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• W2045367107 abstract "Activation through the T-cell receptor and the costimulatory receptor CD28 supports efficient HIV transcription as well as reactivation of latent provirus. To characterize critical signals associated with CD28 that regulate HIV-1 transcription, we generated a library of chimeric CD28 receptors that harbored different combinations of key tyrosine residues in the cytoplasmic tail, Tyr-173, Tyr-188, Tyr-191, and Tyr-200. We found that Tyr-191 and Tyr-200 induce HIV-1 transcription via the activation of NF-κB and its recruitment to the HIV-long terminal repeat. Tyr-188 modifies positive and negative signals associated with CD28. Importantly, signaling through Tyr-188, Tyr-191, and Tyr-200 is required to overcome the inhibition posed by Tyr-173. CD28 also regulates P-TEFb activity, which is necessary for HIV-1 transcription processivity, by limiting the release of P-TEFb from the HEXIM1–7SK inhibitory complex in response to T-cell receptor signaling. Our studies reveal that CD28 regulates HIV-1 provirus transcription through a complex interplay of positive and negative signals that may be manipulated to control HIV-1 transcription and replication. Activation through the T-cell receptor and the costimulatory receptor CD28 supports efficient HIV transcription as well as reactivation of latent provirus. To characterize critical signals associated with CD28 that regulate HIV-1 transcription, we generated a library of chimeric CD28 receptors that harbored different combinations of key tyrosine residues in the cytoplasmic tail, Tyr-173, Tyr-188, Tyr-191, and Tyr-200. We found that Tyr-191 and Tyr-200 induce HIV-1 transcription via the activation of NF-κB and its recruitment to the HIV-long terminal repeat. Tyr-188 modifies positive and negative signals associated with CD28. Importantly, signaling through Tyr-188, Tyr-191, and Tyr-200 is required to overcome the inhibition posed by Tyr-173. CD28 also regulates P-TEFb activity, which is necessary for HIV-1 transcription processivity, by limiting the release of P-TEFb from the HEXIM1–7SK inhibitory complex in response to T-cell receptor signaling. Our studies reveal that CD28 regulates HIV-1 provirus transcription through a complex interplay of positive and negative signals that may be manipulated to control HIV-1 transcription and replication. One of the major blocks to eradicating human immunodeficiency virus (HIV) 2The abbreviations used are: HIVhuman immunodeficiency virusHIV-1HIV type 1TCRT-cell receptorLTRlong terminal repeatPI3Kphosphatidylinositol 3-kinaseNFATnuclear factor of activated T cellsAP-1activator protein 1NF-κBnuclear factor-κBP-TEFbpositive transcription elongation factor bpolpolymeraseCsAcyclosporin AWTwild typeERKextracellular signal-regulated kinaseJNKc-Jun N-terminal kinaseMAPKmitogen-activated protein kinaseIL-2interleukin-27SK RNP7SK RNA particle complexTARtransactivating response element. infections with highly active anti-retroviral therapy has been the inability of this treatment to eliminate cellular reservoirs harboring latent provirus.(1Coiras M. Lüpez-Huertas M.R. Pérez-Olmeda M. Alcamí J. Nat. Rev. Microbiol. 2009; 7: 798-812Crossref PubMed Scopus (226) Google Scholar, 2Bagasra O. Expert. Opin. Biol. Ther. 2006; 6: 1135-1149Crossref PubMed Scopus (28) Google Scholar, 3Richman D.D. Margolis D.M. Delaney M. Greene W.C. Hazuda D. Pomerantz R.J. Science. 