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• W2021986727 abstract "Human immunodeficiency virus type 1 (HIV-1) infection is associated with severe psoriasis, B cell lymphoma, and Kaposi's sarcoma. A deregulated production of interleukin-6 (IL6) has been implicated in the pathogenesis of these diseases. The molecular mechanisms underlying the abnormal IL6 secretion of HIV-1-infected cells may include transactivation of the IL6 gene by HIV-1. Here we report the molecular mechanisms of Tat activity on the expression of the IL6 gene. By using 5′ deletion mutants of pIL6Pr-CAT and using IL6:HIV-1-LTR hybrid constructs where discrete regions of the IL6 promoter replaced the TAR sequence in HIV-1 LTR, we identified a short sequence of the 5′-untranslated region of the IL6 mRNA that is required for Tat to trans-activate the IL6 promoter. This sequence acquires a stem-loop structure and includes a UCU sequence that binds to Tat and is necessary for full trans-activation. In addition, we provide the evidence that Tat can function by enhancing the CAAT enhancer-binding protein (C/EBP) DNA binding activity and is able to complex with in vitro translated C/EBPβ, which is a major mediator of IL6 promoter function. By using the yeast two-hybrid system and immunoprecipitation, we observed that the interaction of Tat with C/EBP proteins also occurred in vivo. The data are consistent with the possibility that Tat may function on heterologous genes by interacting with RNA structures possibly present in a large number of cellular and viral genes. In addition, Tat may function by protein-protein interactions, leading to the generation of heterodimers with specific transcription factors. Human immunodeficiency virus type 1 (HIV-1) infection is associated with severe psoriasis, B cell lymphoma, and Kaposi's sarcoma. A deregulated production of interleukin-6 (IL6) has been implicated in the pathogenesis of these diseases. The molecular mechanisms underlying the abnormal IL6 secretion of HIV-1-infected cells may include transactivation of the IL6 gene by HIV-1. Here we report the molecular mechanisms of Tat activity on the expression of the IL6 gene. By using 5′ deletion mutants of pIL6Pr-CAT and using IL6:HIV-1-LTR hybrid constructs where discrete regions of the IL6 promoter replaced the TAR sequence in HIV-1 LTR, we identified a short sequence of the 5′-untranslated region of the IL6 mRNA that is required for Tat to trans-activate the IL6 promoter. This sequence acquires a stem-loop structure and includes a UCU sequence that binds to Tat and is necessary for full trans-activation. In addition, we provide the evidence that Tat can function by enhancing the CAAT enhancer-binding protein (C/EBP) DNA binding activity and is able to complex with in vitro translated C/EBPβ, which is a major mediator of IL6 promoter function. By using the yeast two-hybrid system and immunoprecipitation, we observed that the interaction of Tat with C/EBP proteins also occurred in vivo. The data are consistent with the possibility that Tat may function on heterologous genes by interacting with RNA structures possibly present in a large number of cellular and viral genes. In addition, Tat may function by protein-protein interactions, leading to the generation of heterodimers with specific transcription factors. Human immunodeficiency virus type 1 (HIV-1) 1The abbreviations used are:HIV-1human immunodeficiency virus type 1IL6interleukin-6TARtransactivating responsive elementC/EBPCAAT enhancer-binding proteinEMSAelectrophoretic mobility shift assayCATchloramphenicol acetyltransferaseLTRlong terminal repeatGSTglutathioneS-transferasePBSphosphate-buffered salineDTTdithiothreitolPMSFphenylmethylsulfonyl fluoridex-gal5-bromo-4-chloro-3-indolyl β-d-galactoside is the etiologic agent for acquired immunodeficiency syndrome (AIDS) and causes various clinical and immunological abnormalities, including activation of polyclonal B cells that manifests as hypergammaglobulinemia and autoantibody production, lymphadenopathy, Kaposi's sarcoma, and lymphoma of the B cell phenotype (1Fauci A.S. Macher A.M. Longo D.L. Lane H.C. Rook A.H. Masur H. Gelman E.P. Ann. Intern. Med. 1984; 100: 92-103Google Scholar, 2Beral V. Peterman A. Berrkelman R.L. Jaffe H.W. Lancet. 