc and 0.1 μg/ml mouse anti-human CD3 and 1.0 μg/ml anti-human CD28 or 1.0 μg/ml anti-human CD8α antibodies (BD Pharmingen, San Diego, CA). Following stimulation, Jurkat cells were harvested and luciferase activity measured. Transient Transfections—1.2 × 107 Jurkat cells were resuspended in 400 μl of RPMI 1640 and placed on ice for 15 min along with 15 μg HIV-luc, LTR-luc, –205LTR-luc, –158LTR-luc, or –205mκB-luc and then electroporated using a T280 square electroporation system (BTX, San Diego, CA) (39Henderson A.J. Zou X. Calame K.L. J. Virol. 1995; 69: 5337-5344Crossref PubMed Google Scholar). For some experiments, 12 μg of LTR-luc and 10 μg of RacN17 (generously provided by Dr. M. A. Schwartz, The Scripps Research Institute, La Jolla, CA) or 10 μg 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+ cells were stimulated for 12 h with 10 ng/ml PMA and 2 μg/ml PHA prior to electroporation. Following stimulation, 1.2 × 107 primary CD4+ T cells were resuspended in 400 μl of 10% fetal calf serum RPMI 1640 and placed on ice for 15 min with 20 μg of HIV-luc, and 20 μg of pMHneo 8WT or F200 (17Teng J.M.C. King P.D. Sadra A. Liu X. Han A. Selvakumar A. August A. Dupont B. Tissue Antigens. 1996; 48: 255-264Crossref PubMed Scopus (22) Google Scholar). 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-CD8α. 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 μg/ml mouse anti-human CD3, 1.0 μg/ml anti-human CD28, or 1.0 μg/ml anti-human CD8α antibodies (BD Pharmingen) and cross-linked using 5.0 μg/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 μg of anti-Vav (New England Biolabs), washed twice in lysis buffer before adding to precleared whole cell extracts (5 × 106 cells/sample to 20 μl of beads) for 15 h at 4 °C. Protein-bound beads were washed three times in lysis buffer before adding 1× 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 + —Retroviral expression vectors for CD8α/28 8WT and CD8α/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 μg of the viral DNA with 3 μg of VSV-G envelope, 3 μg of pECO, and 3 μg of Tat by CaPO4 transfection (42Pear W.S. Nolan G.P. Scott M.L. Baltimore D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8392-8396Crossref PubMed Scopus (2291) Google Scholar). Supernatants were collected and filtered through a 0.45-micron disc prior to infection. Typically, the multiplicity of infection of infectious supernatants was ∼0.1–0.5. Cells were infected by culturing in the presence of virus stock and 10 ng/ml PMA and 2 μg/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 μg/ml leupeptin, 10 μg/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 1× 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 (43Schreiber E. Matthias P. Muller M.M. Schaffner W. Nucleic Acids Res. 1989; 17: 6419Crossref PubMed Scopus (3912) Google Scholar). Electrophoretic mobility shift assays (EMSAs) were performed by incubating 4 μg of protein from nuclear extracts with 4 μg 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 MgCl2. Reaction mixtures were preincubated with 50-fold excess specific or nonspecific competitors, or 0.5 μg of polyclonal antibodies against NF-κB subunits or C/EBP for 20 min at room temperature. Samples were loaded onto a 6% polyacrylamide gel and electrophoresed at 120 V in 0.5× Tris borate-EDTA. Probes for EMSA were generated by annealing oligonucleotides representing the HIV-1 NF-κB sites 5′-AGCTCCTGGAAAGTCCCCAGCGGAAAGTCCCTT-3′ and 5′-AGCTAAGGGACTTTCCGCTGGGGACTTTCCAGG-3′ and C/EBP site 5′-GATCGCCTAGCATTTCATCACACGT-3′ and 5′-GATCACGTGTGATGAAATGCTAGGC-3′. Probes were generated by end filling with the Klenow fragment of Escherichia coli polymerase in the presence of [α-P32]dCTP (44Maniatis T. Fritsch E.F. Sambrook J. Molecular Cloning. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1982Google Scholar). Tyr200 Is Necessary for CD28-induced HIV-1 Transcription—We have previously shown that Tyr173 within the cytoplasmic tail of CD28 initiates a negative signal in CD28-mediated costimulation of HIV-1 transcription (18Cook J.A. August A. Henderson A.J. J. Immunol. 