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• W2055296721 abstract "Tat, the transactivator protein of human immunodeficiency virus-1, has the unusual capacity of being internalized by cells when present in the extracellular milieu. This property can be exploited for the cellular delivery of heterologous proteins fused to Tat both in cell culture and in living animals. Here we provide genetic and biochemical evidence that cell membrane heparan sulfate (HS) proteoglycans act as receptors for extracellular Tat uptake. Cells genetically defective in the biosynthesis of fully sulfated HS are selectively impaired in the internalization of recombinant Tat fused to the green fluorescent protein, as evaluated by both flow cytometry and functional assays. In wild type cells, Tat uptake is competitively inhibited by soluble heparin and by treatment with glycosaminoglycan lyases specifically degrading HS chains. Cell surface HS proteoglycans also mediate physiological internalization of Tat green fluorescent protein released from neighboring producing cells. In contrast to extracellular Tat uptake, both wild type cells and cells genetically impaired in proteoglycan synthesis are equally proficient in the extracellular release of Tat, thus indicating that proteoglycans are not required for this process. The ubiquitous distribution of HS proteoglycans is consistent with the efficient intracellular delivery of heterologous proteins fused with Tat to different mammalian cell types. Tat, the transactivator protein of human immunodeficiency virus-1, has the unusual capacity of being internalized by cells when present in the extracellular milieu. This property can be exploited for the cellular delivery of heterologous proteins fused to Tat both in cell culture and in living animals. Here we provide genetic and biochemical evidence that cell membrane heparan sulfate (HS) proteoglycans act as receptors for extracellular Tat uptake. Cells genetically defective in the biosynthesis of fully sulfated HS are selectively impaired in the internalization of recombinant Tat fused to the green fluorescent protein, as evaluated by both flow cytometry and functional assays. In wild type cells, Tat uptake is competitively inhibited by soluble heparin and by treatment with glycosaminoglycan lyases specifically degrading HS chains. Cell surface HS proteoglycans also mediate physiological internalization of Tat green fluorescent protein released from neighboring producing cells. In contrast to extracellular Tat uptake, both wild type cells and cells genetically impaired in proteoglycan synthesis are equally proficient in the extracellular release of Tat, thus indicating that proteoglycans are not required for this process. The ubiquitous distribution of HS proteoglycans is consistent with the efficient intracellular delivery of heterologous proteins fused with Tat to different mammalian cell types. human immunodeficiency virus-1 long terminal repeat heparan sulfate Chinese hamster ovary glycosaminoglycan chloramphenicol transferase green fluorescent protein fluorescence-activated cell sorter phosphate-buffered saline glutathione S-transferase The Tat protein of human immunodeficiency virus-1 (HIV-1)1 is a powerful transcriptional activator of the integrated viral genome. The protein binds to a highly structured RNA element located at the 5′ end of all viral transcripts (1Berkhout B. Silverman R.H. Jeang K.T. Cell. 1989; 59: 273-282Abstract Full Text PDF PubMed Scopus (513) Google Scholar), and from there it increases the rates of both transcriptional initiation and elongation from the long terminal repeat (LTR) promoter. These two functions are mediated by the interaction of Tat with nuclear proteins possessing chromatin remodeling activity (2Benkirane M. Chun R.F. Xiao H. Ogryzko V.V. Howard B.H. Nakatani Y. Jeang K.T. J. Biol. Chem. 1998; 273: 24898-24905Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar, 3Marzio G. Tyagi M. Gutierrez M.I. Giacca M. Proc. Natl. Acad. Sci. U. S. A. 1998; 23: 13519-13524Crossref Scopus (266) Google Scholar, 4Hottiger M.O. Nabel G.J. J. Virol. 