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COUP-TF and Sp1 Interact and Cooperate in the Transcriptional Activation of the Human Immunodeficiency Virus Type 1 Long Terminal Repeat in Human Microglial Cells

1997; Elsevier BV; Volume: 272; Issue: 49 Linguagem: Inglês

10.1074/jbc.272.49.31149

1083-351X

Olivier Rohr, Dominique Aunis, Evelyne Schaeffer,

RNA Interference and Gene Delivery

We have recently reported that chicken ovalbumin upstream promoter transcription factor (COUP-TF) activates human immunodeficiency virus type 1 (HIV-1) gene transcription in glial and neuronal cells. Here, we have examined the role of COUP-TF in microglial cells, the major target cells for HIV-1 infection in brain. We show that COUP-TF activates gene expression from both the lymphotropic LAI and the macrophage-tropic JR-FL HIV-1 strains. Although COUP-TF binds to the −352/−320 nuclear receptor responsive element of the long terminal repeat, it functions as a transcriptional activator by acting on the −68/+29 minimal promoter. This region is a direct target of transcription factors Sp1 and Sp3. We report the discovery and features of a physical and functional interplay between COUP-TF and Sp1. Our cotransfection experiments provide evidence for a functional synergism between Sp1 and COUP-TF leading to enhanced transcriptional activity of the HIV-1 long terminal repeat through the Sp1 element. In contrast, Sp3 functions as a repressor of Sp1- or COUP-TF-induced activation. We further demonstrate that COUP-TF and Sp1 are capable of physically interacting, via the DNA-binding domain of COUP-TF, in vitro and in the cell. These findings reveal how the novel interplay of Sp1 and COUP-TF families of transcription factors regulate HIV-1 gene expression. We have recently reported that chicken ovalbumin upstream promoter transcription factor (COUP-TF) activates human immunodeficiency virus type 1 (HIV-1) gene transcription in glial and neuronal cells. Here, we have examined the role of COUP-TF in microglial cells, the major target cells for HIV-1 infection in brain. We show that COUP-TF activates gene expression from both the lymphotropic LAI and the macrophage-tropic JR-FL HIV-1 strains. Although COUP-TF binds to the −352/−320 nuclear receptor responsive element of the long terminal repeat, it functions as a transcriptional activator by acting on the −68/+29 minimal promoter. This region is a direct target of transcription factors Sp1 and Sp3. We report the discovery and features of a physical and functional interplay between COUP-TF and Sp1. Our cotransfection experiments provide evidence for a functional synergism between Sp1 and COUP-TF leading to enhanced transcriptional activity of the HIV-1 long terminal repeat through the Sp1 element. In contrast, Sp3 functions as a repressor of Sp1- or COUP-TF-induced activation. We further demonstrate that COUP-TF and Sp1 are capable of physically interacting, via the DNA-binding domain of COUP-TF, in vitro and in the cell. These findings reveal how the novel interplay of Sp1 and COUP-TF families of transcription factors regulate HIV-1 gene expression. Human immunodeficiency virus type 1 (HIV-1) 1The abbreviations used are: HIV-1, human immunodeficiency virus type 1; CNS, central nervous system; LTR, long terminal repeat; COUP-TF, chicken ovalbumin upstream promoter transcription factor; NRRE, nuclear receptor responsive element; CAT, chloramphenicol acetyltransferase; RSV, Rous sarcoma virus; EMSA, electrophoretic mobility shift assay; GST, glutathioneS-transferase; PAGE, polyacrylamide gel electrophoresis; TFII, transcription factor II. 1The abbreviations used are: HIV-1, human immunodeficiency virus type 1; CNS, central nervous system; LTR, long terminal repeat; COUP-TF, chicken ovalbumin upstream promoter transcription factor; NRRE, nuclear receptor responsive element; CAT, chloramphenicol acetyltransferase; RSV, Rous sarcoma virus; EMSA, electrophoretic mobility shift assay; GST, glutathioneS-transferase; PAGE, polyacrylamide gel electrophoresis; TFII, transcription factor II. infects the central nervous system (CNS) and plays a direct role in the pathogenesis of AIDS dementia (1Fauci A.S. Science. 1988; 239: 617-622Crossref PubMed Scopus (1198) Google Scholar, 2Portegies P. Brew B.J. AIDS. 1991; 5: S211-S217Crossref PubMed Scopus (12) Google Scholar), but how the infection leads to brain damage has been poorly understood. The CNS resident macrophages or microglial cells (3Perry V.H. Lawson L.J. Reid D.M. J. Leukocyte Biol. 1994; 56: 399-406Crossref PubMed Scopus (42) Google Scholar) are the primary target of HIV-1 infection in brain (4Wiley C.A. Schrier R.D. Nelson J.A. Lampert P.W. Oldstone M.B. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 7089-7093Crossref PubMed Scopus (1053) Google Scholar, 5Price R.W. Brew B. Sidtis J. Rosenblum M. Scheck A.C. Cleary P. Science. 1988; 239: 586-592Crossref PubMed Scopus (1081) Google Scholar, 6Watkins B.A. Dorn H.H. Kelly W.B. Armstrong R.C. Potts B.J. Michaels F. Kufta C.V. Dubois-Dalcq M. Science. 1990; 249: 549-553Crossref PubMed Scopus (294) Google Scholar, 7Peudenier S. Hery C. Montagnier L. Tardieu M. Annu. Neurol. 1991; 29: 152-161Crossref PubMed Scopus (134) Google Scholar). Glial and neuronal cells are capable of harboring a restricted infection with HIV-1 (8Hatch W.C. Pousada E. Rashbaum W.K. Lyman W.D. AIDS Res. Hum. Retroviruses. 1994; 10: 1597-1607Crossref PubMed Scopus (19) Google Scholar, 9Nuovo G.J. Gallery F. MacConnell P. Braun A. Am. J. Pathol. 1994; 144: 659-666PubMed Google Scholar, 10Albright A.V. Strizki J. Harouse J.M. Lavi E. O'Connor M. Gonzalez-Scarano F. Virology. 1996; 217: 211-219Crossref PubMed Scopus (65) Google Scholar). HIV-1 infection is established in the CNS by viruses present early in infection (11Stritzki J.M. Albright A.V. Sheng H. O'Connor M. Perrin L. Gonzalez-Scarano F. J. Virol. 1996; 70: 7654-7662Crossref PubMed Google Scholar). Macrophage-tropic isolates infect microglial cells more efficiently then do T cell tropic isolates and predominate early in the infection (12He J. Chen Y. Farzan M. Choe H. Ohagen A. Gartner S. Busciglio J. Yang X. Hofman W. Newman W. Mackay C.R. Sodroski J. Gabuzda D. Nature. 1997; 385: 645-649Crossref PubMed Scopus (812) Google Scholar). Therefore we have performed our studies with the macrophage-tropic JR-FL strain, compared with the T-tropic LAI strain.HIV-1 gene expression is controlled by a combination of viral and host cell transcription factors interacting with the long terminal repeat (LTR) region (for review, see Refs. 13Garcia J.A. Gaynor R.B. Prog. Nucleic Acid Res. Mol. Biol. 1994; 49: 157-196Crossref PubMed Scopus (33) Google Scholar and 14Kingsman S.M. Kingsman A.J. Eur. J. Biochem. 