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• W2078009198 abstract "HIV infection and the progression to AIDS are characterized by the depletion of CD4+ T cells through apoptosis of the uninfected bystander cells and the direct killing of HIV-infected cells. This is mediated in part by the human immunodeficiency virus, type 1 Tat protein, which is secreted by virally infected cells and taken up by uninfected cells and CD178 gene expression, which is critically involved in T cell apoptosis. The differing ability of HIV strains to induce death of infected and uninfected cells may play a role in the clinical and biological differences displayed by HIV strains. We chemically synthesized the 86-residue truncated short variant of Tat and its full-length form. We show that the trans-activation ability of Tat at the long terminal repeat does not correlate with T cell apoptosis but that the ability of Tat to up-regulate CD178 mRNA expression and induce apoptosis in T cells is critically dependent on the C terminus of Tat. Moreover, the greater 86-residue Tat-induced apoptosis is via the extrinsic pathway of CD95-CD178. HIV infection and the progression to AIDS are characterized by the depletion of CD4+ T cells through apoptosis of the uninfected bystander cells and the direct killing of HIV-infected cells. This is mediated in part by the human immunodeficiency virus, type 1 Tat protein, which is secreted by virally infected cells and taken up by uninfected cells and CD178 gene expression, which is critically involved in T cell apoptosis. The differing ability of HIV strains to induce death of infected and uninfected cells may play a role in the clinical and biological differences displayed by HIV strains. We chemically synthesized the 86-residue truncated short variant of Tat and its full-length form. We show that the trans-activation ability of Tat at the long terminal repeat does not correlate with T cell apoptosis but that the ability of Tat to up-regulate CD178 mRNA expression and induce apoptosis in T cells is critically dependent on the C terminus of Tat. Moreover, the greater 86-residue Tat-induced apoptosis is via the extrinsic pathway of CD95-CD178. The induction of apoptosis in uninfected bystander cells has been postulated as a mechanism of CD4+ T cell depletion and immune dys-regulation during the clinical course of HIV 2The abbreviations used are: HIV-1, human immunodeficiency virus, type 1; Tat, trans-acting regulatory protein; LTR, long terminal repeat; PBS, phosphate-buffered saline; Fmoc, 9-fluorenylmethoxycarbonyl; BSA, bovine serum albumin; HPLC, high performance liquid chromatography.2The abbreviations used are: HIV-1, human immunodeficiency virus, type 1; Tat, trans-acting regulatory protein; LTR, long terminal repeat; PBS, phosphate-buffered saline; Fmoc, 9-fluorenylmethoxycarbonyl; BSA, bovine serum albumin; HPLC, high performance liquid chromatography. infection (1.Ameisen J.C. Capron A. Immunol. Today. 1991; 12: 102-105Abstract Full Text PDF PubMed Scopus (490) Google Scholar, 2.Finkel T.H. Tudor-Williams G. Banda N.K. Cotton M.F. Curiel T. Monks C. Baba T.W. Ruprecht R.M. Kupfer A. Nat. Med. 1995; 1: 129-134Crossref PubMed Scopus (811) Google Scholar), leading to the loss of immune competence (3.Meyaard L. Otto S.A. Jonker R.R. Mijnster M.J. Keet R.P. Miedema F. Science. 1992; 257: 217-219Crossref PubMed Scopus (852) Google Scholar, 4.Fauci A.S. Science. 1993; 262: 1011-1018Crossref PubMed Scopus (732) Google Scholar). Apoptotic cell death is a process characterized by cell shrinkage, loss of membrane integrity, DNA fragmentation, and formation of apoptotic bodies (5.Green D.R. Cell. 2000; 102: 1-4Abstract Full Text Full Text PDF PubMed Scopus (882) Google Scholar). The key apoptotic effectors in mammals are a family of cysteine-containing, aspartate-specific proteases called caspases (6.Alnemri E.S. Livingston D.J. Nicholson D.W. Salvesen G. Thornberry N.A. Wong W.W. Yuan J. Cell. 1996; 87: 171Abstract Full Text Full Text PDF PubMed Scopus (2129) Google Scholar). The CD95/CD178 (Fas/Fas ligand) system also has a well known role in mediating apoptosis and has been shown to play a possible role of variable significance in the induction of apoptosis in bystander cells in in vitro HIV systems (7.Alimonti J.B. Ball T.B. Fowke K.R. J. Gen. Virol. 