2009; 323: 1304-1307Crossref PubMed Scopus (706) Google Scholar). T cells are a major target for HIV-1 infection, and T-cell signal transduction has been demonstrated to impact multiple steps of HIV-1 replication, including provirus transcription (4Tyagi M. Karn J. EMBO J. 2007; 26: 4985-4995Crossref PubMed Scopus (187) Google Scholar, 5Strasner A.B. Natarajan M. Doman T. Key D. August A. Henderson A.J. J. Immunol. 2008; 181: 3706-3713Crossref PubMed Scopus (24) Google Scholar, 6Readinger J.A. Schiralli G.M. Jiang J.K. Thomas C.J. August A. Henderson A.J. Schwartzberg P.L. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 6684-6689Crossref PubMed Scopus (59) Google Scholar, 7Gruters R.A. Otto S.A. Al B.J. Verhoeven A.J. Verweij C.L. Van Lier R.A. Miedema F. Eur. J. Immunol. 1991; 21: 167-172Crossref PubMed Scopus (38) Google Scholar). Characterizing T-cell signaling regulatory networks that govern T-cell function and HIV-1 transcription is critical for understanding the molecular mechanisms that directly contribute to the establishment, maintenance, and breaking of proviral transcription latency (8Williams S.A. Kwon H. Chen L.F. Greene W.C. J. Virol. 2007; 81: 6043-6056Crossref PubMed Scopus (115) Google Scholar, 9Brooks D.G. Arlen P.A. Gao L. Kitchen C.M. Zack J.A. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 12955-12960Crossref PubMed Scopus (89) Google Scholar). human immunodeficiency virus HIV type 1 T-cell receptor long terminal repeat phosphatidylinositol 3-kinase nuclear factor of activated T cells activator protein 1 nuclear factor-κB positive transcription elongation factor b polymerase cyclosporin A wild type extracellular signal-regulated kinase c-Jun N-terminal kinase mitogen-activated protein kinase interleukin-2 7SK RNA particle complex transactivating response element. HIV provirus transcription is controlled by the upstream long terminal repeat (LTR), which includes cis-elements that are recognized by cellular transcription factors, including NF-κB, AP-1, and NFAT that are induced in response to T-cell receptor (TCR)/CD28 engagement (10Rohr O. Marban C. Aunis D. Schaeffer E. J. Leukoc. Biol. 2003; 74: 736-749Crossref PubMed Scopus (112) Google Scholar, 11Pierson T. McArthur J. Siliciano R.F. Annu. Rev. Immunol. 2000; 18: 665-708Crossref PubMed Scopus (461) Google Scholar). These transcription factors recruit coactivators, including histone acetyltransferases and the ATP-dependent chromatin-remodeling Swi/Snf complexes that influence the chromatin structure of integrated provirus (12Van Lint C. Emiliani S. Ott M. Verdin E. EMBO J. 1996; 15: 1112-1120Crossref PubMed Scopus (485) Google Scholar, 13Pumfery A. Deng L. Maddukuri A. de la Fuente C. Li H. Wade J.D. Lambert P. Kumar A. Kashanchi F. Curr. HIV Res. 2003; 1: 343-362Crossref PubMed Scopus (68) Google Scholar, 14Lusic M. Marcello A. Cereseto A. Giacca M. EMBO J. 2003; 22: 6550-6561Crossref PubMed Scopus (197) Google Scholar, 15Lee E.S. Sarma D. Zhou H. Henderson A.J. Virology. 2002; 299: 20-31Crossref PubMed Scopus (29) Google Scholar, 16Henderson A. Holloway A. Reeves R. Tremethick D.J. Mol. Cell Biol. 2004; 24: 389-397Crossref PubMed Scopus (69) Google Scholar). Furthermore, the LTR forms an RNA stem loop structure, TAR, which the HIV transactivator, Tat, binds. Tat enhances RNA polymerase II (pol II) processivity by recruiting P-TEFb to the HIV-LTR (17Zhang J. Tamilarasu N. Hwang S. Garber M.E. Huq I. Jones K.A. Rana T.M. J. Biol. Chem. 2000; 275: 34314-34319Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 18Bieniasz P.D. Grdina T.A. Bogerd H.P. Cullen B.R. Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 7791-7796Crossref PubMed Scopus (155) Google Scholar). The availability of P-TEFb, which is negatively regulated through association with the HEXIM1/7SK RNA particle, is also controlled by cellular signals (19Yik J.H. Chen R. Pezda A.C. Samford C.S. Zhou Q. Mol. Cell Biol. 2004; 24: 5094-5105Crossref PubMed Scopus (107) Google Scholar, 20Barboric M. Yik J.H. Czudnochowski N. Yang Z. Chen R. Contreras X. Geyer M. Matija Peterlin B. Zhou Q. Nucleic Acids Res. 2007; 35: 2003-2012Crossref PubMed Scopus (146) Google Scholar). Therefore, it may be possible to manipulate specific signaling cascades to control HIV transcription and improve the efficacy of current anti-viral regimens. Efficient T-cell activation requires signals from the TCR as well costimulatory molecules, including CD28, which enhances TCR activation, promotes cell survival and increases cytokine production (21Ward S.G. Biochem. J. 1996; 318: 361-377Crossref PubMed Scopus (139) Google Scholar, 22Wang S. Chen L. Microbes Infect. 2004; 6: 759-766Crossref PubMed Scopus (107) Google Scholar, 23Slavik J.M. Hutchcroft J.E. Bierer B.E. Immunol. Res. 1999; 19: 1-24Crossref PubMed Scopus (151) Google Scholar, 24Lenschow D.J. Walunas T.L. Bluestone J.A. Annu. Rev. Immunol. 1996; 14: 233-258Crossref PubMed Scopus (2367) Google Scholar). CD28 possesses no enzymatic activity and mediates signaling by recruiting other proteins to tyrosines and proline-rich motifs within its cytoplasmic domain. CD28 has four signaling tyrosine residues (Y) in the cytoplasmic tail of CD28 at positions Tyr-173, Tyr-188, Tyr-191, and Tyr-200, which are required for appropriate T-cell activation, induction of cytokine gene expression, cytoskeleton reorganization, and immunological synapse formation (25Teng J.M. 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, 26Sadra A. Cinek T. Imboden J.B. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 11422-11427Crossref PubMed Scopus (25) Google Scholar). Key signal transduction events associated with CD28 include activation of Itk, Vav, and Rho/Rac GTPases, protein kinase C θ, and transcription factors such as NF-κB, AP-1, and NFAT (6Readinger J.A. Schiralli G.M. Jiang J.K. Thomas C.J. August A. Henderson A.J. Schwartzberg P.L. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 6684-6689Crossref PubMed Scopus (59) Google Scholar, 27Park S.G. Schulze-Luehrman J. Hayden M.S. Hashimoto N. Ogawa W. Kasuga M. Ghosh S. Nat. Immunol. 2009; 10: 158-166Crossref PubMed Scopus (97) Google Scholar, 28Nolz J.C. Fernandez-Zapico M.E. Billadeau D.D. J. Immunol. 2007; 179: 1104-1112Crossref PubMed Scopus (20) Google Scholar, 29Hehner S.P. Hofmann T.G. Dienz O. Droge W. Schmitz M.L. J. Biol. Chem. 2000; 275: 18160-18171Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 30Cook J.A. Albacker L. August A. Henderson A.J. J. Biol. Chem. 2003; 278: 35812-35818Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 31August 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, 32August A. Dupont B. Int. Immunol. 1994; 6: 769-774Crossref PubMed Scopus (97) Google Scholar). We have previously shown that signaling associated with CD28 positively and negatively regulates HIV-1 provirus transcription. Specifically, we demonstrated that Tyr-200 positively regulated HIV transcription by initiating Vav-1 and NF-κB signaling, whereas recruitment of PI3K to the Tyr-173 residue inhibited the ability of Tat to bind P-TEFb and HIV-1 transcription (30Cook J.A. Albacker L. August A. Henderson A.J. J. Biol. Chem. 2003; 278: 35812-35818Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 33Cook J.A. August A. Henderson A.J. J. Immunol. 