1996; 240: 491-507Crossref PubMed Scopus (106) Google Scholar). Recent studies have focused on transcription factors that regulate HIV-1 expression in brain cells. Analysis of HIV-1 directed gene expression in transgenic mice derived from CNS-isolated HIV-1 strains (15Koyanagi Y. Miles S. Mitsuyasu R.T. Merrill J.E. Vinters H.V. Chen I.S.Y. Science. 1987; 236: 819-822Crossref PubMed Scopus (622) Google Scholar), suggests that HIV-1 replication in the CNS uses transcription factors different from those in non-neural tissues (16Corboy J.R. Busy J.M. Zink M.C. Clements J.E. Science. 1992; 258: 1804-1808Crossref PubMed Scopus (69) Google Scholar, 17Busy J.M. Lindstrom L.M. Zink M.C. Clements J.E. Virology. 1995; 210: 361-371Crossref PubMed Scopus (13) Google Scholar, 18Kurth J. Busy J.M. Lindstrom L. Clements J.E. J. Virol. 1996; 70: 7686-7694Crossref PubMed Google Scholar). Transcriptional activity of the HIV-1 promoter is mediated by κB regulatory sequences of the LTR, through the action of the transcription factor NF-κB, both in neurons (19Rattner A. Korner M. Walker M.D. Citri Y. EMBO J. 1993; 12: 4261-4267Crossref PubMed Scopus (78) Google Scholar,20Kaltschmidt C. Kaltschmidt B. Neumann H. Wekerle H. Baeuerle P.A. Mol. Cell. Biol. 1994; 14: 3981-3992Crossref PubMed Google Scholar) and in astrocytes (21Taylor J.P. Khalili K. Adv. Neuroimm. 1994; 4: 291-303Abstract Full Text PDF PubMed Scopus (20) Google Scholar). Besides the κB regulatory element, our recent data have highlighted the importance of the nuclear receptor-responsive element (NRRE), which appears to be the point of convergence of a network of physiological signals which modulate HIV-1 gene expression in brain cells. We have described that the orphan nuclear receptors COUP-TF/Ear3 (22Wang L.-H. Tsai S.Y. Sagami I. Tsai M.-J. O'Malley B.W. J. Biol. Chem. 1987; 262: 16080-16086Abstract Full Text PDF PubMed Google Scholar, 23Wang L.H. Tsai S.Y. Cook R.G. Beattie W.G. Tsai M.J. O'Malley B.W. Nature. 1989; 340: 163-166Crossref PubMed Scopus (385) Google Scholar, 24Miyajima N.Y. Kadowaki S.-I. Fukushige S.-I. Shimizu K. Semba Y. Yamanashi K.-I. Matsubara K. Yoyoshima K. Yamamoto T. Nucleic Acids Res. 1988; 16: 11057-11069Crossref PubMed Scopus (173) Google Scholar) are present in three human brain cell lines, oligodendroglioma TC-620, astrocytoma U373-MG, and neuroblastoma SK-N-MC cells. Our data have also demonstrated the importance of COUP-TF as a potent transcriptional activator in oligodendroglioma cells, of both the lymphotropic LAI and the neurotropic JR-CSF HIV-1 LTR. They revealed the action of the dopamine transduction pathway, which coupled to COUP-TF, contributes to enhance HIV-1 gene transcription in neuronal cells (25Sawaya B.E. Rohr O. Aunis D. Schaeffer E. J. Biol. Chem. 1996; 271: 23572-23576Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Our findings have further established the importance of the retinoic acid signaling pathway in HIV-1 gene transcription in glial and neuronal cells (26Sawaya B.E. Rohr O. Aunis D. Schaeffer E. J. Biol. Chem. 1996; 271: 22895-22900Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). Except for retinoid receptors, the role of transcription factors belonging to the steroid/thyroid/retinoid receptor superfamily has not yet been described in microglial cells.In this report, we have investigated the functional effect and the molecular mechanisms by which the orphan nuclear receptor COUP-TF regulates HIV-1 gene expression in a human microglial cell line (27Janabi N. Peudenier S. Héron B. Ng K.H. Tardieu M. Neurosci. Lett. 1995; 195: 105-108Crossref PubMed Scopus (195) Google Scholar). Our findings reveal the importance of COUP-TF as an activator of LTR-driven transcription of both the T cell line-tropic LAI and the macrophage-tropic JR-FL HIV-1 strains. We provide evidence for a novel physiological and functional interaction between the DNA-binding domain of COUP-TF and the transcription factor Sp1, leading to enhanced transactivation of the HIV-1 LTR. In addition, our data reveal that the COUP-TF- and Sp1-induced stimulation is repressed by the transcription factor Sp3. These data describe novel molecular mechanisms which govern the network of interactions between the HIV-1 LTR, the transcriptional activators COUP-TF and Sp1, and the repressor Sp3, in the regulation of HIV-1 gene expression.DISCUSSIONIn this report we have investigated the regulation of HIV-1 gene transcription in human microglial cells, which represent the primary target of HIV-1 infection in the central nervous system. Since we have previously shown that the orphan nuclear receptor COUP-TF is expressed in brain cells and leads to a transcriptional stimulation of the HIV-1 genome in oligodendroglioma and neuronal cells (25Sawaya B.E. Rohr O. Aunis D. Schaeffer E. J. Biol. Chem. 1996; 271: 23572-23576Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), we have focused our studies on the regulation of HIV-1 gene transcription by COUP-TF in microglial cells. It is now well established that COUP-TF is able to exert both positive and negative effects on gene expression, depending upon the promoter and the cell contexts (29Schaeffer E. Guillou F. Part D. Zakin M.M. J. Biol. Chem. 1993; 268: 23399-23408Abstract Full Text PDF PubMed Google Scholar, 35Kimura A. Nishiyori A. Murakami T. Tsukamoto T. Hata S. Osumi T. Okamura R. Mori M. Takiguchi M. J. Biol. Chem. 1993; 268: 11125-11133Abstract Full Text PDF PubMed Google Scholar, 36Kadowaki Y. Toyoshima K. Yamamoto T. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4432-4436Crossref PubMed Scopus (23) Google Scholar, 37Sawaya B.E. Schaeffer E. Nucleic Acids Res. 1995; 23: 2206-2211Crossref PubMed Scopus (19) Google Scholar, 38Leng X. Cooney A.J. Tsai S.Y. Tsai M.