1990; 335: 123-128Google Scholar, 3Levine A.M. Blood. 1992; 80: 8-15Google Scholar). Studies on small cohorts of subjects who were exposed to HIV-1 and did not develop HIV-1 infection and individuals who harbored HIV-1 but remained disease-free for long periods (4Nowak M.A. Anderson R.M. McLeann A.R. Wolfs T.F.W. Goudsmit J. May R.M. Science. 1991; 254: 963-969Google Scholar, 5Biggar R.J. AIDS. 1990; 4: 1059-1065Google Scholar) strongly suggest that the development of AIDS may depend on a dynamic interplay between viral and host cellular gene products. Accordingly, in HIV-1-infected subjects there is a deregulated production of cytokines, including the proinflammatory interleukin-6 (IL6) (6Nakajima N. Martinez-Maza O. Hirano T. Breen E.C. Nishanian P.G. Sazar-Gonzalez J.F. Fahey J.L. Kishimoto T. J. Immunol. 1989; 142: 144-155Google Scholar), which affects the growth and differentiation of lymphoid and mesenchymal cells (7Kishimoto T. Akira S. Taga T. Science. 1992; 258: 593-597Google Scholar) and may contribute to the development of the clinical features of AIDS. Accordingly, IL6 gene transcription is induced in cells infected by HIV-1 (8Breen E.C. Rezal A.R. Nakajima K. Beall G.N. Mitsuyasu R.T. Hirano T. Kishimoto T. Martinez-Maza O. J. Immunol. 1990; 144: 480-487Google Scholar), and increased levels of IL6 have been reported in serum and cerebral spinal fluid of HIV-1-infected patients (9Gallo P., K. Frei K. Rordorf C. Lazdins J. Tavolato B. Fontana A. J. Neuroimmunol. 1989; 23: 109-115Google Scholar).The Tat protein of HIV-1 is required for efficient viral gene expression (10Rosen C. Sodroski J.G. Haseltine W.A. Cell. 1985; 41: 813-823Google Scholar, 11Sharp P.A. Marciniak R.A. Cell. 1989; 59: 229-230Google Scholar, 12Gatignol A. Buckler-White A. Berkhout B. Jeang K.-T. Science. 1991; 251: 1597-1600Google Scholar, 13Cullen B.R. Cell. 1993; 73: 417-420Google Scholar, 14Rice A.P. Matthews M.B. Nature. 1988; 332: 551-555Google Scholar, 15Berkhout B. Silverman R.H. Jeang K.T. Cell. 1989; 9: 273-282Google Scholar). Tat increases the initiation of transcription from the HIV-1 LTR (14Rice A.P. Matthews M.B. Nature. 1988; 332: 551-555Google Scholar) and affects RNA processing and utilization by interacting with a transactivating responsive element (TAR) located between nucleotides +1 and + 44 with respect to the initiation site (+1) of viral transcription (16Lapsia M.F. Rice A.P. Matthews M.B. Cell. 1989; 59: 283-292Google Scholar, 17Arya S.K. Guo C. Josephs S.F. Wong-Staal F. Science. 1985; 229: 69-73Google Scholar). TAR contains a 6-nucleotide loop and a 3-nucleotide pyrimidine bulge that are essential for Tat activity (18Sodroski J. Patarca R. Rosen C. Wong-Staal F. Haseltine W.A. Science. 1985; 229: 74-77Google Scholar, 19Garcia J.A. Harrich D. Soultanakis E.W.F. Mitsuyasu R. Gaynor R.B. EMBO J. 1989; 8: 765-778Google Scholar, 20Frankel A.D. Pabo C.O. Cell. 1988; 55: 1189-1193Google Scholar, 21Dingwall C. Ernberg I. Gait M.J. Green S.M. Heaphy S. Karn J. Lowe A.D. Singh M. Skinner M.A. Valerio R. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6925-6929Google Scholar). Tat binds to the bulge and appears to require cellular factors binding to the loop sequence to efficiently transactivate the HIV-1 LTR (22Roy S. Delling U. Chen C.H. Rosen C.A. Sonenberg N. Genes & Dev. 1990; 4: 1365-1373Google Scholar, 23Nelbock P. Dillon P.J. Perkins A. Rosen C.A. Science. 