2002; 169: 254-260Crossref PubMed Scopus (16) Google Scholar). However, CD28 signaling overall increases HIV-1 transcription suggesting that other residues must be required for this activity. We tested the role of Tyr200 in CD28-dependent HIV-1 transcription using CD8α/28 chimeric receptors in which the extracellular and transmembrane domains derived from CD8α were fused to the CD28 cytoplasmic domain, which is sufficient for T cell costimulation (17Teng J.M.C. King P.D. Sadra A. Liu X. Han A. Selvakumar A. August A. Dupont B. Tissue Antigens. 1996; 48: 255-264Crossref PubMed Scopus (22) Google Scholar). Jurkat cell lines were generated that express CD8α/28 chimeric receptors, in which all four tyrosine residues are maintained (8WT), or the Tyr200 has been mutated to phenylalanine (F200) (17Teng J.M.C. King P.D. Sadra A. Liu X. Han A. Selvakumar A. August A. Dupont B. Tissue Antigens. 1996; 48: 255-264Crossref PubMed Scopus (22) Google Scholar). CD8α/28 receptor expression was confirmed using antibodies directed against CD8α 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. 18Cook J.A. August A. Henderson A.J. J. Immunol. 2002; 169: 254-260Crossref PubMed Scopus (16) Google Scholar, Fig. 1A). In order to determine if Tyr200 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 (39Henderson A.J. Zou X. Calame K.L. J. Virol. 1995; 69: 5337-5344Crossref PubMed Google Scholar). These cells were stimulated with different combinations of antibodies against CD3, CD8α, and CD28 and cross-linked 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. 18Cook J.A. August A. Henderson A.J. J. Immunol. 2002; 169: 254-260Crossref PubMed Scopus (16) Google Scholar, 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, CD8α, 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+ T cells. Expression of the chimeric receptors was confirmed by flow cytometry with antibodies directed against CD8α (Fig. 1C). Receptors were stimulated with antibodies against CD3 plus CD28 or CD8α 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-CD8α were unable to induce HIV-1 transcription over background levels (Fig. 1D). These data show that the CD8α/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+ T cells and the Jurkat T cell lines demonstrate that Tyr200 is necessary for CD28-dependent HIV-1 activation. Tyr200 Mediates Vav Phosphorylation and Rac1 Activity—To identify molecules downstream of Tyr200, changes in tyrosine phosphorylation were examined following stimulation with anti-CD3 plus anti-CD28 or anti-CD8α. A decrease in the phosphorylation of an ∼98 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 ∼95 kDa that has been shown to be phosphorylated post-CD28 ligation (15August A. Gibson S. Kawakami Y. Kawakami T. Mills G.B. Dupont B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9347-9351Crossref PubMed Scopus (210) Google Scholar, 36Marinari B. Costanzo A. Viola A. Michel F. Mangino G. Acuto O. Levrero M. Piccolella E. Tuosto L. Eur. J. Immunol. 2002; 32: 447-456Crossref PubMed Scopus (59) Google Scholar, 45Salojin K.V. Zhang J. Delovitch T.L. J. Immunol. 1999; 163: 844-853PubMed Google Scholar). 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 Tyr200 and plays a role in the activation of HIV-1 transcription. Vav mediates guanine nucleotide exchange of Rac1 downstream of CD28 signals (36Marinari B. Costanzo A. Viola A. Michel F. Mangino G. Acuto O. Levrero M. Piccolella E. Tuosto L. Eur. J. Immunol. 2002; 32: 447-456Crossref PubMed Scopus (59) Google Scholar, 45Salojin K.V. Zhang J. Delovitch T.L. J. Immunol. 1999; 163: 844-853PubMed Google Scholar, 46Kaga S. Ragg S. Rogers K.A. Ochi A. J. Immunol. 1998; 160: 4182-4189PubMed Google Scholar). To determine whether Rac1 was a critical signaling intermediate for CD28-dependent HIV-1 transcription, a Rac1 dominant negative mutant (RacN17) was transiently tran
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