-J. Mol. Cell. Biol. 1996; 16: 2332-2340Crossref PubMed Scopus (84) Google Scholar). We show here that COUP-TF proteins are present in human microglial cells and interact directly with the NRRE, spanning the −356/−320 LTR region of the lymphotropic HIV-1 LAI isolate, but are unable to bind to the mutant NRRE present in the macrophage-tropic HIV-1 JR-FL isolate. Interestingly, our transient expression data reveal that COUP-TF/Ear3 is able to activate HIV-1 LTR-driven transcription in microglial cells, independently of the NRRE sequence. This shows that COUP-TF is able to transactivate the LTR from the lymphotropic LAI and the macrophage-tropic JR-FL isolates.We have already reported that distinct cell-type specific and sequence-dependent mechanisms govern COUP-TF-induced stimulation. In oligodendrocytes this transcription factor stimulates HIV-1 gene transcription, either by direct interactions with its NRRE target site or also, depending on the LTR sequence, by cross-coupling interactions with downstream-located proteins. In neuronal SK-N-MC cells, the transcriptional stimulation induced by COUP-TF in the presence of dopamine is mediated by the minimal −68/+29 LTR region (25Sawaya B.E. Rohr O. Aunis D. Schaeffer E. J. Biol. Chem. 1996; 271: 23572-23576Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Similarly the NRRE site in the JR-CSF LTR is not indispensable for the stimulation induced by the retinoic acid receptor, since the retinoid action appears to be mediated by downstream-located elements, such as the −247/−222 AP-1 region and, to a lesser extent, the NF-κB region (26Sawaya B.E. Rohr O. Aunis D. Schaeffer E. J. Biol. Chem. 1996; 271: 22895-22900Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar).We show here that the minimal −68/+29 LTR region, containing two Sp1-binding sites, is sufficient for COUP-TF-mediated stimulation. Our cotransfection and in vitro experiments demonstrate the ability of COUP-TF to transactivate the LTR indirectly via the Sp1 sites, by direct interaction with the Sp1 protein. We present evidence for a direct association between the N-terminal part of COUP-TF, containing the DNA-binding domain, with Sp1. Our data show that COUP-TF can interact with Sp1 both in vitro and in microglial cells. This association leads to a functional cooperation between the two proteins, which is detected with the full-length LTR as well as the −68/+80 LTR region. Moreover we show that the C-terminal domain of COUP-TF is involved in the transcriptional activation.Previous studies have already described that the action of COUP-TFs can be mediated not only by direct interaction with their DNA-binding site, but also by protein-protein interaction. COUP-TFI/Ear3 and ARP-1/COUP-TFII are able to directly target components of the basal transcription machinery, such as the basal transcription factor TFIIB (39Ing N.H. Beckman J.M. Tsai S.Y. Tsai M.J. O'Malley B.M. J. Biol. Chem. 1992; 267: 17617-17623Abstract Full Text PDF PubMed Google Scholar, 40Malik S. Karathanasis S. Nucleic Acids Res. 1995; 23: 1536-1543Crossref PubMed Scopus (60) Google Scholar). TFIIB recognizes and associates with COUP-TF and two other members of the steroid hormone receptor family, via their activation domain (39Ing N.H. Beckman J.M. Tsai S.Y. Tsai M.J. O'Malley B.M. J. Biol. Chem. 1992; 267: 17617-17623Abstract Full Text PDF PubMed Google Scholar). In contrast, our findings reveal that COUP-TF associates with Sp1 via its N-terminal part, containing the DNA-binding domain. A direct interaction between COUP-TF and the transcription factors Oct1 and Oct2 has also been demonstrated in the regulation of the vHNFI promoter (30Power S.C. Cereghini S. Mol. Cell. Biol. 1996; 16: 778-791Crossref PubMed Google Scholar).It has been well established that the Sp1 transcription factor (41Kadonaga J.T. Carner K.R. Masiarz F.R. Tjian R. Cell. 1987; 51: 1079-1090Abstract Full Text PDF PubMed Scopus (1246) Google Scholar) plays an essential role in the regulation of basal transcription as well as in Tat-mediated transactivation of the HIV-1 LTR (42Jones K.A. Kadonaga J.T. Luciw P.A. Tjian R. Science. 1986; 232: 755-759Crossref PubMed Scopus (444) Google Scholar, 43Sune C. Garcia-Blanco M.A. J. Virol. 1995; 69: 6572-6576Crossref PubMed Google Scholar, 44Harrich D. Garcia J. Wu F. Mitsuyasu R. Gonazalez J. Gaynor R. J. Virol. 1989; 63: 2585-2591Crossref PubMed Google Scholar). Recent reports describe that Sp1 is critical for in vivotranscriptional regulation of HIV, through its interaction with other DNA-binding proteins. A cooperative interaction between Sp1 and NF-κB, bound to the two adjacent binding sites, is required for optimal HIV-1 enhancer activation and inducible HIV-1 gene expression (32Majello B. De Luca P. Hagen G. Suske G. Lania L. Nucleic Acids Res. 1994; 22 (1921): 4914Crossref PubMed Scopus (202) Google Scholar, 45Perkins N.D. Edwards N.L. Duckett C.S. Agranoff A.B. Schmid R.M. Nabel G.J. EMBO J. 1993; 12: 3551-3558Crossref PubMed Scopus (399) Google Scholar, 46Li Y. Mak G. Franza Jr., B.R. J. Biol. Chem. 1994; 269: 30616-30619Abstract Full Text PDF PubMed Google Scholar). A physical interaction between Sp1 and the p53 tumor-suppressor gene has been described in the tumor necrosis factor-induced transcriptional activation of the HIV-1 LTR (47Gualberto A. Baldwin Jr., A.S. J. Biol. Chem. 1995; 270: 19680-19683Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Moreover in vitro and in vivo data suggest a direct interaction between Sp1 and Tat during transactivation (48Kamine J. Chinnadurai G. J. Virol. 1992; 66: 3932-3936Crossref PubMed Google Scholar,49Jeang K.T. Chun R. Lin N.H. Gatignol A. Glabe C.G. Fan H. J. Virol. 1993; 67: 6224-6233Crossref PubMed Google Scholar).Here our findings reveal a novel direct association between two zinc finger proteins, Sp1 and COUP-TF, which leads to enhanced transcriptional activation of the HIV-1 genome. These data suggest a more general model for gene activation by the orphan receptor COUP-TF or steroid/thyroid/retinoid receptors, in the presence of Sp1-binding sites. As shown here, Sp1 bound to its DNA-binding site is able to associate directly with the N-terminal DNA-binding domain of the nuclear receptor. It is well known that the adjacent TATA element binds the general transcription factor TFIID, which directly interacts with TFIIB, which itself is able to bind COUP-TF. In such a situation, the nuclear receptor may function as an adaptor protein, bringing together Sp1 and TFIIB, thereby enhancing communication between the general transcription machinery and Sp1. Alternatively, COUP-TF may bring together Sp1 and a cell type-specific nuclear factor, leading to various levels of activation induced by COUP-TF in different cell types. The precise mechanisms which account for our observations in the case of HIV-1 and SV40 gene transcription need to be further investigated.The Sp1 multigene family contains three closely related members