1990; 248: 1650-1653Google Scholar, 24Desai K. Loewestein P.M. Green M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8875-8879Google Scholar). In addition, Tat interacts with upstream regulatory DNA sequences circumscribed within the NF-κB/Sp1 sites of the HIV-1 promoter (25Berkhout B. Gatignol A. Rabson A.B. Jeang K.T. Cell. 1990; 62: 757-767Google Scholar) and with host cell proteins (12Gatignol A. Buckler-White A. Berkhout B. Jeang K.-T. Science. 1991; 251: 1597-1600Google Scholar, 24Desai K. Loewestein P.M. Green M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8875-8879Google Scholar). The 86-amino acid-long Tat contains a highly conserved cysteine-rich region, which mediates the formation of metal-linked dimers in vitro and is essential for Tat function (16Lapsia M.F. Rice A.P. Matthews M.B. Cell. 1989; 59: 283-292Google Scholar, 17Arya S.K. Guo C. Josephs S.F. Wong-Staal F. Science. 1985; 229: 69-73Google Scholar, 18Sodroski J. Patarca R. Rosen C. Wong-Staal F. Haseltine W.A. Science. 1985; 229: 74-77Google Scholar). A conserved basic region with 6 arginines and 2 lysines in nine residues, stretching from amino acid 47 to 58, is crucial for nuclear localization, mediates the specific binding of Tat to TAR RNA, and is required for the full activity of Tat (26Shibuya H. Irie K. Ninomiya-Tsuji J. Goebl M. Taniguchi T. Matsumoto K. Nature. 1992; 357: 700-704Google Scholar, 27Dang C.V. Lee W.M.F. J. Biol. Chem. 1989; 264: 18019-18023Google Scholar, 28Siomi H. Shida H. Maki M. Hatanaka M. J. Virol. 1990; 64: 1803-1807Google Scholar, 29Weeks K.M. Ampe C. Schultz S.C. Steitz T.A. Crothers D.M. Science. 1990; 249: 1281-1285Google Scholar).In addition to its role in HIV-1 transcription, Tat may participate in the development of AIDS by modulating the expression of heterologous genes. In support of this possibility, Tat has been shown to increase the expression of cellular genes, such as the IL6 (30Scala G. Ruocco M.R. Ambrosino C. Mallardo M. Giordano V. Baldassarre F. Dragonetti E. Quinto I. Venuta S. J. Exp. Med. 1994; 179: 961-971Google Scholar) and tumor necrosis factor-β genes (31Sastry K.J. Reddy R.H.R. Pandita R. Totpal K. Aggarwal B.B. J. Biol. Chem. 1990; 265: 20091-20093Google Scholar, 32Buonaguro L. Barillari G. Chang H.K. Bohan C.A. Kao V. Morgan R. Gallo R.C. Ensoli B. J. Virol. 1992; 68: 2677-2682Google Scholar), and to activate the life cycle of some AIDS-associated viruses (33Chowdhury M. Taylor J.P. Chang C.F. Rappaport J. Khalili K. J. Virol. 1992; 66: 7355-7361Google Scholar). The mechanisms of the Tat-mediated activation of non-HIV-1 genes are obscure. Here, we describe the mechanisms for Tat-mediated induction of the IL6 gene expression. We find that Tat is tethered to the IL6 transcription start site by specific binding to a UCU sequence present in the stem-loop structure of IL6 leader RNA. Tat physically interacts with C/EBPβ and increases selectively the nuclear pool of C/EBP factors binding to the C/EBPcis sequence in the IL6 promoter. This interaction was confirmed to occur in vivo by immunoprecipitation and by using the yeast two-hybrid system.DISCUSSIONDespite the intensive investigation of the immunopathogenesis of AIDS, many questions concerning the molecular mechanisms of HIV-1 primary infection and progression remain unanswered (5Biggar R.J. AIDS. 1990; 4: 1059-1065Google Scholar, 51Pantaleo G. Graziosi C. Fauci A.S. N. Engl. J. Med. 1993; 328: 327-335Google Scholar, 52Paul W.E. Cell. 1995; 82: 177-182Google Scholar). Recently, the identification of cohorts of HIV-exposed individuals who remain free of infection over a long period of viral exposure (53Salk J. Bretscher P.A. Salk P.M. Clerici M. Shearer G.M. Science. 