1998; 72: 8252-8256Crossref PubMed Google Scholar) and with cellular kinases phosphorylating the C-terminal domain of RNA polymerase II (5Wei P. Garber M.E. Fang S.-M. Fisher W.H. Jones K.A. Cell. 1998; 92: 451-462Abstract Full Text Full Text PDF PubMed Scopus (1055) Google Scholar, 6Parada C.A. Roeder R.G. Nature. 1996; 384: 375-378Crossref PubMed Scopus (237) Google Scholar, 7Gold M.O. Yang X. Herrmann C.H. Rice A.P. J. Virol. 1998; 72: 4448-4453Crossref PubMed Google Scholar, 8Cujec T.P. Okamoto H. Fujinaga K. 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Campioni D. Rossi C. Albini A. Possati L. Rusnati M. Gazzanelli G. Benelli R. Masiello L. Sparacciari V. Presta M. Mannello F. Fontanini G. Barbanti-Brodano G. AIDS. 1996; 10: 701-710Crossref PubMed Scopus (42) Google Scholar); inhibits antigen-specific lymphocyte proliferation (28Subramanyam M. Gutheil W.G. Bachovchin W.W. Huber B.T. J. Immunol. 1993; 150: 2544-2553PubMed Google Scholar, 29Chirmule N. Than S. Khan S.A. Pahwa S. J. Virol. 1995; 69: 492-498Crossref PubMed Google Scholar, 30Viscidi R.P. Mayur K. Lederman H.M. Frankel A.D. Science. 1989; 246: 1606-1608Crossref PubMed Scopus (319) Google Scholar); and induces neurotoxicity in the central nervous system (31Sabatier J.M. Vives E. Mabrouk K. Benjouad A. Rochat H. Duval A. Hue B. Bahraoui E. J. Virol. 1991; 65: 961-967Crossref PubMed Google Scholar, 32Taylor J.P. Cupp C. Diaz A. Chowdhury M. Khalili K. Jimenez S.A. Amini S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9617-9621Crossref PubMed Scopus (90) Google Scholar, 33Philippon V. Vellutini C. Gambarelli D. Harkiss G. Arbuthnott G. Metzger D. Roubin R. Filippi P. Virology. 1994; 205: 519-529Crossref PubMed Scopus (133) Google Scholar, 34Weeks B.S. Lieberman D.M. Johnson B. Roque E. Green M. Loewenstein P. Oldfield E.H. Kleinman H.K. J. Neurosci. Res. 1995; 42: 34-40Crossref PubMed Scopus (68) Google Scholar, 35Hofman F.M. Dohadwala M.M. Wright A.D. Hinton D.R. Walker S.M. J. Neuroimmunol. 1994; 54: 19-28Abstract Full Text PDF PubMed Scopus (92) Google Scholar, 36Nath A. Psooy K. Martin C. Knudsen B. Magnuson D.S. Haughey N. Geiger J.D. J. Virol. 1996; 70: 1475-1480Crossref PubMed Google Scholar). Since a growing body of evidence exists that Tat could be released by producing cells (37Helland D.E. Welles J.L. Caputo A. Haseltine W.A. J. Virol. 1991; 65: 4547-4549Crossref PubMed Google Scholar, 38Thomas C.A. Dobkin J. Weinberger O.K. J. Immunol. 1994; 153: 3831-3839PubMed Google Scholar, 39Chang H.C. Samaniego F. Nair B.C. Buonaguro L. Ensoli B. AIDS. 1997; 11: 1421-1431Crossref PubMed Scopus (393) Google Scholar, 40Re M.C. Furlini G. Vignoli M. Ramazzotti E. Roderigo G. De Rosa V. Zauli G. Lolli S. Capitani S. La Placa M. J. Acquired Immune Deficiency Syndrome. 1995; 10: 408-416Crossref Scopus (116) Google Scholar), it is likely that some of the above-mentioned effects of extracellular Tat could have important implications for the pathogenesis of HIV disease in an autocrine or paracrine fashion. Besides its interaction with cell surface receptors and the consequent activation of intracellular signal transduction pathways (41Albini A. Soldi R. Giunciuglio D. Giraudo E. Benelli R. Primo L. Noonan D. Salio M. Camussi G. Rockl W. Bussolino F. Nat. Med. 1996; 2: 1371-1375Crossref PubMed Scopus (349) Google Scholar, 42Albini A. Ferrini S. Benelli R. Sforzini S. Giunciuglio D. Aluigi M.G. Proudfoot A.E.I. Alouani S. Wells T.N.C. Mariani G. Rabin R.L. Farber J.M. Noonan D.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13153-13158Crossref PubMed Scopus (245) Google Scholar, 43Mitola S. Sozzani S. Luini W. Primo L. Borsatti A. Weich H. Bussolino F. Blood. 