Sp1, Sp3, and Sp4 which recognize the GC box and the GT motif (33Hagen G. Müller S. Beato M. Suske G. EMBO J. 1994; 13: 3843-3851Crossref PubMed Scopus (650) Google Scholar). While Sp1 and Sp4 function as transcriptional activators, Sp3 is a bifunctional transcription regulator, whose activity is dependent upon the promoter and the cellular contexts (33Hagen G. Müller S. Beato M. Suske G. EMBO J. 1994; 13: 3843-3851Crossref PubMed Scopus (650) Google Scholar, 34Majello B. De Luca P. Lania L. J. Biol. Chem. 1997; 272: 4021-4026Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar). Recent reports have established that different members of the Sp1 family exert opposite transcriptional regulation of the HIV LTR. While Sp1 and Sp4 stimulate transcription of the LTR, Sp3 markedly represses the HIV promoter activity in HeLa and Drosophila SL2 cells. Sp3-dependent repression is dependent on the presence of the DNA-binding domain, indicating that repression occurs through interference with Sp1 binding to the GC motifs (32Majello B. De Luca P. Hagen G. Suske G. Lania L. Nucleic Acids Res. 1994; 22 (1921): 4914Crossref PubMed Scopus (202) Google Scholar). Our data confirm that Sp1 and Sp3 are present in microglial cells, and exert an antagonist action on LTR-driven transcription. Moreover they show that Sp3 is able to mediate transcriptional repression not only of the Sp1 action, but also of the COUP-TF action exerted via Sp1.Only a limited number of studies have been devoted to HIV-1 gene transcription in macrophages and microglial cells. A differential role of LTR elements for the regulation of basal and Tat-mediated transcription of HIV-1 has been reported in stimulated and unstimulated primary human macrophages (50Moses A.V. Ibanez C. Gaynor R. Ghazal P. Nelson J.A. J. Virol. 1994; 68: 298-307Crossref PubMed Google Scholar). Our findings establish the essential role of the orphan nuclear receptor COUP-TF as a transcriptional activator of LTR-directed HIV-1 gene expression in microglial cells. They further reveal a novel mechanism of HIV-1 gene transactivation by direct association of COUP-TF with Sp1, thus enhancing transcription via the minimal LTR region. Our present and previous studies (25Sawaya B.E. Rohr O. Aunis D. Schaeffer E. J. Biol. Chem. 1996; 271: 23572-23576Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) reveal the remarkable diverse mechanisms by which COUP-TF regulates gene transcription of distinct HIV-1 strains and in different brain cells. Human immunodeficiency virus type 1 (HIV-1) 1The abbreviations used are: HIV-1, human immunodeficiency virus type 1; CNS, central nervous system; LTR, long terminal repeat; COUP-TF, chicken ovalbumin upstream promoter transcription factor; NRRE, nuclear receptor responsive element; CAT, chloramphenicol acetyltransferase; RSV, Rous sarcoma virus; EMSA, electrophoretic mobility shift assay; GST, glutathioneS-transferase; PAGE, polyacrylamide gel electrophoresis; TFII, transcription factor II. 1The abbreviations used are: HIV-1, human immunodeficiency virus type 1; CNS, central nervous system; LTR, long terminal repeat; COUP-TF, chicken ovalbumin upstream promoter transcription factor; NRRE, nuclear receptor responsive element; CAT, chloramphenicol acetyltransferase; RSV, Rous sarcoma virus; EMSA, electrophoretic mobility shift assay; GST, glutathioneS-transferase; PAGE, polyacrylamide gel electrophoresis; TFII, transcription factor II. infects the central nervous system (CNS) and plays a direct role in the pathogenesis of AIDS dementia (1Fauci A.S. Science. 1988; 239: 617-622Crossref PubMed Scopus (1198) Google Scholar, 2Portegies P. Brew B.J. AIDS. 1991; 5: S211-S217Crossref PubMed Scopus (12) Google Scholar), but how the infection leads to brain damage has been poorly understood. The CNS resident macrophages or microglial cells (3Perry V.H. Lawson L.J. Reid D.M. J. Leukocyte Biol. 1994; 56: 399-406Crossref PubMed Scopus (42) Google Scholar) are the primary target of HIV-1 infection in brain (4Wiley C.A. Schrier R.D. Nelson J.A. Lampert P.W. Oldstone M.B. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 7089-7093Crossref PubMed Scopus (1053) Google Scholar, 5Price R.W. Brew B. Sidtis J. Rosenblum M. Scheck A.C. Cleary P. Science. 1988; 239: 586-592Crossref PubMed Scopus (1081) Google Scholar, 6Watkins B.A. Dorn H.H. Kelly W.B. Armstrong R.C. Potts B.J. Michaels F. Kufta C.V. Dubois-Dalcq M. Science. 1990; 249: 549-553Crossref PubMed Scopus (294) Google Scholar, 7Peudenier S. Hery C. Montagnier L. Tardieu M. Annu. Neurol. 1991; 29: 152-161Crossref PubMed Scopus (134) Google Scholar). Glial and neuronal cells are capable of harboring a restricted infection with HIV-1 (8Hatch W.C. Pousada E. Rashbaum W.K. Lyman W.D. AIDS Res. Hum. Retroviruses. 1994; 10: 1597-1607Crossref PubMed Scopus (19) Google Scholar, 9Nuovo G.J. Gallery F. MacConnell P. Braun A. Am. J. Pathol. 1994; 144: 659-666PubMed Google Scholar, 10Albright A.V. Strizki J. Harouse J.M. Lavi E. O'Connor M. Gonzalez-Scarano F. Virology. 1996; 217: 211-219Crossref PubMed Scopus (65) Google Scholar). HIV-1 infection is established in the CNS by viruses present early in infection (11Stritzki J.M. Albright A.V. Sheng H. O'Connor M. Perrin L. Gonzalez-Scarano F. J. Virol. 1996; 70: 7654-7662Crossref PubMed Google Scholar). Macrophage-tropic isolates infect microglial cells more efficiently then do T cell tropic isolates and predominate early in the infection (12He J. Chen Y. Farzan M. Choe H. Ohagen A. Gartner S. Busciglio J. Yang X. Hofman W. Newman W. Mackay C.R. Sodroski J. Gabuzda D. Nature. 1997; 385: 645-649Crossref PubMed Scopus (812) Google Scholar). Therefore we have performed our studies with the macrophage-tropic JR-FL strain, compared with the T-tropic LAI strain. HIV-1 gene expression is controlled by a combination of viral and host cell transcription factors interacting with the long terminal repeat (LTR) region (for review, see Refs. 13Garcia J.A. Gaynor R.B. Prog. Nucleic Acid Res. Mol. Biol. 1994; 49: 157-196Crossref PubMed Scopus (33) Google Scholar and 14Kingsman S.M. Kingsman A.J. Eur. J. Biochem. 1996; 240: 491-507Crossref PubMed Scopus (106) Google Scholar). Recent studies have focused on transcription factors that regulate HIV-1 expression in brain cells. Analysis of HIV-1 directed gene expression in transgenic mice derived from CNS-isolated HIV-1 strains (15Koyanagi Y. Miles S. Mitsuyasu R.T. Merrill J.E. Vinters H.V. Chen I.S.Y. Science. 