2002; 169: 254-260Crossref PubMed Scopus (16) Google Scholar). How these apparently opposing signals are coordinated to lead to induction of HIV-1 transcription, as well as the role of the other tyrosines in modulating HIV-1 transcription in response to CD28, has not been extensively investigated. Using chimeric CD28 receptors harboring mutations in different key tyrosines in the cytoplasmic domain, we show that CD28 induces HIV transcription through distinct but cooperative activities associated with the individual tyrosines. Jurkat E6.1 T cells were purchased from American Type Culture Collection (ATCC, Manassas, VA) and grown in RPMI 1640 medium supplemented with 5% fetal calf serum, 100 units/ml penicillin, 100 μg/ml streptomycin, and 0.2 ml-glutamine. Human embryonic kidney 293T cells were also obtained from ATCC and cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. Peripheral blood mononuclear cells were isolated from whole blood by Ficoll/histopaque gradient (Sigma-Aldrich), and CD4+ T cells were positively selected using the Dynal isolation kit (Invitrogen, 113.21D). The 8WT, YFFF, FFYF, FFFY, YFFF, YFYY, and YYFY expression vectors have been described previously (25Teng J.M. 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, 30Cook J.A. Albacker L. August A. Henderson A.J. J. Biol. Chem. 2003; 278: 35812-35818Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 33Cook J.A. August A. Henderson A.J. J. Immunol. 2002; 169: 254-260Crossref PubMed Scopus (16) Google Scholar, 34King P.D. Sadra A. Teng J.M. Xiao-Rong L. Han A. Selvakumar A. August A. Dupont B. J. Immunol. 1997; 158: 580-590PubMed Google Scholar), and these key residues are shown in Fig. 1. To generate receptors, FYYF and YFFY, the plasmids corresponding to pMHneo FFFY and pMHneo YFFF were digested with ApaI and HindIII (New England Biolabs). Two fragments were generated, a 400-bp fragment containing CD8α and the nucleotide sequence coding for tyrosine 173 of the cytoplasmic tail of CD28, and a 6.8-kb fragment containing the rest of CD28 and the pMHneo backbone. The 400-bp fragment from pMHneo FFFY and 6.8-kb fragment from pMHneo YFFF were gel-purified and ligated to generate pMHneo FYYF using T4 DNA ligase (Invitrogen). The pMHneo YFFY was generated similarly by ligating the 400-bp fragment isolated from pMHneo YYYF and the 6.8-kb fragment from pMHneo FFFY vectors. Additional mutants were generated by site-directed mutagenesis. Primers were designed (Table 1) to mutate key tyrosines to phenylalanines. Primers are listed in Table 1. For PCR reactions template plasmid DNA and appropriate primers were amplified using Vent polymerase (New England Biolabs) following standard protocols. The PCR products were digested with DpnI to eliminate donor plasmid and transformed into competent Escherichia coli DH5α cells. Positive clones were confirmed by sequencing.TABLE 1Primers for CD28 site-directed mutagenesisInitial templateFinal chimeric mutantForward primerReverse primerFYYFFYFF5′-GCAAGCATTACCAGCCCTTTGCCCCACC-3′5′-GGTGGGGCAAAGGGCTGGTAATGCTTGC-3′FYFFFFFF5′-GCAAGCATTTCCAGCCCTTTGCCCCACC-3′5′-GGTGGGGCAAAGGGCTGGAAATGCTTGC-3′FFYFYFYF5′-GCTCCTGCACAGTGACTACATGAACATGACTCC-3′5′-GGAGTCATGTTCATGTAGTCACTGTGCAGGAGC-3′FYFFYYFF5′ GCTCCTGCACAGTGACTACATGAACATGACTCC-3′5′ GGAGTCATGTTCATGTAGTCACTGTGCAGGAGC-3′YYFYFYFY5′ GCACAGTGACTTCATGAACATGACTCC-3′5′ GGAGTCATGTTCATGTAGTCACTGTGC-3′YFYYFFYY5′ GCACAGTGACTTCATGAACATGACTCC-3′5′ GGAGTCATGTTCATGTAGTCACTGTGC-3′ Open table in a new tab CD8/28 expression constructs were introduced in Jurkat E6.1 T cells using electroporation. 3 × 107 cells were washed and resuspended in 750 μl of serum-free RPMI containing 20 mm HEPES. 