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• W2067335145 abstract "The human immunodeficiency virus (HIV) Nef protein plays a critical role in AIDS pathogenesis by enhancing replication and survival of the virus within infected cells and by facilitating its spread in vivo. Most of the data obtained so far have been in experiments with endogenous Nef protein, so far overlooking the effects of exogenous soluble Nef protein. We used recombinant exogenous Nef proteins to activate nuclear transcription factors NF-κB and AP-1 in the promonocytic cell line U937. Exogenous SIV and HIV-1 Nef proteins activated NF-κB and AP-1 in a dose- and time-dependent manner. Activation of NF-κB by exogenous Nef was concomitant to the degradation of the inhibitor of NF-κB, IκBα. In agreement with increased AP-1 activation, a time- and dose-dependent increase in JNK activation was observed following treatment of U937 cells with exogenous Nef. Since exogenous Nef activates the transcription factors NF-κB and AP-1, which bind to the HIV-1 long terminal repeat (LTR), we investigated the effect of exogenous Nef on HIV-1 replication. We observed that exogenous Nef stimulated HIV-1 LTR via NF-κB activation in U937 cells and enhanced viral replication in the chronically infected promonocytic cells U1. Therefore, our results suggest that exogenous Nef could fuel the progression of the disease via stimulation of HIV-1 provirus present in such cellular reservoirs as mononuclear phagocytes in HIV-infected patients. The human immunodeficiency virus (HIV) Nef protein plays a critical role in AIDS pathogenesis by enhancing replication and survival of the virus within infected cells and by facilitating its spread in vivo. Most of the data obtained so far have been in experiments with endogenous Nef protein, so far overlooking the effects of exogenous soluble Nef protein. We used recombinant exogenous Nef proteins to activate nuclear transcription factors NF-κB and AP-1 in the promonocytic cell line U937. Exogenous SIV and HIV-1 Nef proteins activated NF-κB and AP-1 in a dose- and time-dependent manner. Activation of NF-κB by exogenous Nef was concomitant to the degradation of the inhibitor of NF-κB, IκBα. In agreement with increased AP-1 activation, a time- and dose-dependent increase in JNK activation was observed following treatment of U937 cells with exogenous Nef. Since exogenous Nef activates the transcription factors NF-κB and AP-1, which bind to the HIV-1 long terminal repeat (LTR), we investigated the effect of exogenous Nef on HIV-1 replication. We observed that exogenous Nef stimulated HIV-1 LTR via NF-κB activation in U937 cells and enhanced viral replication in the chronically infected promonocytic cells U1. Therefore, our results suggest that exogenous Nef could fuel the progression of the disease via stimulation of HIV-1 provirus present in such cellular reservoirs as mononuclear phagocytes in HIV-infected patients. human immunodeficiency virus activator protein-1 inhibitor of NF-κB Jun N-terminal kinase nuclear factor κB Nef-associated kinase p21-activated kinase mitogen-activated protein kinase long terminal repeat tumor necrosis factor IκBα kinase signal transducer and activator of transcription 1 secreted alkaline phosphatase lipopolysaccharide fluorescein isothiocyanate electrophoretic mobility shift assay Nef is a 27-kDa HIV1protein that is produced early during infection and translated from multiply spliced viral mRNAs (1Kim S.Y. Byrn R. Groopman J. Baltimore D. J. Virol. 1989; 63: 3708-3713Crossref PubMed Google Scholar). Information is beginning to emerge that suggests that endogenous Nef may have evolved a number of different, independent functional activities to enhance the replication and survival of the virus within infected cells and to facilitate its spread in vivo (2Lama J. Mangasarian A. Trono D. Curr. Biol. 1999; 9: 622-631Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, 3Ross T.M. Oran A.E. Cullen B.R. Curr. Biol. 1999; 9: 613-621Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar). Nef enhances virion infectivity (4Aiken C. Trono D. J. Virol. 