1987; 236: 819-822Crossref PubMed Scopus (622) Google Scholar), suggests that HIV-1 replication in the CNS uses transcription factors different from those in non-neural tissues (16Corboy J.R. Busy J.M. Zink M.C. Clements J.E. Science. 1992; 258: 1804-1808Crossref PubMed Scopus (69) Google Scholar, 17Busy J.M. Lindstrom L.M. Zink M.C. Clements J.E. Virology. 1995; 210: 361-371Crossref PubMed Scopus (13) Google Scholar, 18Kurth J. Busy J.M. Lindstrom L. Clements J.E. J. Virol. 1996; 70: 7686-7694Crossref PubMed Google Scholar). Transcriptional activity of the HIV-1 promoter is mediated by κB regulatory sequences of the LTR, through the action of the transcription factor NF-κB, both in neurons (19Rattner A. Korner M. Walker M.D. Citri Y. EMBO J. 1993; 12: 4261-4267Crossref PubMed Scopus (78) Google Scholar,20Kaltschmidt C. Kaltschmidt B. Neumann H. Wekerle H. Baeuerle P.A. Mol. Cell. Biol. 1994; 14: 3981-3992Crossref PubMed Google Scholar) and in astrocytes (21Taylor J.P. Khalili K. Adv. Neuroimm. 1994; 4: 291-303Abstract Full Text PDF PubMed Scopus (20) Google Scholar). Besides the κB regulatory element, our recent data have highlighted the importance of the nuclear receptor-responsive element (NRRE), which appears to be the point of convergence of a network of physiological signals which modulate HIV-1 gene expression in brain cells. We have described that the orphan nuclear receptors COUP-TF/Ear3 (22Wang L.-H. Tsai S.Y. Sagami I. Tsai M.-J. O'Malley B.W. J. Biol. Chem. 1987; 262: 16080-16086Abstract Full Text PDF PubMed Google Scholar, 23Wang L.H. Tsai S.Y. Cook R.G. Beattie W.G. Tsai M.J. O'Malley B.W. Nature. 1989; 340: 163-166Crossref PubMed Scopus (385) Google Scholar, 24Miyajima N.Y. Kadowaki S.-I. Fukushige S.-I. Shimizu K. Semba Y. Yamanashi K.-I. Matsubara K. Yoyoshima K. Yamamoto T. Nucleic Acids Res. 1988; 16: 11057-11069Crossref PubMed Scopus (173) Google Scholar) are present in three human brain cell lines, oligodendroglioma TC-620, astrocytoma U373-MG, and neuroblastoma SK-N-MC cells. Our data have also demonstrated the importance of COUP-TF as a potent transcriptional activator in oligodendroglioma cells, of both the lymphotropic LAI and the neurotropic JR-CSF HIV-1 LTR. They revealed the action of the dopamine transduction pathway, which coupled to COUP-TF, contributes to enhance HIV-1 gene transcription in neuronal cells (25Sawaya B.E. Rohr O. Aunis D. Schaeffer E. J. Biol. Chem. 1996; 271: 23572-23576Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Our findings have further established the importance of the retinoic acid signaling pathway in HIV-1 gene transcription in glial and neuronal cells (26Sawaya B.E. Rohr O. Aunis D. Schaeffer E. J. Biol. Chem. 1996; 271: 22895-22900Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). Except for retinoid receptors, the role of transcription factors belonging to the steroid/thyroid/retinoid receptor superfamily has not yet been described in microglial cells. In this report, we have investigated the functional effect and the molecular mechanisms by which the orphan nuclear receptor COUP-TF regulates HIV-1 gene expression in a human microglial cell line (27Janabi N. Peudenier S. Héron B. Ng K.H. Tardieu M. Neurosci. Lett. 1995; 195: 105-108Crossref PubMed Scopus (195) Google Scholar). Our findings reveal the importance of COUP-TF as an activator of LTR-driven transcription of both the T cell line-tropic LAI and the macrophage-tropic JR-FL HIV-1 strains. We provide evidence for a novel physiological and functional interaction between the DNA-binding domain of COUP-TF and the transcription factor Sp1, leading to enhanced transactivation of the HIV-1 LTR. In addition, our data reveal that the COUP-TF- and Sp1-induced stimulation is repressed by the transcription factor Sp3. These data describe novel molecular mechanisms which govern the network of interactions between the HIV-1 LTR, the transcriptional activators COUP-TF and Sp1, and the repressor Sp3, in the regulation of HIV-1 gene expression. DISCUSSIONIn this report we have investigated the regulation of HIV-1 gene transcription in human microglial cells, which represent the primary target of HIV-1 infection in the central nervous system. Since we have previously shown that the orphan nuclear receptor COUP-TF is expressed in brain cells and leads to a transcriptional stimulation of the HIV-1 genome in oligodendroglioma and neuronal cells (25Sawaya B.E. Rohr O. Aunis D. Schaeffer E. J. Biol. Chem. 1996; 271: 23572-23576Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), we have focused our studies on the regulation of HIV-1 gene transcription by COUP-TF in microglial cells. It is now well established that COUP-TF is able to exert both positive and negative effects on gene expression, depending upon the promoter and the cell contexts (29Schaeffer E. Guillou F. Part D. Zakin M.M. J. Biol. Chem. 1993; 268: 23399-23408Abstract Full Text PDF PubMed Google Scholar, 35Kimura A. Nishiyori A. Murakami T. Tsukamoto T. Hata S. Osumi T. Okamura R. Mori M. Takiguchi M. J. Biol. Chem. 1993; 268: 11125-11133Abstract Full Text PDF PubMed Google Scholar, 36Kadowaki Y. Toyoshima K. Yamamoto T. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4432-4436Crossref PubMed Scopus (23) Google Scholar, 37Sawaya B.E. Schaeffer E. Nucleic Acids Res. 1995; 23: 2206-2211Crossref PubMed Scopus (19) Google Scholar, 38Leng X. Cooney A.J. Tsai S.Y. Tsai M.-J. Mol. Cell. Biol. 1996; 16: 2332-2340Crossref PubMed Scopus (84) Google Scholar). We show here that COUP-TF proteins are present in human microglial cells and interact directly with the NRRE, spanning the −356/−320 LTR region of the lymphotropic HIV-1 LAI isolate, but are unable to bind to the mutant NRRE present in the macrophage-tropic HIV-1 JR-FL isolate. Interestingly, our transient expression data reveal that COUP-TF/Ear3 is able to activate HIV-1 LTR-driven transcription in microglial cells, independently of the NRRE sequence. This shows that COUP-TF is able to transactivate the LTR from the lymphotropic LAI and the macrophage-tropic JR-FL isolates.We have already reported that distinct cell-type specific and sequence-dependent mechanisms govern COUP-TF-induced stimulation. In oligodendrocytes this transcription factor stimulates HIV-1 gene transcription, either by direct interactions with its NRRE target site or also, depending on the LTR sequence, by cross-coupling interactions with downstream-located proteins. In neuronal SK-N-MC cells, the transcriptional stimulation induced