1997; 90: 1365-1372Crossref PubMed Google Scholar), most of the activities of extracellular Tat are mediated by its unique property of being rapidly internalized by a variety of cell types, as originally shown more than 10 years ago (44Green M. Loewenstein P.M. Cell. 1988; 55: 1179-1188Abstract Full Text PDF PubMed Scopus (1291) Google Scholar, 45Frankel A.D. Pabo C.O. Cell. 1988; 55: 1189-1193Abstract Full Text PDF PubMed Scopus (2353) Google Scholar, 46Mann D.A. Frankel A.D. EMBO J. 1991; 10: 1733-1739Crossref PubMed Scopus (451) Google Scholar). One of the consequences of Tat internalization is the activation of cellular transcription factor NF-κB; several of the pleiotropic functions of extracellular Tat could be mediated by this pathway (47Demarchi F. Gutierrez M.I. Giacca M. J. Virol. 1999; 73: 7080-7086Crossref PubMed Google Scholar, 48Demarchi F. d'Adda di Fagagna F. Falaschi A. Giacca M. J. Virol. 1996; 70: 4427-4437Crossref PubMed Google Scholar, 49Biswas D.K. Salas T.R. Wang F. Ahlers C.M. Dezube B.J. Pardee A.B. J. Virol. 1995; 69: 7437-7444Crossref PubMed Google Scholar, 50Westendorp M.O. Shatrov V.A. Schulze-Osthoff K. Frank R. Kraft M. Los M. Krammer P.H. Droge W. Lehmann V. EMBO J. 1995; 14: 546-554Crossref PubMed Scopus (367) Google Scholar). The uptake, internalization, and nuclear translocation of extracellular Tat can also be exploited as a biotechnological tool for intracellular protein delivery. Chemical cross-linking of Tat peptides with heterologous proteins (51Fawell S. Seery J. Daikh Y. Moore C. Chen L.L. Pepinsky B. Barsoum J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 664-668Crossref PubMed Scopus (1107) Google Scholar) or, more efficiently, production of recombinant proteins containing the protein transduction domain of Tat (52Nagahara H. Vocero-Akbani A.M. Snyder E.L. Ho A. Latham D.G. Lissy N.A. Becker-Hapak M. Ezhevsky S.A. Dowdy S.F. Nat. Med. 1998; 4: 1449-1452Crossref PubMed Scopus (887) Google Scholar, 53Schwarze S.R. Ho A. Vocero-Akbani A. Dowdy S.F. Science. 1999; 285: 1569-1572Crossref PubMed Scopus (2210) Google Scholar) facilitate the intracellular delivery of these proteins. In particular, it has been recently reported that the intraperitoneal injection of the 120-kDa β-galactosidase protein fused to 11 amino acids encompassing the arginine-rich region of Tat results in delivery of the fusion protein to virtually all tissues in mice (53Schwarze S.R. Ho A. Vocero-Akbani A. Dowdy S.F. Science. 1999; 285: 1569-1572Crossref PubMed Scopus (2210) Google Scholar). Despite the large body of evidence available about the functions of extracellular Tat and its recent use, a biotechnological vector for protein transduction, the cellular mechanisms for Tat uptake and internalization, are still largely unexplored. Inspired by the observation that Tat can enter a variety of different cell types bothin vitro and in vivo and by the possibility of modulating several of the biological properties of extracellular Tat by soluble heparin (54Rusnati M. Coltrini D. Oreste P. Zoppetti G. Albini A. Noonan D. d'Adda di Fagagna F. Giacca M. Presta M. J. Biol. Chem. 1997; 272: 11313-11320Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 55Rusnati M. Tulipano G. Urbinati C. Tanghetti E. Giuliani R. Giacca M. Ciomei M. Corallini A. Presta M. J. Biol. Chem. 1998; 273: 16027-16037Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 56Rusnati M. Tulipano G. Spillmann D. Tanghetti E. Oreste P. Zoppetti G. Giacca M. Presta M. J. Biol. Chem. 1999; 274: 28198-28205Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar), we started investigating the role of cell surface proteoglycans in the process of Tat translocation through the plasma membrane. Here we provide genetic and biochemical evidences that cell membrane heparan sulfate (HS) proteoglycans are the receptors for extracellular Tat internalization. The wild type CHO K1 and CHO K1 mutants deficient in proteoglycan biosynthesis (57Rostand K.S. Esko J.D. Infect. Immun. 