1993; 260: 1270-1272Google Scholar) as well as the existence of a small subgroup of HIV-1-infected subjects who are long term nonprogressors were described (54Pantaleo G. Menzo S. Vaccerezze M. Graziosi C. Cohen O.J. Demorest J.F. Montefiori D. Orenstein J.M. Fox C. Schrager L.K. Fauci A.S. N. Engl. J. Med. 1995; 332: 209-216Google Scholar). Together with recent reports on viral life cycle (55Wey X. Ghosh S.K. Taylor M.E. Johnson V.A. Emini E.A. Deutsch P. Lifson J.D. Bonhoeffer S. Nowak M.A. Hahn B.H. Saag M.S. Shaw G.M. Nature. 1995; 373: 117-122Google Scholar, 56Ho D. Neumann A.U. Perelson A.S. Chen W. Leonard J.M. Markowitz M. Nature. 1995; 373: 123-126Google Scholar), the above evidence argues that HIV infection and disease progression may ultimately result from a complex interplay between viral and host cellular factors involved in the immunological response to the viral infection and in the clinical evolution of AIDS.HIV-1 Tat is a potent transactivator of HIV-1 LTR, acting on nascent TAR RNA and promoting full-length gene transcription (10Rosen C. Sodroski J.G. Haseltine W.A. Cell. 1985; 41: 813-823Google Scholar, 11Sharp P.A. Marciniak R.A. Cell. 1989; 59: 229-230Google Scholar, 12Gatignol A. Buckler-White A. Berkhout B. Jeang K.-T. Science. 1991; 251: 1597-1600Google Scholar, 13Cullen B.R. Cell. 1993; 73: 417-420Google Scholar). Accordingly, Tat-defective HIV-1 is not viable (57Dayton A.I. Sodroski J.C. Rosen C.A. Goh W.C. Haseltine W.A. Cell. 1986; 44: 941-947Google Scholar, 58Fisher A.G. Feinberg M.B. Josephs S.F. Harper M.E. Marselle L.M. Gallo R.C. Wong-Staal F. Nature. 1986; 320: 367-371Google Scholar). Emerging evidence shows that, in addition to its role on HIV-1 gene expression, Tat may exert additional functions. Tat is released in some extent extracellularly (20Frankel A.D. Pabo C.O. Cell. 1988; 55: 1189-1193Google Scholar, 59Ensoli B. Buonaguro L. Barillari G. Fiorelli V. Gendelman R. Morgan R.A. Wingfield P. Gallo R.C. J. Virol. 1993; 67: 277-287Google Scholar) and can function as a cytokine. In fact, Tat promotes the growth of endothelial cells and Kaposi's sarcoma cells directly or synergistically with basic fibroblast growth factor (Ref.60Ensoli B. Gendelman R. Markham P. Fiorelli V. Colombini S. Raffeld M. Cafaro A. Chang H. Brady J.N. Gallo R.C. Nature. 1994; 371: 674-680Google Scholar and references therein) and enhances cell survival intat-expressing cells (61Zauli G. Gibellini D. Milani D. Mazzoni M. Borgatti P. La Placa M. Capitani S. Cancer Res. 1993; 53: 4481-4485Google Scholar). Constitutive expression oftat in transgenic mice results in tumor development, including Kaposi's-like sarcomas and B cell lymphomas (62Corallini A. Altavilla G. Pozzi L. Bignozzi F. Negrini M. Rimessi P. Gualandi F. Barbanti-Brodano G. Cancer Res. 1993; 53: 5569-5575Google Scholar). Accordingly, stable expression of tat in IL6-dependent cells results in growth factor-independent growth and in tumorigenicity (30Scala G. Ruocco M.R. Ambrosino C. Mallardo M. Giordano V. Baldassarre F. Dragonetti E. Quinto I. Venuta S. J. Exp. Med. 1994; 179: 961-971Google Scholar). Moreover, data in support of a nontranscriptional function of Tat in virion infectivity has been reported (63Huang L. Joshi A. Willey R. Orenstein J. Jeang K.T. EMBO J. 1994; 13: 2886-2896Google Scholar). The above evidence strongly suggests that Tat may participate in the establishment of HIV-1 infection and in the development of AIDS clinical features by promoting the expression of host cellular genes. In support of this possibility, Tat has been shown to activate the expression of the proinflammatory cytokines IL6 and tumor necrosis factor-β (30Scala G. Ruocco M.R. Ambrosino C. Mallardo M. Giordano V. Baldassarre F. Dragonetti E. Quinto I. Venuta S. J. Exp. Med. 1994; 179: 961-971Google Scholar, 31Sastry K.J. Reddy R.H.R. Pandita R. Totpal K. Aggarwal B.B. J. Biol. Chem. 1990; 265: 20091-20093Google Scholar, 32Buonaguro L. Barillari G. Chang H.K. Bohan C.A. Kao V. Morgan R. Gallo R.C. Ensoli B. J. Virol. 