by COUP-TF in the presence of dopamine is mediated by the minimal −68/+29 LTR region (25Sawaya B.E. Rohr O. Aunis D. Schaeffer E. J. Biol. Chem. 1996; 271: 23572-23576Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Similarly the NRRE site in the JR-CSF LTR is not indispensable for the stimulation induced by the retinoic acid receptor, since the retinoid action appears to be mediated by downstream-located elements, such as the −247/−222 AP-1 region and, to a lesser extent, the NF-κB region (26Sawaya B.E. Rohr O. Aunis D. Schaeffer E. J. Biol. Chem. 1996; 271: 22895-22900Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar).We show here that the minimal −68/+29 LTR region, containing two Sp1-binding sites, is sufficient for COUP-TF-mediated stimulation. Our cotransfection and in vitro experiments demonstrate the ability of COUP-TF to transactivate the LTR indirectly via the Sp1 sites, by direct interaction with the Sp1 protein. We present evidence for a direct association between the N-terminal part of COUP-TF, containing the DNA-binding domain, with Sp1. Our data show that COUP-TF can interact with Sp1 both in vitro and in microglial cells. This association leads to a functional cooperation between the two proteins, which is detected with the full-length LTR as well as the −68/+80 LTR region. Moreover we show that the C-terminal domain of COUP-TF is involved in the transcriptional activation.Previous studies have already described that the action of COUP-TFs can be mediated not only by direct interaction with their DNA-binding site, but also by protein-protein interaction. COUP-TFI/Ear3 and ARP-1/COUP-TFII are able to directly target components of the basal transcription machinery, such as the basal transcription factor TFIIB (39Ing N.H. Beckman J.M. Tsai S.Y. Tsai M.J. O'Malley B.M. J. Biol. Chem. 1992; 267: 17617-17623Abstract Full Text PDF PubMed Google Scholar, 40Malik S. Karathanasis S. Nucleic Acids Res. 1995; 23: 1536-1543Crossref PubMed Scopus (60) Google Scholar). TFIIB recognizes and associates with COUP-TF and two other members of the steroid hormone receptor family, via their activation domain (39Ing N.H. Beckman J.M. Tsai S.Y. Tsai M.J. O'Malley B.M. J. Biol. Chem. 1992; 267: 17617-17623Abstract Full Text PDF PubMed Google Scholar). In contrast, our findings reveal that COUP-TF associates with Sp1 via its N-terminal part, containing the DNA-binding domain. A direct interaction between COUP-TF and the transcription factors Oct1 and Oct2 has also been demonstrated in the regulation of the vHNFI promoter (30Power S.C. Cereghini S. Mol. Cell. Biol. 1996; 16: 778-791Crossref PubMed Google Scholar).It has been well established that the Sp1 transcription factor (41Kadonaga J.T. Carner K.R. Masiarz F.R. Tjian R. Cell. 1987; 51: 1079-1090Abstract Full Text PDF PubMed Scopus (1246) Google Scholar) plays an essential role in the regulation of basal transcription as well as in Tat-mediated transactivation of the HIV-1 LTR (42Jones K.A. Kadonaga J.T. Luciw P.A. Tjian R. Science. 1986; 232: 755-759Crossref PubMed Scopus (444) Google Scholar, 43Sune C. Garcia-Blanco M.A. J. Virol. 1995; 69: 6572-6576Crossref PubMed Google Scholar, 44Harrich D. Garcia J. Wu F. Mitsuyasu R. Gonazalez J. Gaynor R. J. Virol. 1989; 63: 2585-2591Crossref PubMed Google Scholar). Recent reports describe that Sp1 is critical for in vivotranscriptional regulation of HIV, through its interaction with other DNA-binding proteins. A cooperative interaction between Sp1 and NF-κB, bound to the two adjacent binding sites, is required for optimal HIV-1 enhancer activation and inducible HIV-1 gene expression (32Majello B. De Luca P. Hagen G. Suske G. Lania L. Nucleic Acids Res. 1994; 22 (1921): 4914Crossref PubMed Scopus (202) Google Scholar, 45Perkins N.D. Edwards N.L. Duckett C.S. Agranoff A.B. Schmid R.M. Nabel G.J. EMBO J. 1993; 12: 3551-3558Crossref PubMed Scopus (399) Google Scholar, 46Li Y. Mak G. Franza Jr., B.R. J. Biol. Chem. 1994; 269: 30616-30619Abstract Full Text PDF PubMed Google Scholar). A physical interaction between Sp1 and the p53 tumor-suppressor gene has been described in the tumor necrosis factor-induced transcriptional activation of the HIV-1 LTR (47Gualberto A. Baldwin Jr., A.S. J. Biol. Chem. 1995; 270: 19680-19683Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Moreover in vitro and in vivo data suggest a direct interaction between Sp1 and Tat during transactivation (48Kamine J. Chinnadurai G. J. Virol. 1992; 66: 3932-3936Crossref PubMed Google Scholar,49Jeang K.T. Chun R. Lin N.H. Gatignol A. Glabe C.G. Fan H. J. Virol. 1993; 67: 6224-6233Crossref PubMed Google Scholar).Here our findings reveal a novel direct association between two zinc finger proteins, Sp1 and COUP-TF, which leads to enhanced transcriptional activation of the HIV-1 genome. These data suggest a more general model for gene activation by the orphan receptor COUP-TF or steroid/thyroid/retinoid receptors, in the presence of Sp1-binding sites. As shown here, Sp1 bound to its DNA-binding site is able to associate directly with the N-terminal DNA-binding domain of the nuclear receptor. It is well known that the adjacent TATA element binds the general transcription factor TFIID, which directly interacts with TFIIB, which itself is able to bind COUP-TF. In such a situation, the nuclear receptor may function as an adaptor protein, bringing together Sp1 and TFIIB, thereby enhancing communication between the general transcription machinery and Sp1. Alternatively, COUP-TF may bring together Sp1 and a cell type-specific nuclear factor, leading to various levels of activation induced by COUP-TF in different cell types. The precise mechanisms which account for our observations in the case of HIV-1 and SV40 gene transcription need to be further investigated.The Sp1 multigene family contains three closely related members Sp1, Sp3, and Sp4 which recognize the GC box and the GT motif (33Hagen G. Müller S. Beato M. Suske G. EMBO J. 1994; 13: 3843-3851Crossref PubMed Scopus (650) Google Scholar). While Sp1 and Sp4 function as transcriptional activators, Sp3 is a bifunctional transcription regulator, whose activity is dependent upon the promoter and the cellular contexts (33Hagen G. Müller S. Beato M. Suske G. EMBO J. 1994; 13: 3843-3851Crossref PubMed Scopus (650) Google Scholar, 34Majello B. De Luca P. Lania L. J. Biol. Chem. 1997; 272: 4021-4026Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar). Recent reports have established that different