1997; 65: 1-8Crossref PubMed Google Scholar) were obtained from the American Type Culture Collection (Manassas, VA). The pgs A-745 cell line does not produce detectable levels of proteoglycans since it lacks xylosyltransferase, an enzyme necessary for the initiation of glycosaminoglycan (GAG) synthesis. Mutant pgs B-618 has a defect in the galactosyltransferase-I enzyme gene and produces about 15% the amount of proteoglycans synthesized by wild type cells. Cell line pgs E-606 is partially deficient in HS N-sulfotransferase and produces an undersulfated form of HS proteoglycan. The cell line pgs D-677 has a single mutation that affects bothN-acetylglucosaminyltransferase and glucuronosyltransferase activities, which are necessary for the polymerization of HS disaccharide chains and does not synthesize any HS proteoglycan. This mutant cell line also produces approximately three times more chondroitin sulfate than wild type cells. Finally, mutant cell line pgs C-605 has a defect in a saturable, 4-acetamido-4-isothiocyanostilbene-2,2′-disulfonic acid-sensitive transport system required for sulfate uptake. Despite a dramatic reduction in 35SO4 incorporation, the mutant synthesizes sulfated heparan and chondroitin chains by using the inorganic sulfate produced from oxidative metabolism of cysteine and methionine (58Esko J.D. Elgavish A. Prasthofer T. Taylor W.H. Weinke J.L. J. Biol. Chem. 1986; 261 (15333): 15725Abstract Full Text PDF PubMed Google Scholar). HL3T1 cells (a HeLa derivative containing an integrated LTR-CAT cassette) were a kind gift of B. Felber. Cell lines constitutively expressing Tat-green fluorescent protein (GFP) were obtained by selection for neomycin-resistant clones with 500 μg/ml G418 (Life Technologies, Inc.) after transfection of pCDNA3-Tat-GFP; single clones were collected and propagated. Recombinant glutathioneS-transferase (GST)-Tat containing the 86-amino acid Tat protein of HIV-1 clone HXB2 fused to glutathioneS-transferase and its mutated derivative containing alanine substitutions at arginines 49, 52, 53, 55, 56, and 57, GST-Tat R (49Biswas D.K. Salas T.R. Wang F. Ahlers C.M. Dezube B.J. Pardee A.B. J. Virol. 1995; 69: 7437-7444Crossref PubMed Google Scholar, 50Westendorp M.O. Shatrov V.A. Schulze-Osthoff K. Frank R. Kraft M. Los M. Krammer P.H. Droge W. Lehmann V. EMBO J. 1995; 14: 546-554Crossref PubMed Scopus (367) Google Scholar, 51Fawell S. Seery J. Daikh Y. Moore C. Chen L.L. Pepinsky B. Barsoum J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 664-668Crossref PubMed Scopus (1107) Google Scholar, 52Nagahara H. Vocero-Akbani A.M. Snyder E.L. Ho A. Latham D.G. Lissy N.A. Becker-Hapak M. Ezhevsky S.A. Dowdy S.F. Nat. Med. 1998; 4: 1449-1452Crossref PubMed Scopus (887) Google Scholar, 53Schwarze S.R. Ho A. Vocero-Akbani A. Dowdy S.F. Science. 1999; 285: 1569-1572Crossref PubMed Scopus (2210) Google Scholar, 54Rusnati M. Coltrini D. Oreste P. Zoppetti G. Albini A. Noonan D. d'Adda di Fagagna F. Giacca M. Presta M. J. Biol. Chem. 1997; 272: 11313-11320Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 55Rusnati M. Tulipano G. Urbinati C. Tanghetti E. Giuliani R. Giacca M. Ciomei M. Corallini A. Presta M. J. Biol. Chem. 1998; 273: 16027-16037Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 56Rusnati M. Tulipano G. Spillmann D. Tanghetti E. Oreste P. Zoppetti G. Giacca M. Presta M. J. Biol. Chem. 1999; 274: 28198-28205Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 57Rostand K.S. Esko J.D. Infect. Immun. 