1995; 69: 5048-5056Crossref PubMed Google Scholar,5Harris M. Curr. Biol. 1999; 9: R459-R461Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) and increases viral replication in primary lymphocytes and macrophages (6Miller M.D. Warmerdam M.T. Gaston I. Greene W.C. Feinberg M.B. J. Exp. Med. 1994; 179: 101-113Crossref PubMed Scopus (479) Google Scholar, 7Spina C.A. Kwoh T.J. Chowers M.Y. Guatelli J.C. Richman D.D. J. Exp. Med. 1994; 179: 115-123Crossref PubMed Scopus (363) Google Scholar). Nef can mediate down-regulation of CD4 cell surface expression, a phenomenon shown to be important for the release of HIV-1 from the cell (2Lama J. Mangasarian A. Trono D. Curr. Biol. 1999; 9: 622-631Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, 3Ross T.M. Oran A.E. Cullen B.R. Curr. Biol. 1999; 9: 613-621Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar). Nef can also downregulate the cell surface expression of major histocompatibility complex class I (MHC-I) molecules, (8Schwartz O. Marechal V., Le Gall S. Lemonnier F. Heard J.M. Nat. Med. 1996; 2: 338-342Crossref PubMed Scopus (877) Google Scholar), an effect found to protect infected cells from killing by cytotoxic T cells (9Collins K.L. Chen B.K. Kalams S.A. Walker B.D. Baltimore D. Nature. 1998; 391: 397-401Crossref PubMed Scopus (851) Google Scholar). Nef prevents apoptosis of HIV-1-infected T cells (10Geleziunas R., Xu, W. Takeda K. Ichijo H. Greene W.C. Nature. 2001; 410: 834-838Crossref PubMed Scopus (292) Google Scholar, 11Mahlknecht U. Deng C., Lu, M.C. Greenough T.C. Sullivan J.L. O'Brien W.A. Herbein G. J. Immunol. 2000; 165: 6437-6446Crossref PubMed Scopus (54) Google Scholar, 12Wolf D. Witte V. Laffert B. Blume K. Stromer E. Trapp S. d'Aloja P. Schurmann A. Baur A.S. Nat. Med. 2001; 7: 1217-1224Crossref PubMed Scopus (250) Google Scholar). Nef expression within macrophages has been reported to favor the recruitment of resting T cells via the secretion of C-C chemokines and to subsequently favor their activation, suggesting a role for Nef in lymphocyte recruitment and activation at sites of viral replication (13Swingler S. Mann A. Jacque J. Brichacek B. Sasseville V.G. Williams K. Lackner A.A. Janoff E.N. Wang R. Fisher D. Stevenson M. Nat. Med. 1999; 5: 997-1003Crossref PubMed Scopus (322) Google Scholar). In vivo, several studies have demonstrated the importance of Nef for the efficiency of viral replication and for the maintenance of high viral loads (14Kestler 3rd, H.W. Ringler D.J. Mori K. Panicali D.L. Sehgal P.K. Daniel M.D. Desrosiers R.C. Cell. 1991; 65: 651-662Abstract Full Text PDF PubMed Scopus (1431) Google Scholar, 15Deacon N.J. Tsykin A. Solomon A. Smith K. Ludford-Menting M. Hooker D.J. McPhee D.A. Greenway A.L. Ellett A. Chatfield C. Science. 1995; 270: 988-991Crossref PubMed Scopus (1026) Google Scholar). Nef can alter T-cell signaling pathways (16Niederman T.M. Garcia J.V. Hastings W.R. Luria S. Ratner L. J. Virol. 1992; 66: 6213-6219Crossref PubMed Google Scholar). Nef has been found to interact with several signaling molecules: with a serine kinase (17Baur A.S. Sass G. Laffert B. Willbold D. Cheng-Mayer C. Peterlin B.M. Immunity. 1997; 6: 283-291Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar), with a distinct serine/threonine kinase, the Nef-associated kinase (NAK) identified as a member of the p21-activated kinase (PAK) family (18Lu X., Wu, X. Plemenitas A., Yu, H. Sawai E.T. Abo A. Peterlin B.M. Curr. Biol. 1996; 6: 1677-1684Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 19Luo T. Garcia J.V. J. Virol. 1996; 70: 6493-6496Crossref PubMed Google Scholar, 20Nunn M.F. Marsh J.W. J. Virol. 1996; 70: 6157-6161Crossref PubMed Google Scholar, 21Sawai E.T. Khan I.H. Montbriand P.M. Peterlin B.M. Cheng-Mayer C. Luciw P.A. Curr. Biol. 1996; 6: 1519-1527Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar), with members of the Src-family of tyrosine kinases, notably Lck (17Baur A.S. Sass G. Laffert B. Willbold D. Cheng-Mayer C. Peterlin B.M. Immunity. 1997; 6: 283-291Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 22Collette Y. 