15 μg of plasmid DNA was then added to these cells and electroporated using a T280 square electroporation system (BTX, San Diego, CA). Cells were given 1 pulse for 65 ms at 215 V in a 4-mm cuvette and then recovered in complete RPMI. 48 h post-transfections cells were put on selection by including 1 mg/ml G418 in the growth media. After 3 weeks cells expressing the chimeric receptors were positively selected for CD8 using the Dynal isolation kit (Invitrogen) to generate a polyclonal pool of cells. Several independent pools for each receptor were generated to assure that there was no bias from an individual transfection and selection protocol. We also generated clonal cell lines and these behaved identically to the CD8/CD28 pooled cell lines (Refs. 30Cook J.A. Albacker L. August A. Henderson A.J. J. Biol. Chem. 2003; 278: 35812-35818Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 33Cook J.A. August A. Henderson A.J. J. Immunol. 2002; 169: 254-260Crossref PubMed Scopus (16) Google Scholar and data not shown). The expression of the chimeric receptors was verified by Western blot (data not shown) and flow cytometry. For flow cytometry 2 × 106 cells were washed and resuspended in 100 μl of staining media (phosphate-buffered saline containing 2% serum). Cells were incubated with 2 μl of anti-CD8α-phycoerythrin (BD 555635) and anti-CD28-FITC (BD 555728) for 45 min on ice. Cells were washed three times with staining media and fixed with 2% paraformaldehyde. Fluorescence was measured using a BD Biosciences FACScan at the Flow Core Facility at Boston Medical Center. 0.5 × 106 293T cells were plated in a 6-well plate 24 h prior to calcium phosphate transfections, which were performed using 15 μg of pNL4-3-Luc(+) Env(−) Nef(−) (35Henderson A.J. Zou X. Calame K.L. J. Virol. 1995; 69: 5337-5344Crossref PubMed Google Scholar) or pHXB-PLAP-Env Nef(+) (36Chen B.K. Gandhi R.T. Baltimore D. J. Virol. 1996; 70: 6044-6053Crossref PubMed Google Scholar) (obtained from the National Institutes of Health AIDS Research and Reference Reagent Program) and 3 μg of RSV-Rev, 3 μg of LTR VSV-G. 293T transfection efficiency for pNL4-3-Luc was assessed by determining luciferase activity using a luciferase kit (Promega Madison, WI), whereas p24 enzyme-linked immunosorbent assays were performed for the pHXB-PLAP virus. Supernatants were collected and filtered through a 0.45-μm disc prior to infection. Jurkat cells were infected with this virus for 12–16 h. Cells were then recovered and cultured in complete RPMI. Jurkat T cells were washed and resuspended in 5% fetal calf serum RPMI. 1 × 106 cells were plated in each well of a 24-well plate. Cells were either left unactivated, or activated with 0.1 μg/ml anti-human CD3 alone (BD 555336), anti-CD3, and 1.0 μg/ml anti-human CD28 (BD 555725) or 1.0 μg/ml anti-human CD8α antibodies (BD 555630) for 30 min. 5 μg/ml of goat anti-mouse antibody (Sigma M 4280) was added to cross-link the receptors. Following 8 h of stimulation, Jurkat cells were harvested, and luciferase activity was measured. In experiments using cyclosporin A (CsA), infected Jurkat T cells were recovered and activated in the presence of 500 ng/ml CsA or vehicle control. Jurkat T cells were serum-starved for 12–16 h, activated with antibodies as described above for 5 min prior to preparing protein extracts 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% Triton X-100, 1.0 mm phenylmethylsulfonyl fluoride, and protease inhibitor mixture III (Calbiochem). Lysates were precleared by incubating with protein A/G beads (Santa Cruz Biotechnology, sc-2003) for 30 min at 4 °C before incubating with primary anti-Vav (Santa Cruz Biotechnology, sc-132). Protein A/G beads were added to the antibody-lysate