1997; 65: 1-8Crossref PubMed Google Scholar)A, were produced and purified by glutathione-agarose affinity chromatography as already described (3Marzio G. Tyagi M. Gutierrez M.I. Giacca M. Proc. Natl. Acad. Sci. U. S. A. 1998; 23: 13519-13524Crossref Scopus (266) Google Scholar, 48Demarchi F. d'Adda di Fagagna F. Falaschi A. Giacca M. J. Virol. 1996; 70: 4427-4437Crossref PubMed Google Scholar). The plasmid expressing GST-Tat-GFP was obtained by cloning a polymerase chain reaction-amplified fragment into the Bam HI andEco RI sites of the commercial vector pGEX2T (Amersham Pharmacia Biotech). The fragment was obtained by the separate amplifications of HXB2 Tat using primers 5′-GTGGATCCATGGAGCCAGTAGATCCTA-3′ and 5′-CCCTTGCTCACCATAAGCTTTTCCTTCGGGCC-3′ and of enhanced green fluorescent protein (GFP) using primers 5′-GGCCCGAAGGAAAAGCTTATGGTGAGCAAGGG-3′ and 5′-GGCGAATTCTCTAGAGTCGCGGCCGCTTTA-3′. Templates for amplification were plasmid pGEX2T-Tat (3Marzio G. Tyagi M. Gutierrez M.I. Giacca M. Proc. Natl. Acad. Sci. U. S. A. 1998; 23: 13519-13524Crossref Scopus (266) Google Scholar) and pEGFP-N1 (CLONTECH, Palo Alto CA) respectively. The two amplification products contain complementary sequences at the 3′ and 5′ regions of the coding strands of Tat and GFP, respectively. They were gel purified, mixed, annealed, and amplified with the external primers to obtain a single amplification products, which contains Bam HI andEco RI sites at the extremities. To study the kinetics of recombinant Tat internalization, HL3T1 cells were seeded in 24-well or 10-cm-diameter dishes at the density of 1–2 × 104cells/cm2 in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. After 24 h, cell cultures were washed twice and incubated for an additional 24 h in fresh medium containing 10% fetal calf serum, 100 μm chloroquine, and recombinant Tat protein. Incubation in the presence of chloroquine favors Tat uptake by modifying the pH of endolysosomal vesicles and preventing protein degradation (46Mann D.A. Frankel A.D. EMBO J. 1991; 10: 1733-1739Crossref PubMed Scopus (451) Google Scholar). After 24 h, the medium was changed to Dulbecco's modified Eagle's medium, 10% fetal calf serum, and cells were incubated for an additional 24 h. At the end of incubation, cells were extracted, and the amount of CAT protein present in the cell extracts was determined by the CAT enzyme-linked immunosorbent assay kit (Roche Molecular Biochemicals) according to the manufacturer's instructions. Alternatively, cells were collected and analyzed by FACS (see below). Recombinant GST-Tat was labeled with 125I (17 Ci/mg, PerkinElmer Life Sciences) using iodogen (Pierce) to a specific radioactivity of 400 cpm/fmol as described previously (55Rusnati M. Tulipano G. Urbinati C. Tanghetti E. Giuliani R. Giacca M. Ciomei M. Corallini A. Presta M. J. Biol. Chem. 1998; 273: 16027-16037Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). HL3T1 cells were seeded in 24-well dishes at the density of 45,000 cells/cm2 in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. After 24 h, cell cultures were washed twice with Tris-buffered saline and incubated for 16 h at 37 °C in binding medium (serum-free medium containing 0.15% gelatin and 20 mmHepes buffer, pH 7.5) with the addition of 20 ng/ml [125I]GST-Tat plus 200 ng/ml unlabeled GST-Tat as a carrier and in the presence of 100 μm chloroquine. After incubation, medium was removed, cells were washed three times with cold Tris-buffered saline and lysed by incubation with 0.5% Triton X-100 in 0.1 m sodium phosphate, pH 8.1. Radioactivity of the cell lysates was measured, and nonspecific binding, determined in the presence of a 200-fold molar excess of unlabeled GST-Tat (4 μg/ml), was subtracted. For immunocytochemistry, HL3T1 cells were grown to about 60% confluency on glass coverslips. The GST-Tat protein (1 μg/ml) was added to the cell culture medium in the presence of 100 μm chloroquine. After different time intervals, cells were washed 6 times with PBS and fixed with a cold acetone:methanol