2003; 84: 1649-1661Crossref PubMed Scopus (262) Google Scholar) but not in the apoptosis of activated primary CD4+ T cells (8.Noraz N. Gozlan J. Corbeil J. Brunner T. Spector S.A. AIDS. 1997; 11: 1671-1680Crossref PubMed Scopus (46) Google Scholar). CD95-mediated apoptosis is irreversible and is, thus, tightly regulated both pre- and post-CD95 engagement (9.Tschopp J. Irmler M. Thome M. Curr. Opin. Immunol. 1998; 10: 552-558Crossref PubMed Scopus (467) Google Scholar). For T cells, one of the major forms of regulation is exerted at the level of CD178 expression. CD178 mRNA is not expressed in resting T cells but is induced shortly after an activating stimulus (10.Suda T. Takahashi T. Golstein P. Nagata S. Cell. 1993; 75: 1169-1178Abstract Full Text PDF PubMed Scopus (2431) Google Scholar). In HIV-infected individuals, both CD4+ and CD8+ T cells are more susceptible to CD178-induced apoptosis, and this is related to the regulation of surface levels of both CD95 and CD178. It has been shown that T cells from HIV-infected individuals overexpress CD178 (11.Silvestris F. Cafforio P. Frassanito M.A. Tucci M. Romito A. Nagata S. Dammacco F. AIDS. 1996; 10: 131-141Crossref PubMed Scopus (98) Google Scholar), the proportion of these T cells increases with disease progression (12.Aries S.P. Schaaf B. Muller C. Dennin R.H. Dalhoff K. J. Mol. Med. 1995; 73: 591-593Crossref PubMed Scopus (63) Google Scholar, 13.Baumler C.B. Bohler T. Herr I. Benner A. Krammer P.H. Debatin K.M. Blood. 1996; 88: 1741-1746Crossref PubMed Google Scholar, 14.Estaquier J. Tanaka M. Suda T. Nagata S. Golstein P. Ameisen J.C. Blood. 1996; 87: 4959-4966Crossref PubMed Google Scholar), and the rate of apoptosis is correlated with disease progression (15.Moretti S. Marcellini S. Boschini A. Famularo G. Santini G. Alesse E. Steinberg S.M. Cifone M.G. Kroemer G. De Simone C. Clin. Exp. Immunol. 2000; 122: 364-373Crossref PubMed Scopus (47) Google Scholar). The HIV-1 trans-acting regulatory protein (Tat) has been shown to up-regulate CD178 expression in infected (16.Li-Weber M. Laur O. Dern K. Krammer P.H. Eur. J. Immunol. 2000; 30: 661-670Crossref PubMed Scopus (85) Google Scholar) and non-infected bystander cells (17.Westendorp M.O. Frank R. Ochsenbauer C. Stricker K. Dhein J. Walczak H. Debatin K.M. Krammer P.H. Nature. 1995; 375: 497-500Crossref PubMed Scopus (910) Google Scholar, 18.Campbell G.R. Pasquier E. Watkins J. Bourgarel-Rey V. Peyrot V. Esquieu D. Barbier P. de Mareuil J. Braguer D. Kaleebu P. Yirrell D.L. Loret E.P. J. Biol. Chem. 2004; 279: 48197-48204Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). However, it has also been shown that Tat does not require the CD178-CD95 interaction to induce apoptosis and that Tat binds to tubulin and LIS1, perturbing microtubule dynamics, leading to apoptosis through the intrinsic mitochondrial pathway (19.Chen D. Wang M. Zhou S. Zhou Q. EMBO J. 2002; 24: 6801-6810Crossref Scopus (166) Google Scholar, 20.de Mareuil J. Carre M. Barbier P. Campbell G.R. Lancelot S. Opi S. Esquieu D. Watkins J.D. Prevot C. Braguer D. Peyrot V. Loret E.P. Retrovirology. 2005; 2: 5Crossref PubMed Scopus (53) Google Scholar, 21.Epie N. Ammosova T. Sapir T. Voloshin Y. Lane W.S. Turner W. Reiner O. Nekhai S. Retrovirology. 2005; 2: 6Crossref PubMed Scopus (27) Google Scholar).Tat is an 86-101 residue regulatory protein (9-11 kDa) produced early in HIV-1 infection whose primary role is in regulating productive and processive transcription from the HIV-1 long terminal repeat (LTR) (22.Loret E.P. Georgel P. Johnson W.C. Ho P.S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9734-9738Crossref PubMed Scopus (78) Google Scholar, 23.Karn J. J. Mol. Biol. 1999; 293: 235-254Crossref PubMed Scopus (388) Google Scholar, 24.Gatignol A. Jeang K.T. Adv. Pharmacol. 