members of the Sp1 family exert opposite transcriptional regulation of the HIV LTR. While Sp1 and Sp4 stimulate transcription of the LTR, Sp3 markedly represses the HIV promoter activity in HeLa and Drosophila SL2 cells. Sp3-dependent repression is dependent on the presence of the DNA-binding domain, indicating that repression occurs through interference with Sp1 binding to the GC motifs (32Majello B. De Luca P. Hagen G. Suske G. Lania L. Nucleic Acids Res. 1994; 22 (1921): 4914Crossref PubMed Scopus (202) Google Scholar). Our data confirm that Sp1 and Sp3 are present in microglial cells, and exert an antagonist action on LTR-driven transcription. Moreover they show that Sp3 is able to mediate transcriptional repression not only of the Sp1 action, but also of the COUP-TF action exerted via Sp1.Only a limited number of studies have been devoted to HIV-1 gene transcription in macrophages and microglial cells. A differential role of LTR elements for the regulation of basal and Tat-mediated transcription of HIV-1 has been reported in stimulated and unstimulated primary human macrophages (50Moses A.V. Ibanez C. Gaynor R. Ghazal P. Nelson J.A. J. Virol. 1994; 68: 298-307Crossref PubMed Google Scholar). Our findings establish the essential role of the orphan nuclear receptor COUP-TF as a transcriptional activator of LTR-directed HIV-1 gene expression in microglial cells. They further reveal a novel mechanism of HIV-1 gene transactivation by direct association of COUP-TF with Sp1, thus enhancing transcription via the minimal LTR region. Our present and previous studies (25Sawaya B.E. Rohr O. Aunis D. Schaeffer E. J. Biol. Chem. 1996; 271: 23572-23576Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) reveal the remarkable diverse mechanisms by which COUP-TF regulates gene transcription of distinct HIV-1 strains and in different brain cells. In this report we have investigated the regulation of HIV-1 gene transcription in human microglial cells, which represent the primary target of HIV-1 infection in the central nervous system. Since we have previously shown that the orphan nuclear receptor COUP-TF is expressed in brain cells and leads to a transcriptional stimulation of the HIV-1 genome in oligodendroglioma and neuronal cells (25Sawaya B.E. Rohr O. Aunis D. Schaeffer E. J. Biol. Chem. 1996; 271: 23572-23576Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), we have focused our studies on the regulation of HIV-1 gene transcription by COUP-TF in microglial cells. It is now well established that COUP-TF is able to exert both positive and negative effects on gene expression, depending upon the promoter and the cell contexts (29Schaeffer E. Guillou F. Part D. Zakin M.M. J. Biol. Chem. 1993; 268: 23399-23408Abstract Full Text PDF PubMed Google Scholar, 35Kimura A. Nishiyori A. Murakami T. Tsukamoto T. Hata S. Osumi T. Okamura R. Mori M. Takiguchi M. J. Biol. Chem. 1993; 268: 11125-11133Abstract Full Text PDF PubMed Google Scholar, 36Kadowaki Y. Toyoshima K. Yamamoto T. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4432-4436Crossref PubMed Scopus (23) Google Scholar, 37Sawaya B.E. Schaeffer E. Nucleic Acids Res. 1995; 23: 2206-2211Crossref PubMed Scopus (19) Google Scholar, 38Leng X. Cooney A.J. Tsai S.Y. Tsai M.-J. Mol. Cell. Biol. 1996; 16: 2332-2340Crossref PubMed Scopus (84) Google Scholar). We show here that COUP-TF proteins are present in human microglial cells and interact directly with the NRRE, spanning the −356/−320 LTR region of the lymphotropic HIV-1 LAI isolate, but are unable to bind to the mutant NRRE present in the macrophage-tropic HIV-1 JR-FL isolate. Interestingly, our transient expression data reveal that COUP-TF/Ear3 is able to activate HIV-1 LTR-driven transcription in microglial cells, independently of the NRRE sequence. This shows that COUP-TF is able to transactivate the LTR from the lymphotropic LAI and the macrophage-tropic JR-FL isolates. We have already reported that distinct cell-type specific and sequence-dependent mechanisms govern COUP-TF-induced stimulation. In oligodendrocytes this transcription factor stimulates HIV-1 gene transcription, either by direct interactions with its NRRE target site or also, depending on the LTR sequence, by cross-coupling interactions with downstream-located proteins. In neuronal SK-N-MC cells, the transcriptional stimulation induced by COUP-TF in the presence of dopamine is mediated by the minimal −68/+29 LTR region (25Sawaya B.E. Rohr O. Aunis D. Schaeffer E. J. Biol. Chem. 1996; 271: 23572-23576Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Similarly the NRRE site in the JR-CSF LTR is not indispensable for the stimulation induced by the retinoic acid receptor, since the retinoid action appears to be mediated by downstream-located elements, such as the −247/−222 AP-1 region and, to a lesser extent, the NF-κB region (26Sawaya B.E. Rohr O. Aunis D. Schaeffer E. J. Biol. Chem. 1996; 271: 22895-22900Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). We show here that the minimal −68/+29 LTR region, containing two Sp1-binding sites, is sufficient for COUP-TF-mediated stimulation. Our cotransfection and in vitro experiments demonstrate the ability of COUP-TF to transactivate the LTR indirectly via the Sp1 sites, by direct interaction with the Sp1 protein. We present evidence for a direct association between the N-terminal part of COUP-TF, containing the DNA-binding domain, with Sp1. Our data show that COUP-TF can interact with Sp1 both in vitro and in microglial cells. This association leads to a functional cooperation between the two proteins, which is detected with the full-length LTR as well as the −68/+80 LTR region. Moreover we show that the C-terminal domain of COUP-TF is involved in the transcriptional activation. Previous studies have already described that the action of COUP-TFs can be mediated not only by direct interaction with their DNA-binding site, but also by protein-protein interaction. COUP-TFI/Ear3 and ARP-1/COUP-TFII are able to directly target components of the basal transcription machinery, such as the basal transcription factor TFIIB (39Ing N.H. Beckman J.M. Tsai S.Y. Tsai M.J. O'Malley B.M. J. Biol. Chem. 1992; 267: 17617-17623Abstract Full Text PDF PubMed Google Scholar, 40Malik S. Karathanasis S. Nucleic Acids Res. 1995; 23: 1536-1543Crossref PubMed Scopus (60) Google Scholar). TFIIB recognizes and associates with COUP-TF and two other members of the steroid hormone receptor family, via their activation domain (39Ing N.H. Beckman J.M. Tsai S.Y. Tsai M.J. O'Malley B.M. J. Biol. Chem. 1992; 267: 17617-17623Abstract Full Text PDF PubMed Google Scholar). In contrast, our findings reveal that COUP-TF associates with Sp1 via its N-terminal part, containing the DNA-binding domain. A direct interaction between COUP-TF and the transcription factors Oct1 and Oct2 has also been demonstrated in the regulation of the vHNFI promoter (30Power S.C. Cereghini S. Mol. Cell. Biol. 1996; 16: 778-791Crossref PubMed Google Scholar). It has been well established that the Sp1 transcription factor (41Kadonaga J.T. Carner K.R. Masiarz F.R. Tjian R. Cell. 1987; 51: 1079-1090Abstract Full Text PDF PubMed Scopus (1246) Google Scholar) plays an essential role in the regulation of basal transcription as well as in Tat-mediated transactivation of the HIV-1 LTR (42Jones K.A. Kadonaga J.T. Luciw P.A. Tjian R. Science. 