mixture (50:50) for 15 min. Cells were then washed 3 times with PBS containing 0.2% Triton X-100 (PBS-Triton X-100) and then 5 times with PBS for 5 min each. Cells were then incubated with an anti-Tat monoclonal antibody (ADP352/NT3, obtained from the Medical Research Council repository for AIDS research) for 1 h, washed 5 times with warm PBS (25 to 28 °C), and incubated with rhodamine-conjugated secondary antibody (Sigma) for 30 min. Cells were then washed three times with warm PBS/Triton X-100 and with warm PBS for 5 min each time. For each immunostaining, one coverslip was incubated in secondary antibody alone as a negative control for background immunofluorescence. Nuclei were counterstained with Hoechst 33342 (10 μg/ml in PBS) for 5 min, and coverslips were washed three times with PBS and mounted on glass slides. Slides were observed using Zeiss Axiophot fluorescence microscope. To analyze GFP-Tat internalization by cell cytometry, cells were washed four times with PBS, trypsinized, again washed with PBS, and analyzed with a FACScan flow cytometer (Becton Dickinson). A total of 10,000 events per sample were considered. The soluble GAGs (heparin, from porcine intestinal mucosa; chondroitin sulfate A, from bovine trachea; chondroitin sulfate B, from porcine intestinal mucosa; and chondroitin sulfate C, from shark cartilage) and dextran sulfate (molecular weight, 5000) were all purchased from Sigma. In the FACS experiments with soluble GAG analogues, 5 × 105 CHO K1 cells were incubated in fresh culture medium with the addition of 1 μg/ml GST-Tat-GFP protein, 100 μm chloroquine, and the appropriate amount of GAG dissolved in PBS. Inhibition of Tat transactivation by soluble GAGs was studied in 3 × 105 CHO K1 cells transfected with 1 μg of pBlue-LTR-CAT (containing an LTR-CAT cassette, a kind gift of B. Berkhout) using Lipofectin (Life Technologies, Inc.). Forty-eight hours after transfection, cells were treated with different concentrations of GAGs along with 1 μg/ml GST-Tat-GFP and 100 μmchloroquine in fresh culture medium for 14 h. After this time period, cells were washed four times with PBS, and fresh culture medium was added. CAT assays were performed after 36 h according to standard protocols (59Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) and after normalization for transfection efficiency. Each experimental point was performed in triplicate. Enzymatic treatment with GAG lyases was performed as described (60Summerford C. Samulski R.J. J. Virol. 1998; 72: 1438-1445Crossref PubMed Google Scholar). Briefly, 5 × 105 CHO K1 cells were incubated with the GAG lyases (Sigma; dissolved in PBS) in PBS containing 0.1% bovine serum albumin, 0.2% gelatin, and 0.1% glucose for 40 min at 37 °C in a CO2incubator. Cells were then washed gently 6 times with PBS and incubated in Ham's F-10 medium without serum in the presence of 1 μg/ml GST-Tat-GFP protein and 100 μm chloroquine for 5 h. Cells were then washed 4 times with PBS, trypsinized, washed again with complete Ham's F-10 medium, resuspended, and used for FACS analysis. Each experimental point was performed in triplicate. For CAT assays, 1 × 106 cells were seeded in 10-cm-diameter dishes. After 24 h, cells were transfected with either pBlue-LTR-CAT (1 μg) alone or together with pCDNA3-Tat-GFP (1 μg). The latter plasmid was obtained by recovering the Tat-GFP cassette of plasmid pGEX2T-Tat-GFP using Eco RI andBam HI and cloning into the respective sites of vector pCDNA3 (Invitrogen, Carlsbad, CA). In this plasmid, Tat-GFP is expressed under the control of the cytomegalovirus promoter. After lipofection, cells were washed twice with PBS and incubated in fresh culture medium for additional 24 h. Cells transfected with pBlue-LTR-CAT alone were supplied with 1 μg/ml GST-Tat-GFP in the presence of 100 μm chloroquine and incubated for 24 h. Cells were then