2000; 48: 209-227Crossref PubMed Google Scholar). Tat is secreted from infected cells (25.Ensoli B. Barillari G. Salahuddin S.Z. Gallo R.C. Wong-Staal F. Nature. 1990; 345: 84-86Crossref PubMed Scopus (793) Google Scholar, 26.Ensoli B. Buonaguro L. Barillari G. Fiorelli V. Gendelman R. Morgan R.A. Wingfield P. Gallo R.C. J. Virol. 1993; 67: 277-287Crossref PubMed Google Scholar, 27.Chang H.C. Samaniego F. Nair B.C. Buonaguro L. Ensoli B. AIDS. 1997; 11: 1421-1431Crossref PubMed Scopus (391) Google Scholar) and has been detected using weak avidity anti-Tat antibodies in the sera of HIV-1-infected patients at concentrations of up to 40 ng/ml. However, current estimates of Tat concentrations are thought to be an underestimation as Tat is rapidly taken up by cells, local concentrations of Tat in lymphoid tissues might be higher, the antibody used for detection has weak avidity, and Tat in vivo might be sequestered by endogenous anti-Tat antibody and/or by glycosaminoglycans (28.Xiao H. Neuveut C. Tiffany H.L. Benkirane M. Rich E.A. Murphy P.M. Jeang K.T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 11466-11471Crossref PubMed Scopus (316) Google Scholar). Extracellular Tat has a variety of effects on a number of different cell types (for review, see Ref. 29.Huigen M.C. Kamp W. Nottet H.S. Eur. J. Clin. Investig. 2004; 34: 57-66Crossref PubMed Scopus (91) Google Scholar). The viral mRNA for Tat is composed of two exons (30.Arya S.K. Guo C. Josephs S.F. Wong-Staal F. Science. 1985; 229: 69-73Crossref PubMed Scopus (589) Google Scholar). The first exon codes for 72 amino acids (residues 1-72) and contains three important functional regions; they are the cysteine-rich region (amino acids 22-37), the basic region (amino acids 49-57), and the glutamine-rich region (amino acids 58-72). The second exon codes for a variable number of amino acids (residues 14-31) that contribute to viral infectivity and other functions (31.Rana T.M. Jeang K.T. Arch. Biochem. Biophys. 1999; 365: 175-185Crossref PubMed Scopus (161) Google Scholar).Two different forms of Tat are found in clinical isolates; they are an 86-residue form and a longer, more predominant 101-residue form (32.Jeang K.T. Human Retroviruses and AIDS: A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences. Los Alamos National Laboratory, Los Alamos, NM1996: III-3-III-18Google Scholar). In research to date, the most widely used form of Tat is Tat HXB2 (86), which is a truncated 86-residue form from a laboratory-passaged subtype B viral strain (32.Jeang K.T. Human Retroviruses and AIDS: A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences. Los Alamos National Laboratory, Los Alamos, NM1996: III-3-III-18Google Scholar, 33.Ratner L. Haseltine W. Patarca R. Livak K.J. Starcich B. Josephs S.F. Doran E.R. Rafalski J.A. Whitehorn E.A. Baumeister K. Ivanoff L. Petteway S.R. Pearson M.L. Lautenberger J.A. Papas T.S. Ghrayeb J. Chang N.T. Gallo R.C. Wongstaal F. Nature. 1985; 313: 277-284Crossref PubMed Scopus (1720) Google Scholar). Indeed, a single nucleotide change at the putative residue 87 allows the translation of the full-length 101-amino acid form (HXB2 (100)). Therefore, the extreme C terminus of Tat has not been frequently considered in research even though it has been shown to be significant in several biological assays (34.Xiao H. Neuveut C. Benkirane M. Jeang K.T. Biochem. Biophys. Res. Commun. 1998; 244: 384-389Crossref PubMed Scopus (43) Google Scholar, 35.Ott M. Emiliani S. Van Lint C. Herbein G. Lovett J. Chirmule N. McCloskey T. Pahwa S. Verdin E. Science. 