1986; 232: 755-759Crossref PubMed Scopus (444) Google Scholar, 43Sune C. Garcia-Blanco M.A. J. Virol. 1995; 69: 6572-6576Crossref PubMed Google Scholar, 44Harrich D. Garcia J. Wu F. Mitsuyasu R. Gonazalez J. Gaynor R. J. Virol. 1989; 63: 2585-2591Crossref PubMed Google Scholar). Recent reports describe that Sp1 is critical for in vivotranscriptional regulation of HIV, through its interaction with other DNA-binding proteins. A cooperative interaction between Sp1 and NF-κB, bound to the two adjacent binding sites, is required for optimal HIV-1 enhancer activation and inducible HIV-1 gene expression (32Majello B. De Luca P. Hagen G. Suske G. Lania L. Nucleic Acids Res. 1994; 22 (1921): 4914Crossref PubMed Scopus (202) Google Scholar, 45Perkins N.D. Edwards N.L. Duckett C.S. Agranoff A.B. Schmid R.M. Nabel G.J. EMBO J. 1993; 12: 3551-3558Crossref PubMed Scopus (399) Google Scholar, 46Li Y. Mak G. Franza Jr., B.R. J. Biol. Chem. 1994; 269: 30616-30619Abstract Full Text PDF PubMed Google Scholar). A physical interaction between Sp1 and the p53 tumor-suppressor gene has been described in the tumor necrosis factor-induced transcriptional activation of the HIV-1 LTR (47Gualberto A. Baldwin Jr., A.S. J. Biol. Chem. 1995; 270: 19680-19683Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Moreover in vitro and in vivo data suggest a direct interaction between Sp1 and Tat during transactivation (48Kamine J. Chinnadurai G. J. Virol. 1992; 66: 3932-3936Crossref PubMed Google Scholar,49Jeang K.T. Chun R. Lin N.H. Gatignol A. Glabe C.G. Fan H. J. Virol. 1993; 67: 6224-6233Crossref PubMed Google Scholar). Here our findings reveal a novel direct association between two zinc finger proteins, Sp1 and COUP-TF, which leads to enhanced transcriptional activation of the HIV-1 genome. These data suggest a more general model for gene activation by the orphan receptor COUP-TF or steroid/thyroid/retinoid receptors, in the presence of Sp1-binding sites. As shown here, Sp1 bound to its DNA-binding site is able to associate directly with the N-terminal DNA-binding domain of the nuclear receptor. It is well known that the adjacent TATA element binds the general transcription factor TFIID, which directly interacts with TFIIB, which itself is able to bind COUP-TF. In such a situation, the nuclear receptor may function as an adaptor protein, bringing together Sp1 and TFIIB, thereby enhancing communication between the general transcription machinery and Sp1. Alternatively, COUP-TF may bring together Sp1 and a cell type-specific nuclear factor, leading to various levels of activation induced by COUP-TF in different cell types. The precise mechanisms which account for our observations in the case of HIV-1 and SV40 gene transcription need to be further investigated. The Sp1 multigene family contains three closely related members Sp1, Sp3, and Sp4 which recognize the GC box and the GT motif (33Hagen G. Müller S. Beato M. Suske G. EMBO J. 1994; 13: 3843-3851Crossref PubMed Scopus (650) Google Scholar). While Sp1 and Sp4 function as transcriptional activators, Sp3 is a bifunctional transcription regulator, whose activity is dependent upon the promoter and the cellular contexts (33Hagen G. Müller S. Beato M. Suske G. EMBO J. 1994; 13: 3843-3851Crossref PubMed Scopus (650) Google Scholar, 34Majello B. De Luca P. Lania L. J. Biol. Chem. 1997; 272: 4021-4026Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar). Recent reports have established that different members of the Sp1 family exert opposite transcriptional regulation of the HIV LTR. While Sp1 and Sp4 stimulate transcription of the LTR, Sp3 markedly represses the HIV promoter activity in HeLa and Drosophila SL2 cells. Sp3-dependent repression is dependent on the presence of the DNA-binding domain, indicating that repression occurs through interference with Sp1 binding to the GC motifs (32Majello B. De Luca P. Hagen G. Suske G. Lania L. Nucleic Acids Res. 1994; 22 (1921): 4914Crossref PubMed Scopus (202) Google Scholar). Our data confirm that Sp1 and Sp3 are present in microglial cells, and exert an antagonist action on LTR-driven transcription. Moreover they show that Sp3 is able to mediate transcriptional repression not only of the Sp1 action, but also of the COUP-TF action exerted via Sp1. Only a limited number of studies have been devoted to HIV-1 gene transcription in macrophages and microglial cells. A differential role of LTR elements for the regulation of basal and Tat-mediated transcription of HIV-1 has been reported in stimulated and unstimulated primary human macrophages (50Moses A.V. Ibanez C. Gaynor R. Ghazal P. Nelson J.A. J. Virol. 1994; 68: 298-307Crossref PubMed Google Scholar). Our findings establish the essential role of the orphan nuclear receptor COUP-TF as a transcriptional activator of LTR-directed HIV-1 gene expression in microglial cells. They further reveal a novel mechanism of HIV-1 gene transactivation by direct association of COUP-TF with Sp1, thus enhancing transcription via the minimal LTR region. Our present and previous studies (25Sawaya B.E. Rohr O. Aunis D. Schaeffer E. J. Biol. Chem. 1996; 271: 23572-23576Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) reveal the remarkable diverse mechanisms by which COUP-TF regulates gene transcription of distinct HIV-1 strains and in different brain cells. We thank N. Israël and J. Clements for providing the vectors containing the LAI and JR-FL LTR, respectively. We are grateful to R. Tjian and G. Suske for providing the Sp1 and Sp3 expression vectors, respectively, and M. J. Tsai for providing the COUP-TF antibodies.