mix for 1 h at 4 °C, and beads were washed three times with lysis buffer and then suspended in SDS-PAGE loading buffer. The samples were heated for 5 min at 100 °C before being loaded onto a 10% SDS-PAGE gel. Proteins were transferred to a polyvinylidene difluoride membrane (Millipore) by electroblotting. Western blot analysis was carried out using a phosphotyrosine antibody (BD Transduction Laboratories, 610024). The blot was stripped and reprobed with a Vav antibody. CyclinT1 (Santa Cruz Biotechnology, sc-8127) and HEXIM1 (Abcam, ab28016) immunoprecipitations were also carried out with the same protocol; however, nuclear extracts (described in the next section) instead of total protein extracts were used. In the immunoprecipitation experiments done with the PI3K inhibitor, 50 μm LY294002 (Promega) was introduced 30 min before activation to the culture. Western blots were quantified by densitometry. The ratio of CyclinT1 over HEXIM was calculated for all samples in Fig. 3A, and the ratio of HEXIM over CyclinT1 was calculated for Fig. 3 (B–D). The numbers depicted in the figures represent ratio of immunoprecipitation in each lane versus immunoprecipitation from the unactivated lane. Jurkat cells were activated for 8 h, and nuclear extracts were isolated by resuspending 1 × 106 cells in low salt buffer (10 mm HEPES, pH 7.9, 10 mm KCl, 0.1 mm EDTA, 0.1 mm EGTA, 1 mm dithiothreitol, and 0.5 mm phenylmethylsulfonyl fluoride) for 15 min. 10% Nonidet P-40 was added to rupture the cell membranes, and the nuclei were pelleted and incubated in a high salt buffer (20 mm HEPES, pH 7.9, 400 mm NaCl, 1.0 mm EDTA, 1.0 mm EGTA, 1 mm dithiothreitol, and 1.0 mm phenylmethylsulfonyl fluoride) to isolate the nuclear extract. Electrophoretic mobility shift assays were carried out by incubating 5 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 100-fold excess of specific or nonspecific competitors, or 0.5 μg of polyclonal antibodies against NF-κB subunits p50 (Santa Cruz Biotechnology, sc-7178) and p65 (Santa Cruz Biotechnology, sc-109). Samples were loaded onto a 6% polyacrylamide gel and electrophoresed at 120 V in 0.5× Tris borate-EDTA. Probes for electrophoretic mobility shift assay were generated by annealing oligonucleotides representing the HIV-1 NF-κB sites (5′-AGCTCCTGGAAAGTCCCCAGCGGAAAGTCCCTT-3′ and 5′-AGCTAAGGGACTTTCCGCTGGGGACTTTCCAGG-3′). Sp1 probe was used as nonspecific competitor (sense sequence 5′-GATCATTCGATCGGGGCGGGGCGAGC-3′ and antisense sequence 5′-GATCGCTCGCCCCGCCCCGATCGAAT-3′). Probes were generated by end filling with the Klenow fragment of E. coli polymerase in the presence of [α-32P]dCTP. Fifteen micrograms of pGL2 LTR luc and pGL2-mκB-LTR luc constructs (kindly provided by Dr. Suryaram Gummuluru (37Gummuluru S. Emerman M. J. Virol. 1999; 73: 5422-5430Crossref PubMed Google Scholar)) were electroporated into 20 × 106 Jurkat E6.1 cells using the T280 BTX electroporator. The cells were recovered in 5% fetal calf serum RPMI for 16 h. 1 × 106 cells were either left untreated or activated with 0.1 μg/ml anti-human CD3 or 0.1 μg/ml anti-human CD28 and 1.0 μg/ml anti-human CD28, and luciferase assays were performed 6 h post activation as described above. 