1997; 275: 1481-1485Crossref PubMed Scopus (186) Google Scholar, 36.Smith S.M. Pentlicky S. Klase Z. Singh M. Neuveut C. Lu C.Y. Reitz Jr., M.S. Yarchoan R. Marx P.A. Jeang K.T. J. Biol. Chem. 2003; 278: 44816-44825Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar) and is the predominant form present in clinical isolates. In this study we compared the ability of these two forms of Tat to trans-activate the HIV-1 LTR and assessed their ability to induce apoptosis in T cells. We show that the last 14 residues at the C terminus of Tat present in the full-length 100-amino acid form are implicated in both a greater trans-activation ability and in a reduced ability to up-regulate CD178 expression and induce apoptosis via the extrinsic pathway.MATERIALS AND METHODSProtein Synthesis, Purification, and Characterization—The Tat proteins were synthesized in solid phase using Fast Fmoc chemistry according to the method of Barany and Merrifield (37.Barany G. Merrifield R.B. Gross E. Meinhofer J. The Peptide: Analysis, Synthesis, Biology. 2. Academic Press, Inc., New York1980: 1-284Google Scholar) using HMP (4-hydroxymethyl-phenoxymethyl-copolystyrene, 1% divinylbenzene)-preloaded resin (0.5 mmol) (PerkinElmer) on an automated synthesizer (ABI 433A, PerkinElmer) and purified as described elsewhere (18.Campbell G.R. Pasquier E. Watkins J. Bourgarel-Rey V. Peyrot V. Esquieu D. Barbier P. de Mareuil J. Braguer D. Kaleebu P. Yirrell D.L. Loret E.P. J. Biol. Chem. 2004; 279: 48197-48204Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 38.Péloponèse J.M. Collette Y. Grégoire C. Bailly C. Campèse D. Meurs E.F. Olive D. Loret E.P. J. Biol. Chem. 1999; 274: 11473-11478Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 39.Opi S. Péloponèse Jr., J.M. Esquieu D. Watkins J. Campbell G. de Mareuil J. Jeang K.T. Yirrell D.L. Kaleebu P. Loret E.P. Vaccine. 2004; 22: 3105-3111Crossref PubMed Scopus (26) Google Scholar). High pressure liquid chromatography (HPLC) analysis was carried out on a Beckman Coulter HPLC apparatus using a Merck Chromolith™ Performance RP-8e (4.6 × 100 mm) column (Merck). Buffer A was water with 0.1% (v/v) trifluoroacetic acid (Sigma-Aldrich), and Buffer B was acetonitrile (Merck) with 0.1% (v/v) trifluoroacetic acid. The gradient was buffer B from 10 to 50% in 15 min with a 1.8-ml/min flow rate. Amino acid analyses were performed on a model 6300 Beckman analyzer, and mass spectrometry was carried out using an Ettan™ matrix-assisted laser desorption ionization time-of-flight (Amersham Biosciences).Trans-activation with HIV LTR-transfected Cells—HeLa P4 cells containing the bacterial lacZ gene under the control of the HIV-1 LTR, as described by Clavel and Charneau (40.Clavel F. Charneau P. J. Virol. 1994; 68: 1179-1185Crossref PubMed Google Scholar), were used in this study. The activity of the synthetic Tat protein was analyzed by monitoring the production of β-galactosidase after activation of lacZ expression mainly as previously described with some modifications (38.Péloponèse J.M. Collette Y. Grégoire C. Bailly C. Campèse D. Meurs E.F. Olive D. Loret E.P. J. Biol. Chem. 1999; 274: 11473-11478Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Briefly, 2 × 105 cells/well were incubated in 24-well flat-bottomed plates (Falcon) at 37 °C, 5% CO2 in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% (v/v) heat-inactivated fetal calf serum (Invitrogen) and 100 μg/ml neomycin (Invitrogen). After 24 h, cells were washed with phosphate-buffered saline (PBS). Tat protein was dissolved in phosphate buffer at pH 6 (to avoid precipitation that occurs at neutral pH) and was directly mixed with Dulbecco's modified Eagle's medium supplemented with 0.01% (w/v) protamine (Sigma-Aldrich) and 0.1% (w/v) bovine serum albumin (BSA) (Sigma-Aldrich) and added to the cells. After 16 h at 37 °C, 5% CO2 cells were washed with PBS, lysed, the β-galactosidase content was measured with a commercially available antigen capture enzyme-linked immunosorbent assay (β-galactosidase enzyme-linked immunosorbent assay, Roche Diagnostics), and absorbance values (B) were measured at 405 nm. Results were normalized using the Bradford reagent (Sigma-Aldrich). B0 corresponds to the background β-galactosidase expressed by HeLa P4 cells in Dulbecco's modified Eagle's medium supplemented with 0.01% (w/v) protamine (Sigma-Aldrich) and 0.1% (w/v) BSA (Sigma-Aldrich) without