washed twice with PBS and incubated for additional 24 h in fresh medium. Finally, cells were scraped off the dishes using a rubber policeman, and cell extracts were used for CAT assays. Wild type CHO K1 cells, the mutated pgs A-745 clone, and HeLa cells were transfected by Lipofectin with 1 μg/ml pBlue-LTR-CAT. After 48 h, cells were trypsinized and plated in equal number with CHO and A-745 cell clones expressing Tat-GFP, with the combinations reported in Fig. 7A. Cells were incubated in complete medium containing 100 μmchloroquine. Seventy-two hours later, cells were scraped off the plate with a rubber policeman, and cell lysates were analyzed for CAT activity. To explore the mechanisms of HIV-1 LTR transactivation by Tat, in the last few years we have taken advantage of the property of a GST-Tat fusion protein to enter cells when added to the cell culture medium (3Marzio G. Tyagi M. Gutierrez M.I. Giacca M. Proc. Natl. Acad. Sci. U. S. A. 1998; 23: 13519-13524Crossref Scopus (266) Google Scholar, 48Demarchi F. d'Adda di Fagagna F. Falaschi A. Giacca M. J. Virol. 1996; 70: 4427-4437Crossref PubMed Google Scholar). This protein contains the first 86 amino acids of Tat fused at the C terminus of the GST protein (∼34 kDa total). The kinetics of HIV-1 LTR transactivation by GST-Tat and by a fusion protein additionally containing the GFP at the C terminus (∼60 kDa total) are shown in Fig. 1 A. LTR transactivation starts to be observed a few hours after the addition of GST-Tat to the culture medium of HL3T1 cells (containing an integrated LTR-CAT reporter gene that is silent in the absence of stimulation) and peaks at 10–15 h. The kinetics of LTR transactivation by the GST-Tat-GFP protein is delayed by a few hours but still reaches the same levels after 15–20 h. These data are consistent with previous findings showing a peak of LTR-CAT mRNA expression ∼5 h after the addition of exogenous GST-Tat protein to the culture medium (48Demarchi F. d'Adda di Fagagna F. Falaschi A. Giacca M. J. Virol. 1996; 70: 4427-4437Crossref PubMed Google Scholar). As shown in Fig. 1 B, LTR transactivation is already appreciable at a concentration of GST-Tat protein equal to 50 ng/ml (∼1.5 nm) and reaches a plateau at 200 ng/ml. A similar dose response curve is evident for the GST-Tat-GFP protein when considering the molar differences between the two preparations. In contrast to these recombinant proteins, which contain the wild type Tat amino acid sequence, fusion of GST to the Tat R (49Biswas D.K. Salas T.R. Wang F. Ahlers C.M. Dezube B.J. Pardee A.B. J. Virol. 1995; 69: 7437-7444Crossref PubMed Google Scholar, 50Westendorp M.O. Shatrov V.A. Schulze-Osthoff K. Frank R. Kraft M. Los M. Krammer P.H. Droge W. Lehmann V. EMBO J. 1995; 14: 546-554Crossref PubMed Scopus (367) Google Scholar, 51Fawell S. Seery J. Daikh Y. Moore C. Chen L.L. Pepinsky B. Barsoum J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 664-668Crossref PubMed Scopus (1107) Google Scholar, 52Nagahara H. Vocero-Akbani A.M. Snyder E.L. Ho A. Latham D.G. Lissy N.A. Becker-Hapak M. Ezhevsky S.A. Dowdy S.F. Nat. Med. 1998; 4: 1449-1452Crossref PubMed Scopus (887) Google Scholar, 53Schwarze S.R. Ho A. Vocero-Akbani A. Dowdy S.F. Science. 1999; 285: 1569-1572Crossref PubMed Scopus (2210) Google Scholar, 54Rusnati M. Coltrini D. Oreste P. Zoppetti G. Albini A. Noonan D. d'Adda di Fagagna F. Giacca M. Presta M. J. Biol. Chem. 1997; 272: 11313-11320Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 55Rusnati M. Tulipano G. Urbinati C. Tanghetti E. Giuliani R. Giacca M. Ciomei M. Corallini A. Presta M. J. Biol. Chem. 1998; 273: 16027-16037Abstract" @default.
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• W2055296721 title "Internalization of HIV-1 Tat Requires Cell Surface Heparan Sulfate Proteoglycans" @default.
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