1 × 108 cells were infected with pHXB-PLAP virus for 5 days. Cells were then activated with 0.1 μg/ml anti-CD3 and 1.0 μg/ml anti-CD8 or anti-CD28 antibodies for 30 min. 5.0 μg/ml of goat anti-mouse was then added, and cells were activated for 6 h. Cells were cross-linked using 11% formaldehyde solution (prepared from 37% formaldehyde, 10% methanol) in 0.1 m NaCl, 1 mm EDTA, 0.5 mm EGTA, 50 mm Tris-HCl (pH 8) to the final concentration of 1% for 10 min at room temperature. The reaction is quenched by adding 2 m glycine to a final concentration of 240 mm. Cells were washed with phosphate-buffered saline and resuspended in 1 ml of sonication buffer (10 mm Tris-HCl, pH 8.0, 1 mm EDTA, 0.5 mm EGTA, 0.5 mm phenylmethylsulfonyl fluoride) and sonicated on ice for 30 cycles, 10 s on, 30 s off. 100 μl of sonicated chromatin was diluted 10-fold with dilution buffer and incubated with 1 μg of antibody pol II (sc-899) p65 (sc-109) for 16 h at 4 °C. Protein A/G beads were then added for 2 h. The beads were then washed twice each with low salt (0.1% SDS, 1% Triton X-100, 2 mm EDTA, 20 mm Tris-HCl, pH 8.0, 150 mm NaCl), high salt (0.1% SDS, 1% Triton X-100, 2 mm EDTA, 20 mm Tris-HCl, pH 0.1, 500 mm NaCl), and LiCl wash buffer (0.25 m LiCl, 1% Nonidet P-40, 1% sodium deoxycholate, 1 mm EDTA, 10 mm Tris-HCl, pH 8.0) and 10 mm Tris and 1 mm EDTA. Complexes were eluted with 1% SDS, 0.1 m NaHCO3. The complexes were reverse cross-linked at 65 °C for 4 h, followed by addition of proteinase K for 1 h at 45 °C. The DNA was extracted using phenol chloroform and precipitated with ethanol. Quantitative real-time PCR analysis was carried out using SYBR green reagents and the primers 5′-TGCATCCGGAGTACTTCAAGA-3′ and 5′-GAGGCTTAAGCAGTGGGTTC-3′, which amplify the −150 to +76 region of HIV-LTR, and 5′-GACTAGAGCCCTGGAAGCA-3′ and 5′-GCTTCTTCCTGCCATAGGAG-3′, which amplify the +5396 to +5531 region of HIV. Statistical analysis was carried out using Student t test. A two-tailed distribution was performed on paired samples, comparing CD3 responses to CD3 plus CD8 responses. Values of <0.01 were considered significant. To study the role of CD28 in regulating HIV transcription, we employed a strategy that was previously described (25Teng J.M. 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, 30Cook J.A. Albacker L. August A. Henderson A.J. J. Biol. Chem. 2003; 278: 35812-35818Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 33Cook J.A. August A. Henderson A.J. J. Immunol. 2002; 169: 254-260Crossref PubMed Scopus (16) Google Scholar), in which we generated CD8/28 chimeric receptors with mutations in key tyrosine residues. The chimeric receptors were designed such that the cytoplasmic domain of CD28 was fused to the transmembrane and extracellular domain of CD8α, which we refer to henceforth as CD8/CD28. This chimeric receptor forms a dimer similar to CD28 and functions identical to endogenous CD28 (25Teng J.M. 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). In addition, the generation and expression of these CD8/CD28 chimeras allow their expression in cells along with endogenous CD28, and the direct comparison of mutant and WT CD28 signals in the same cells (25Teng J.M. 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, 30Cook J.A. Albacker L. August A. Henderson A.J. J. Biol. Chem. 2003; 278: 35812-35818Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 33Cook J.A. August A. Henderson A.J. J. Immunol. 2002; 169: 254-260Crossref PubMed Scopus (16) Google Scholar). As shown in Fig. 1A, we changed individual or different combinations of tyrosines in the cytoplasmic tail of CD28 to phenylalanine (F). Polyclonal populations of the Jurkat E6.1 T cell line expressing these chimeric receptors were generated, and stable expression of CD8/CD28, as well as endogenous CD28 receptors, was evaluated by flow cytometric analysis (Fig. 1B). This panel of Jurkat T cells expressing CD8/CD28 chimeras was infected with NL4-3 luciferase virus to evaluate the function of different tyrosines in HIV-1 transcription. Using