Tat. Concentrations of Tat used are noted in Fig. 1.Entry of Tat into the Cells—HeLa P4 cells were cultured and treated with Tat as for the trans-activation assay. After treatment with Tat, the culture medium was removed, and cells were washed with cold PBS and lysed for 30 min at 4 °C in radioimmunoprecipitation assay buffer (20 mm Tris-HCl, pH 8.0, 200 mm NaCl, 1% Triton, 1 mm EDTA, and protease inhibitors) without BSA. We obtained a cytoplasmic supernatant and a pellet corresponding to the nuclear extract by centrifuging the lysates at 4000 × g for 15 min at 4 °C. All supernatants were stored at -80 °C. After a brief sonication, the lysates were clarified by centrifugation at 10,000 rpm, and protein content was measured by the Bradford method (Bio-Rad). 40 μg/sample was then heated at 100 °C for 5 min in Laemmli sample buffer containing reducing agent, separated by 15% SDS-polyacrylamide gel, and blotted onto Protran® nitrocellulose membranes (Schleicher & Schuell). Nonspecific sites were blocked by 1 h of incubation at room temperature with PBS, 0.1% (v/v) Tween 20 (Sigma-Aldrich), and 10% (w/v) dried nonfat milk (10% MPBS). Membranes were then incubated with anti-HXB2 (100) and anti-HXB2 (86) rabbit sera raised as previously described (39.Opi S. Péloponèse Jr., J.M. Esquieu D. Watkins J. Campbell G. de Mareuil J. Jeang K.T. Yirrell D.L. Kaleebu P. Loret E.P. Vaccine. 2004; 22: 3105-3111Crossref PubMed Scopus (26) Google Scholar) at 1:500 dilution in 10% MPBS overnight, washed 4 times, and incubated with an anti-rabbit horseradish peroxidase-labeled secondary antibody at 1:1000 dilution) (Sigma-Aldrich) for 1 h at room temperature in 10% MPBS. The bound horseradish peroxidase was revealed with H2O2, 0.1% (w/v) diaminobenzidine tetrahydrochloride (Sigma-Aldrich) in PBS as substrate. The intensity of immunoblot bands was analyzed by densitometric imaging using the freely available Scion Image (Scion Corp., Frederick, MD) on a personal computer.Apoptosis Assay—Human peripheral blood mononuclear cells were isolated from heparinized blood of healthy donors by using density centrifugation over Ficoll-Hypaque (Amersham Biosciences). After 2 washes with PBS supplemented with 10% (v/v) autologous serum and 2 mm EDTA (Sigma Aldrich). CD4+ cells were then isolated using the CD4+ isolation kit using MidiMACs with LS columns (Miltenyi Biotech, Auburn, CA) and cultured in AIM-V® medium with human serum albumin (Invitrogen) until used.The Jurkat clone I9.2 cell line was purchased from American Type Culture Collection and cultured in RPMI 1640 (Invitrogen) supplemented with 10% (v/v) heat-inactivated fetal calf serum (Invitrogen), 10 mm HEPES, 1 mm sodium pyruvate, 2 mm l-glutamine, 100 μg/ml streptomycin, and 100 units/ml penicillin (all from Invitrogen). The Jurkat I9.2 cell line is a clone that is functionally defective for Fas-associated death domain and is completely resistant to both caspase-8- and CD178-induced apoptosis (41.Juo P. Kuo C.J. Yuan J. Blenis J. Curr. Biol. 1998; 8: 1001-1008Abstract Full Text Full Text PDF PubMed Scopus (469) Google Scholar).To induce apoptosis cells were cultured in 24-well plates in RPMI 1640 (Invitrogen) 0.1% (w/v) BSA (Sigma Aldrich) for 19 h without sera in the presence or not of Tat. Tat was prepared as for the trans-activation assay.To differentiate between early apoptosis and late apoptosis/necrosis, cells were harvested at 19 h post-treatment and immediately stained with annexin V/fluorescein isothiocyanate and propidium iodide according to the protocol provided by the manufacturer (BD Pharmingen). The harvest and staining took 1 h, and then stained cells were immediately analyzed by flow cytometry (FACScan, BD Biosciences).To determine cellular caspase-8 and caspase-9 activity, cells were harvested at 14 h post-treatment and immediately stained with the caspase-8 specific detection kit (Red-IETD-fluoromethyl ketone (FMK)) and the caspase-9-specific detection kit (fluorescein isothiocyanate ((FITC)-LEHD-FMK) according to the