1992; 68: 2677-2682Google Scholar) and to increase interleukin-2 and collagen gene expression (64Westendorp M.O. Li-Weber M. Frank R. Kramer P.H. J. Virol. 1994; 68: 4177-4185Google Scholar, 65Taylor J.P. Cupp C. Diaz A. Chowdhury M. Khalili K. Jemenez S.A. Amini S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9617-9621Google Scholar). Tat was also shown to suppress promoter activity of major histocompatibility complex class I genes (66Howcroft T.K. Strebel K. Martin J.L. Wirth P.J. Science. 1992; 269: 1320-1322Google Scholar) and to exert immunosuppressive activity on antigen-induced T cell proliferation (67Viscidi R.P. Mayur K. Lederman H.M. Frankel A.D. Science. 1989; 246: 1606-1608Google Scholar, 68Meyaard L. Otto S.A. Jonker R.R. Mijnster M.J. Keet R.P.M. Miedema F. Science. 1992; 257: 217-219Google Scholar, 69Chirmule N. Than S. Khan S.A. Pahwa S. J. Virol. 1995; 69: 492-498Google Scholar). Moreover, Tat has been shown to promote apoptosis by up-regulating CD95 ligand expression (70Westendorp M.O. Frank R. Ochsenbauer C. Stricker K. Dhein J. Walczak H. Debati K.M. Kramer H.P. Nature. 1995; 375: 497-500Google Scholar) or by activating cyclin-dependent kinases (71Li C.J. Friedman D.J. Wang C. Metelev V. Pardee A.B. Science. 1995; 268: 429-431Google Scholar).The mechanisms of Tat function on the expression of heterologous genes are unknown. In this paper, we address in molecular detail the mechanisms of Tat activity on the expression of IL6, a cytokine with a broad biological activity (7Kishimoto T. Akira S. Taga T. Science. 1992; 258: 593-597Google Scholar, 72Scala G. Quinto I. Ruocco M.R. Arcucci A. Mallardo M. Caretto P. Forni G. Venuta S. J. Exp. Med. 1990; 172: 61-68Google Scholar) whose expression is deregulated in HIV-infected subjects (6Nakajima N. Martinez-Maza O. Hirano T. Breen E.C. Nishanian P.G. Sazar-Gonzalez J.F. Fahey J.L. Kishimoto T. J. Immunol. 1989; 142: 144-155Google Scholar, 8Breen E.C. Rezal A.R. Nakajima K. Beall G.N. Mitsuyasu R.T. Hirano T. Kishimoto T. Martinez-Maza O. J. Immunol. 1990; 144: 480-487Google Scholar, 9Gallo P., K. Frei K. Rordorf C. Lazdins J. Tavolato B. Fontana A. J. Neuroimmunol. 1989; 23: 109-115Google Scholar). By using 5′ deletion mutants of pIL6-CAT plasmid, and IL6:HIV-1-LTR hybrid plasmids where discrete regions of the IL6 promoter replaced the TAR sequence in HIV-1 LTR, we identified a short sequence of the 5′-untranslated region of IL6 mRNA that is required for Tat to transactivate the IL6 promoter. This region can acquire a stem-loop structure including a UCU trinucleotide bulge. Point mutations of the UCU bulge or of the stem resulted in a drastic decrease in Tat responsiveness (shown in Fig. 3) and in the inability of Tat to bind to the IL6 leader RNAs (Fig. 4). The IL6 RNA structure, with an estimated structure energy of −9.1 kcal/mol, is expected to be less stable than the TAR RNA structure. This suggests that Tat could bind with a low affinity to heterologous RNA sequences and may account for the ability of Tat to regulate the expression of multiple genes. Interestingly, Tat was still able to induce a low but significant activation of the bulge mutant pIL6(−596/+15)M1-CAT plasmid (shown in Fig. 3), suggesting that Tat can function, albeit at a lower efficiency, without binding to an RNA tethering structure. In this case, Tat could be directed to the transcription start site of IL6 promoter by associating with specific transcription factors. This possibility is supported by the reports showing that Tat may associate with Sp1, TFIID factors, RNA polymerase II, and RNA polymerase II-associated factors (50Jeang K.T. Chun R. Lin N.H. Gatignol A. Glabe C.G. Fan H. J. Virol. 