the HIV-luciferase clone, which lacks envelope and supports only a single round of infection, allowed us to focus on CD28 signaling and HIV-1 transcription, rather than potential effects CD28 signaling has on virus replication and spread. Infected Jurkat T cells were stimulated through the TCR using anti-CD3 antibodies and either the chimeric CD8/CD28 receptor (using anti-CD8α antibodies) or the endogenous CD28 receptor (using anti-CD28 antibodies), which served as a control to ensure that the different T cell lines were capable of being activated. Controls included stimulating cells through the CD28, CD8/CD28, and CD3 receptors alone. Consistent with our previous studies the chimeric receptor CD8/CD28 WT, which retains all functional tyrosines, leads to transcriptional activation similar to that mediated by the endogenous CD28 receptor (25Teng J.M. 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, 30Cook J.A. Albacker L. August A. Henderson A.J. J. Biol. Chem. 2003; 278: 35812-35818Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 33Cook J.A. August A. Henderson A.J. J. Immunol. 2002; 169: 254-260Crossref PubMed Scopus (16) Google Scholar). Receptors lacking the cytoplasmic domain, del167, and the All F mutant, where all four tyrosines in the cytoplasmic tail of CD28 were mutated to phenylalanines, did not induce HIV-1 transcription confirming an indispensable role for these tyrosines in the cytoplasmic tail of CD28 (Fig. 2) (25Teng J.M. 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, 30Cook J.A. Albacker L. August A. Henderson A.J. J. Biol. Chem. 2003; 278: 35812-35818Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 33Cook J.A. August A. Henderson A.J. J. Immunol. 2002; 169: 254-260Crossref PubMed Scopus (16) Google Scholar). Previous studies have shown that the Tyr-173 residue negatively regulates HIV-1 transcription, whereas signaling through Tyr-200 was necessary for HIV-1 transcription (30Cook J.A. Albacker L. August A. Henderson A.J. J. Biol. Chem. 2003; 278: 35812-35818Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 33Cook J.A. August A. Henderson A.J. J. Immunol. 2002; 169: 254-260Crossref PubMed Scopus (16) Google Scholar). We were interested in determining the functional interplay between these two apparently opposite activities mediated through Tyr-200 and Tyr-173, as well as the integration of signals downstream of other tyrosines within the CD28 cytoplasmic domain. We initially examined the ability of CD28 receptors with one functional tyrosine, Tyr-173 (YFFF), Tyr-188 (FYFF), Tyr-191 (FFYF), or Tyr-200 (FFFY), to support HIV-1 transcription. As expected, the YFFF receptor was unable to activate HIV transcription, consistent with our previous report that implicated Tyr-173 signaling as inhibitory (33Cook J.A. August A. Henderson A.J. J. Immunol. 2002; 169: 254-260Crossref PubMed Scopus (16) Google Scholar). Furthermore, the FYFF receptor did not support HIV-1 transcription indicating that signals downstream of Tyr-188 were not sufficient to induce HIV-1 transcription. However, the FFYF and FFFY receptors activated HIV-1 transcription to levels comparable to the wild-type CD8/CD28 or the endogenous CD28 receptors, indicating that Tyr-191 and Tyr-200 positively regulate HIV transcription in the absence of other tyrosine residues in the signaling domain of CD28. These data" @default.
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• W2045367107 title "Combinatorial Signals from CD28 Differentially Regulate Human Immunodeficiency Virus Transcription in T Cells" @default.
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