manufacturer's instructions (Calbiochem). Positive controls were carried out in the presence of 40 μm ceramide analogue C2-ceramide (Calbiochem) initially dissolved in dimethyl sulfoxide (Me2SO). The harvest and staining took 1.5 h, and then stained cells were immediately analyzed by flow cytometry (FACScan, BD Biosciences). Cytogram analysis was performed with Cell Quest Pro® software (BD Bioscience).Real-time PCR—CD178 mRNA expression in CD4+ T cells was measured by real time PCR. 1 × 106 cells/ml were incubated in 24-well flat-bottomed plates (Falcon) at 37 °C with 2 μm Tat for 14 h. Total cellular RNA was prepared with the RNeasy Mini kit in accordance with the manufacturer's directions (Qiagen, Valencia, CA). 1 μg of total RNA was used for reverse transcription with random primers. CD178 mRNA expression in relation to β2 microglobulin expression (internal standard) was determined using the LightCycler System and the FastStart DNA master SYBR Green I kit (Roche Diagnostics). PCR reactions were carried out in a 20-μl mixture composed of 3 mm MgCl2, 0.5 μm concentrations of each primer, a 5-μl sample, and 1-fold LightCycler FastStart DNA Master SYBR Green I. Primers used were as follows: CD178 sense, 5′-GTAGGATTGGGCCTGGGGAT-3′, and antisense, 5′-AGTTGGACTTGCCTGTTAAA-3′; β2 microglobulin sense, 5′-CCGACATTGAAGTTGACTTAC-3′, and antisense 5′-ATCTTCAAACCTCCATGATG-3′. The reaction mixture was initially incubated at 95 °C for 10 min to denature the cDNA. Amplification was performed for 45 cycles, with the following cycle parameters: 10 s of denaturation at 95 °C, 10 s of primer annealing at 65 °C, and 15 s of fragment elongation at 72 °C. Quantification and melting curve were analyzed with LightCycler analysis software, RelQuant (Roche Diagnostics). All results were expressed as the ratio between the copy number of the target gene and the copy number of β2 microglobulin and normalized so that CD178 expression in the non-treated cells equals 1.00.Statistics—All p values correspond to two-sample t tests assuming unequal variances, unless indicated otherwise.RESULTSFull-length HXB2 Has a Higher Trans-activation Activity than the Truncated 86-Residue Form—In this study we have used the full-length form of Tat HXB2 (HXB2 (100)) as described by Jeang (32.Jeang K.T. Human Retroviruses and AIDS: A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences. Los Alamos National Laboratory, Los Alamos, NM1996: III-3-III-18Google Scholar) and the 86-residue-truncated form of Tat HXB2 (Tat HXB2 (86)). Both proteins were obtained using Fast Fmoc chemistry and, after purification using HPLC, gave a good HPLC homogeneity (Fig. 1A), and the expected mass was observed by mass spectrometry (Fig. 1B).We assessed the ability of these Tat variants to cross the cell membrane of HeLa P4 cells and trans-activate the stably transfected HIV-1 LTR in these cells as used previously by ourselves and others (18.Campbell G.R. Pasquier E. Watkins J. Bourgarel-Rey V. Peyrot V. Esquieu D. Barbier P. de Mareuil J. Braguer D. Kaleebu P. Yirrell D.L. Loret E.P. J. Biol. Chem. 2004; 279: 48197-48204Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 38.Péloponèse J.M. Collette Y. Grégoire C. Bailly C. Campèse D. Meurs E.F. Olive D. Loret E.P. J. Biol. Chem. 1999; 274: 11473-11478Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 40.Clavel F. Charneau P. J. Virol. 1994; 68: 1179-1185Crossref PubMed Google Scholar, 42.Vivès E. Charneau P. van Rietschoten J. Rochat H. Bahraoui E. J. Virol. 1994; 68: 3343-3353Crossref PubMed Google Scholar, 43.Badou A. Bennasser Y. Moreau M. Leclerc C. Benkirane M. Bahraoui E. J. Virol. 2000; 74: 10551-10562Crossref PubMed Scopus (98) Google Scholar, 44.Bres V. Tagami H. Peloponese J.M. Loret E. Jeang K.T. Nakatani Y. Emiliani S. Benkirane M. Kiernan R.E. EMBO J. 2002; 21: 6811-6819Crossref PubMed Scopus (80) Google Scholar, 45.Opi S. Péloponèse Jr., J.M. Esquieu D. Campbell G. de Mareuil J. Walburger A. Solomiac M. Gregoire C. Bouveret E. Yirrell D.L. Loret E.P. J. Biol. Chem. 2002; 277: 35915-35919Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 46.Contreras X. Bennasser Y. Bahraoui E. Microbes Infect. 