1993; 67: 6224-6233Google Scholar, 73Howcroft T.K. Palmer L.A. Brown J. Rellahan B. Kashanchi F. Brady J.N. Singer D.S. Immunity. 1995; 3: 127-138Google Scholar, 74Kashanchi F. Piras G. Radonovich M.F. Duval J.F. Fattaey A. Chiang C. Roeder R.G. Brady J.N. Nature. 1994; 367: 295-299Google Scholar, 75Chiang C.M. Roeder R.G. Science. 1995; 267: 531-535Google Scholar, 76Kashanchi F. Khleif S.N. Duvall J.F. Sadaie M.R. Radonovic M.F. Cho M. Martin M.A. Chen S. Weinmann R. Brady J.N. J. Virol. 1996; 70: 5503-5510Google Scholar, 77Mavankal G. Ignatious S.H. Oliver H. Sigman D. Gaynor R.B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2089-2094Google Scholar, 78Zhou Q. Sharp P.A. Science. 1996; 274: 605-610Google Scholar). In addition, we now provide evidence that Tat can function by cooperating with C/EBP transcription factors. In fact, we observed an increase in the C/EBP DNA binding activity of tat-expressing cells with a selective increase in the amounts of nuclear C/EBPβ factors (Figs. 5and 6). This raises the possibility that Tat may increase the nuclear levels of C/EBP transcription factors by inducing post-translational modifications of C/EBP factors through the activation of specific kinases. Indeed, Tat activity on HIV-1 LTR-driven gene expression requires protein kinase C (79Jakobovits A. Rosenthal A. Capon D.J. EMBO J. 1990; 9: 1165-1170Google Scholar). Moreover, specific interaction of Tat with a cellular protein kinase has been reported (80Yang X. Herrmann C.H. Rice A.P. J. Virol. 1996; 70: 4576-4584Google Scholar), and serine and threonine phosphorylations of C/EBPβ are required for IL6 promoter activation (81Nakajima N. Kinoscita S. Sasagawa K. Sasaki K. Naruto T. Kishimoto T. Akira S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2207-2211Google Scholar). The above data are consistent with and extend the recent observation that Tat enhances the tumor necrosis factor-induced activation of NF-κB binding activity by possibly inducing protein phosphorylation (82Westendorp M.O. Shatrov V.A. Shulze-Osthoff K. Frank R. Kraft M. Los M. Krammer H.P. Droge W. Lehmann V. EMBO J. 1995; 14: 546-554Google Scholar). Since C/EBP and NF-κB factors associate as heterodimers (83LeClair K.P. Blanar M.A. Sharp P.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8145-8149Google Scholar), which are potent activators of HIV-1 LTR (84Ruocco M.R. Chen X. Ambrosino C. Dragonetti E. Liu W. Mallardo M. De Falco G. Palmieri C. Franzoso G. Quinto I. Venuta S. Scala G. J. Biol. Chem. 1996; 271: 22479-22486Google Scholar), the above data suggest that Tat may also promote HIV-1 gene expression by up-regulating the cellular levels of transcription factors acting on the viral LTR. Moreover, Tat was able to complex with in vitro translated C/EBPβ, which is a major mediator of IL6 promoter function (Fig. 7). By immunoprecipitation and by taking advantage of the yeast two-hybrid system, this interaction was proved to occur also in vivo and to result in transcriptional activation of a reporter lacZ gene (shown in Figs. 8 and 9). The Tat association with C/EBPβ suggests that Tat may increase the DNA binding activity of C/EBP dimers by enhancing their affinity for the target DNA. This mechanism accounts for the Tax activity on transcription mediated by bZip proteins (86Perini G. Wagner S. Green M.R. Nature. 1995; 376: 602-605Google Scholar, 87Baranger A.M. Palmer C.R. Hamm K.M. Giebler H.A. Brauweiler A. Nyborg J.K. Schepartz A. Nature. 1995; 376: 606-608Google Scholar). In the EMSA experiments shown in Fig. 5A, Tat could not be detected in the C/EBP-DNA complexes with an anti-Tat antibody (data not shown). This suggests either that Tat does not directly participate in the C/EBP-DNA complexes or that Tat dissociates from the DNA-binding complexes due to the electrical field of EMSAs. These possibilities warrant further studies.The data are consistent with the possibility that Tat may function on heterologous genes by interacting with RNA structures possibly present in a large number of cellular and viral genes, as recently reported (30Scala G. Ruocco M.R. Ambrosino C. Mallardo M. Giordano V. Baldassarre F. Dragonetti E. Quinto I. Venuta S. J. Exp. Med. 1994; 179: 961-971Google Scholar, 31Sastry K.J. Reddy R.H.R. Pandita R. Totpal K. Aggarwal B.B. J. Biol. Chem. 1990; 265: 20091-20093Google Scholar, 32Buonaguro L. Barillari G. Chang H.K. Bohan C.A. Kao V. Morgan R. Gallo R.C. Ensoli B. J. Virol. 1992; 68: 2677-2682Google Scholar, 33Chowdhury M. Taylor J.P. Chang C.F. Rappaport J. Khalili K. J. Virol. 1992; 66: 7355-7361Google Scholar). In addition, Tat may function by forming heterodimers with specific transcription factors. These possibilities dramatically enhance the capacity of Tat to modulate the expression of heterologous genes and to play a major role in the pathogenesis of HIV-associated diseases. Human immunodeficiency virus type 1 (HIV-1) 1The abbreviations used are:HIV-1human immunodeficiency virus type 1IL6interleukin-6TARtransactivating responsive elementC/EBPCAAT enhancer-binding proteinEMSAelectrophoretic mobility shift assayCATchloramphenicol acetyltransferaseLTRlong terminal repeatGSTglutathioneS-transferasePBSphosphate-buffered salineDTTdithiothreitolPMSFphenylmethylsulfonyl fluoridex-gal5-bromo-4-chloro-3-indolyl β-d-galactoside is the etiologic agent for acquired immunodeficiency syndrome (AIDS) and causes various clinical and immunological abnormalities, including activation of polyclonal B cells that manifests as hypergammaglobulinemia and autoantibody production, lymphadenopathy, Kaposi's sarcoma, and lymphoma of the B cell phenotype (1Fauci A.S. Macher A.M. Longo D.L. Lane H.C. Rook A.H. Masur H. Gelman E.P. Ann. Intern. Med. 1984; 100: 92-103Google Scholar, 2Beral V. Peterman A. Berrkelman R.L. Jaffe H.W. Lancet. 1990; 335: 123-128Google Scholar, 3Levine A.M. Blood. 1992; 80: 8-15Google Scholar). Studies on small cohorts of subjects who were exposed to HIV-1 and did not develop HIV-1 infection and individuals who harbored HIV-1 but remained disease-free for long periods (4Nowak M.A. Anderson R.M. McLeann A.R. Wolfs T.F.W. Goudsmit J. May R.M. Science. 1991; 254: 963-969Google Scholar, 5Biggar R.J. AIDS. 1990; 4: 1059-1065Google Scholar) strongly suggest that the development of AIDS may depend on a dynamic interplay between viral and host cellular gene products. Accordingly, in HIV-1-infected subjects there is a deregulated production of cytokines, including the proinflammatory interleukin-6 (IL6) (6Nakajima N. Martinez-Maza O. Hirano T. Breen E.C. Nishanian P.G. Sazar-Gonzalez J.F. Fahey J.L. Kishimoto T. J. Immunol. 1989; 142: 144-155Google Scholar), which affects the growth and differentiation of lymphoid and mesenchymal cells (7Kishimoto T. Akira S. Taga T. Science. 1992; 258: 593-597Google Scholar) and may contribute to the development of the clinical features of AIDS. Accordingly, IL6 gene transcription is induced in cells infected by HIV-1 (8Breen E.C. Rezal A.R. Nakajima K. Beall G.N. Mitsuyasu R.T. Hirano T. Kishimoto T. Martinez-Maza O. J. Immunol. 1990; 144: 480-487Google Scholar), and increased levels of IL6 have been reported in serum and cerebral spinal fluid of HIV-1-infected patients (9Gallo P., K. Frei K. Rordorf C. Lazdins J. Tavolato B. Fontana A. J. Neuroimmunol. 