2004; 6: 1182-1190Crossref PubMed Scopus (20) Google Scholar). Initially, HeLa P4 cells were incubated with 1 μm extracellular synthetic Tat that was added to the culture medium. This is the minimum amount of Tat that can be used due to the avidity of the two specific anti-Tat antibodies used. After immunoblotting, we analyzed the blot using Scion Image. We compared the densitometric ratio between T:C and T:N (T, Tat; C, cytoplasmic fraction; N, nucleic fraction) for each Tat. Using this semiquantitative methodology, we found that both the truncated and the full-length form were taken up in equivalent quantities by the transfected cells and were found predominantly in the cytoplasmic region. Only a small portion of either Tat reached the nucleus to activate transcription, which agrees with previous data (18.Campbell G.R. Pasquier E. Watkins J. Bourgarel-Rey V. Peyrot V. Esquieu D. Barbier P. de Mareuil J. Braguer D. Kaleebu P. Yirrell D.L. Loret E.P. J. Biol. Chem. 2004; 279: 48197-48204Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 26.Ensoli B. Buonaguro L. Barillari G. Fiorelli V. Gendelman R. Morgan R.A. Wingfield P. Gallo R.C. J. Virol. 1993; 67: 277-287Crossref PubMed Google Scholar) (Fig. 2A). Importantly, both the truncated and the full-length form demonstrated a similar efficiency in their cytoplasmic and nuclear uptake. Thus, the C terminus does not play a role in the cellular uptake of Tat, which is again in agreement with previous data (47.Vivès E. J. Mol. Recognit. 2003; 16: 265-271Crossref PubMed Scopus (147) Google Scholar).FIGURE 2Trans-activation assay with HeLa P4 cells transfected with the HIV-1 long terminal repeat lacZ construct (40.Clavel F. Charneau P. J. Virol. 1994; 68: 1179-1185Crossref PubMed Google Scholar). A, entry of Tat into HeLa P4 cells. HXB2 (86) and HXB2 (100) enter the cells with the same efficiency. HeLa P4 cells were incubated with 1 μm Tat. Whole cell lysates were prepared 4 h post-treatment and analyzed by anti-Tat immunoblotting. This blot is representative of three independent experiments. T, 10 ng Tat; C, Cytoplasmic fraction; N, nucleic fraction. The analysis of this blot is discussed in the text. B, trans-activation was measured with the different Tat variants at six concentrations. HXB2 (100) is represented by the light gray bars, and HXB2 (86) is represented by the dark gray bars. Without Tat,β-galactosidase was expressed at a basal level, which was used as a control (white bar). B, absorbance values measured at 405 nm; B0, background β-galactosidase expressed by HeLa P4 cells in Dulbecco's modified Eagle's medium supplemented with 0.01% (w/v) protamine and 0.1% (w/v) BSA without Tat. HXB2 (100) had higher trans-activation activity at all concentrations. p values are displayed above each pairing and are the result of three independent experiments carried out in triplicate.View Large Image Figure ViewerDownload Hi-res image Download (PPT)When we tested for the trans-activation activity of the two variants, we found that at almost all concentrations tested there was a significant difference, with HXB2 (100) having significantly more trans-activation activity than HXB2 (86) (Fig. 2B), which agrees with previously published data (45.Opi S. Péloponèse Jr., J.M. Esquieu D. Campbell G. de Mareuil J. Walburger A. Solomiac M. Gregoire C. Bouveret E. Yirrell D.L. Loret E.P. J. Biol. Chem. 2002; 277: 35915-35919Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 48.Neuveut C. Jeang K.T. J. Virol. 1996; 70: 5572-5581Crossref PubMed Google Scholar). Only at 2 μm was there no statistical difference, but this could be due to a saturation of this system (p = 0.065). We also observed a dose-dependent response in the levels of β-galactosidase being quantified.Tat HXB2 (100) Induces Less Apoptosis than Tat HXB2 (86)—It is known that the addition of extracellular recombinant Tat HXB2 (86) to cultures of both primary CD4+ T cells and Jurkat T cell lines induces apoptosis and increases their sensitivity to apoptotic signals, thus contributing in part to the progressive loss of T cells associated with HIV-1 infection (17.Westendorp M.O. Frank R. Ochsenbauer C. Stricker K. Dhein J. Walczak H. Debatin K.M. Krammer P.H. Nature. 