1989; 23: 109-115Google Scholar). human immunodeficiency virus type 1 interleukin-6 transactivating responsive element CAAT enhancer-binding protein electrophoretic mobility shift assay chloramphenicol acetyltransferase long terminal repeat glutathioneS-transferase phosphate-buffered saline dithiothreitol phenylmethylsulfonyl fluoride 5-bromo-4-chloro-3-indolyl β-d-galactoside The Tat protein of HIV-1 is required for efficient viral gene expression (10Rosen C. Sodroski J.G. Haseltine W.A. Cell. 1985; 41: 813-823Google Scholar, 11Sharp P.A. Marciniak R.A. Cell. 1989; 59: 229-230Google Scholar, 12Gatignol A. Buckler-White A. Berkhout B. Jeang K.-T. Science. 1991; 251: 1597-1600Google Scholar, 13Cullen B.R. Cell. 1993; 73: 417-420Google Scholar, 14Rice A.P. Matthews M.B. Nature. 1988; 332: 551-555Google Scholar, 15Berkhout B. Silverman R.H. Jeang K.T. Cell. 1989; 9: 273-282Google Scholar). Tat increases the initiation of transcription from the HIV-1 LTR (14Rice A.P. Matthews M.B. Nature. 1988; 332: 551-555Google Scholar) and affects RNA processing and utilization by interacting with a transactivating responsive element (TAR) located between nucleotides +1 and + 44 with respect to the initiation site (+1) of viral transcription (16Lapsia M.F. Rice A.P. Matthews M.B. Cell. 1989; 59: 283-292Google Scholar, 17Arya S.K. Guo C. Josephs S.F. Wong-Staal F. Science. 1985; 229: 69-73Google Scholar). TAR contains a 6-nucleotide loop and a 3-nucleotide pyrimidine bulge that are essential for Tat activity (18Sodroski J. Patarca R. Rosen C. Wong-Staal F. Haseltine W.A. Science. 1985; 229: 74-77Google Scholar, 19Garcia J.A. Harrich D. Soultanakis E.W.F. Mitsuyasu R. Gaynor R.B. EMBO J. 1989; 8: 765-778Google Scholar, 20Frankel A.D. Pabo C.O. Cell. 1988; 55: 1189-1193Google Scholar, 21Dingwall C. Ernberg I. Gait M.J. Green S.M. Heaphy S. Karn J. Lowe A.D. Singh M. Skinner M.A. Valerio R. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6925-6929Google Scholar). Tat binds to the bulge and appears to require cellular factors binding to the loop sequence to efficiently transactivate the HIV-1 LTR (22Roy S. Delling U. Chen C.H. Rosen C.A. Sonenberg N. Genes & Dev. 1990; 4: 1365-1373Google Scholar, 23Nelbock P. Dillon P.J. Perkins A. Rosen C.A. Science. 1990; 248: 1650-1653Google Scholar, 24Desai K. Loewestein P.M. Green M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8875-8879Google Scholar). In addition, Tat interacts with upstream regulatory DNA sequences circumscribed within the NF-κB/Sp1 sites of the HIV-1 promoter (25Berkhout B. Gatignol A. Rabson A.B. Jeang K.T. Cell. 1990; 62: 757-767Google Scholar) and with host cell proteins (12Gatignol A. Buckler-White A. Berkhout B. Jeang K.-T. Science. 1991; 251: 1597-1600Google Scholar, 24Desai K. Loewestein P.M. Green M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8875-8879Google Scholar). The 86-amino acid-long Tat contains a highly conserved cysteine-rich region, which mediates the formation of metal-linked dimers in vitro and is essential for Tat function (16Lapsia M.F. Rice A.P. Matthews M.B. Cell. 1989; 59: 283-292Google Scholar, 17Arya S.K. Guo C. Josephs S.F. Wong-Staal F. Science. 1985; 229: 69-73Google Scholar, 18Sodroski J. Patarca R. Rosen C. Wong-Staal F. Haseltine W.A. Science. 1985; 229: 74-77Google Scholar). A conserved basic region with 6 arginines and 2 lysines in nine residues, stretching from amino acid 47 to 58, is crucial for nuclear localization, mediates the specific binding of Tat to TAR RNA, and is required for the full activity of Tat (26Shibuya H. Irie K. Ninomiya-Tsuji J. Goebl M. Taniguchi T. Matsumoto K. Nature. 1992; 357:" @default.
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• W2021986727 title "HIV-1 Tat Induces the Expression of the Interleukin-6 (IL6) Gene by Binding to the IL6 Leader RNA and by Interacting with CAAT Enhancer-binding Protein β (NF-IL6) Transcription Factors" @default.
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• W2021986727 doi "https://doi.org/10.1074/jbc.272.23.14883" @default.
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