1995; 375: 497-500Crossref PubMed Scopus (910) Google Scholar, 49.Nardelli B. Gonzalez C.J. Schechter M. Valentine F.T. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7312-7316Crossref PubMed Scopus (82) Google Scholar, 50.Conti L. Rainaldi G. Matarrese P. Varano B. Rivabene R. Columba S. Sato A. Belardelli F. Malorni W. Gessani S. J. Exp. Med. 1998; 187: 403-413Crossref PubMed Scopus (134) Google Scholar, 51.Bartz S.R. Emerman M. J. Virol. 1999; 73: 1956-1963Crossref PubMed Google Scholar). We investigated the ability of Tat HXB2 (100) and Tat HXB2 (86) to induce apoptosis in primary CD4+ T cells. We used an assay in which primary CD4+ T cells were incubated with the Tat proteins at 2 μm, and the apoptotic effects were evaluated by flow cytometry after labeling the cells with propidium iodide and fluorescein isothiocyanate-conjugated annexin V (Fig. 3). Compared with the control, both HXB2 (100) and HXB2 (86) induced a significant level of apoptosis in these cells (p = 0.0005 and 0.0001, respectively) (Fig. 3B). There was also a significant difference between t" @default.
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• W2078009198 title "The C Terminus of HIV-1 Tat Modulates the Extent of CD178-mediated Apoptosis of T Cells" @default.
• W2078009198 cites W1468961 @default.
• W2078009198 cites W1505880863 @default.
• W2078009198 cites W1513727309 @default.
• W2078009198 cites W1524845305 @default.
• W2078009198 cites W1565329973 @default.
• W2078009198 cites W1576582350 @default.
• W2078009198 cites W1605519822 @default.
• W2078009198 cites W172276776 @default.
• W2078009198 cites W1885847713 @default.
• W2078009198 cites W1950862780 @default.
• W2078009198 cites W1965718105 @default.
• W2078009198 cites W1966319061 @default.
• W2078009198 cites W1966700067 @default.
• W2078009198 cites W1970622835 @default.
• W2078009198 cites W1970833400 @default.
• W2078009198 cites W1974393084 @default.
• W2078009198 cites W1981106016 @default.
• W2078009198 cites W1981680523 @default.
• W2078009198 cites W1994564363 @default.
• W2078009198 cites W1994674319 @default.
• W2078009198 cites W1998421163 @default.
• W2078009198 cites W1998439550 @default.
• W2078009198 cites W1999461028 @default.
• W2078009198 cites W2005133363 @default.
• W2078009198 cites W2014746936 @default.
• W2078009198 cites W2014964914 @default.
• W2078009198 cites W2020464641 @default.
• W2078009198 cites W2028348350 @default.
• W2078009198 cites W2030017358 @default.
• W2078009198 cites W2030892254 @default.
• W2078009198 cites W2045822022 @default.
• W2078009198 cites W2066591348 @default.
• W2078009198 cites W2067244066 @default.
• W2078009198 cites W2067592236 @default.
• W2078009198 cites W2073994420 @default.
• W2078009198 cites W2074192294 @default.
• W2078009198 cites W2074966917 @default.
• W2078009198 cites W2075587912 @default.
• W2078009198 cites W2075809552 @default.
• W2078009198 cites W2075882509 @default.
• W2078009198 cites W2076317085 @default.
• W2078009198 cites W2081200884 @default.
• W2078009198 cites W2083930032 @default.
• W2078009198 cites W2090040582 @default.
• W2078009198 cites W2092946683 @default.
• W2078009198 cites W2123671636 @default.
• W2078009198 cites W2139874251 @default.
• W2078009198 cites W2148595561 @default.
• W2078009198 cites W2149951876 @default.
• W2078009198 cites W2165194856 @default.
• W2078009198 cites W2167742988 @default.
• W2078009198 cites W2169649983 @default.
• W2078009198 cites W2172185515 @default.
• W2078009198 cites W2227001432 @default.
• W2078009198 cites W2314450929 @default.
• W2078009198 cites W2414676408 @default.
• W2078009198 cites W4211084625 @default.
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