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Heidecker G. Lautenberger J.A. Samuel K.P. J. Biol. Chem. 1998; 273: 15727-15733Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), p53 (29Greenway A.L. McPhee D.A. Allen K. Johnstone R. Holloway G. Mills J. Azad A. Sankovich S. Lambert P. J. Virol. 2002; 76: 2692-2702Crossref PubMed Scopus (103) Google Scholar), and protein kinase Cθ (30Smith B.L. Krushelnycky B.W. Mochly-Rosen D. Berg P. J. Biol. Chem. 1996; 271: 16753-16757Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). HIV replication is tightly regulated at the transcriptional level through the specific interaction of viral regulatory proteins, namely, Tat and cellular transcription factors binding to a variety of cis-acting DNA sequences in the HIV long terminal repeat (LTR) (reviewed in Ref. 31Cullen B.R. Faseb J. 1991; 5: 2361-2368Crossref PubMed Scopus (198) Google Scholar). One of the main mediators of HIV LTR transcription is nuclear factor-κB (NF-κB) (32Nabel G. Baltimore D. Nature. 1987; 326: 711-713Crossref PubMed Scopus (1453) Google Scholar). An inducible transcription factor, NF-κB is composed of homo- and heterodimers of Rel family proteins (reviewed in Ref. 33Karin M. Ben-Neriah Y. Annu. Rev. Immunol. 2000; 18: 621-663Crossref PubMed Scopus (4084) Google Scholar). All of the NF-κB proteins contain an N-terminal Rel homology domain, which mediates DNA binding, dimerization, and interaction with the inhibitory proteins, or IκBs. In addition, c-Rel, p65 (RelA), and RelB contain a C-terminal transactivation domain (reviewed in Ref. 33Karin M. Ben-Neriah Y. Annu. Rev. Immunol. 2000; 18: 621-663Crossref PubMed Scopus (4084) Google Scholar). The classic NF-κB complex (p50/p65) is sequestered in the cytoplasm by interaction with a family of inhibitory proteins, or IκBs, including IκBα, IκBβ, IκBε, IκBγ, and the proto-oncogene Bcl-3 (34Baldwin A.S., Jr. Annu. Rev. Immunol. 1996; 14: 649-683Crossref PubMed Scopus (5578) Google Scholar). Following cell activation by a variety of extracellular stimuli, such as tumor necrosis factor α (TNF), IκBα is phosphorylated at the N-terminal residues Ser-32 and Ser-36 by the IκB kinase (IKK) complex, leading to ubiquitination and subsequent proteasome-mediated degradation, which allows NF-κB to translocate to the nucleus, where it activates gene expression. Since the identification of NF-κB elements in the HIV LTR (32Nabel G. Baltimore D. Nature. 1987; 326: 711-713Crossref PubMed Scopus (1453) Google Scholar), multiple studies have assessed the effect of this family of transcription factors on the transcriptional regulation of the HIV LTR and its impact on HIV reactivation from latency (35Chen P. Mayne M. Power C. Nath A. J. Biol. Chem. 1997; 272: 22385-22388Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 36Finzi D. Hermankova M. Pierson T. Carruth L.M. Buck C. Chaisson R.E. Quinn T.C. Chadwick K. Margolick J. Brookmeyer R. Gallant J. Markowitz M., Ho, D.D. Richman D.D. Siliciano R.F. Science. 1997; 278: 1295-1300Crossref PubMed Scopus (2483) Google Scholar, 37Folks T. Powell D.M. Lightfoote M.M. Benn S. Martin M.A. Fauci A.S. Science. 1986; 231: 600-602Crossref PubMed Scopus (205) Google Scholar, 38Folks T.M. Justement J. Kinter A. Dinarello C.A. Fauci A.S. Science. 1987; 238: 800-802Crossref PubMed Scopus (717) Google Scholar). Study of the interaction between NF-κB and HIV in both human monocytic cells and transformed human macrophages has mainly focused on how monocyte differentiation may lead to HIV expression (39Griffin G.E. Leung K. Folks T.M. Kunkel S. Nabel G.J. Nature. 1989; 339: 70-73Crossref PubMed Scopus (441) Google Scholar, 40Schuitemaker H. Kootstra N.A. Koppelman M.H. Bruisten S.M. Huisman H.G. Tersmette M. Miedema F. J. Clin. Invest. 1992; 89: 1154-1160Crossref PubMed Scopus (156) Google Scholar) and how HIV infection leads to NF-κB activation. In the promonocytic cell line U937, HIV activates the inducible pool of NF-κB as a result of enhanced IκBα degradation, which is believed to be secondary to IKK activation (41Bachelerie F. Alcami J. Arenzana-Seisdedos F. Virelizier J.L. Nature. 1991; 350: 709-712Crossref PubMed Scopus (151) Google Scholar, 42Jacque J.M. Fernandez B. Arenzana-Seisdedos F. Thomas D. Baleux F. Virelizier J.L. Bachelerie F. J. Virol. 1996; 70: 2930-2938Crossref PubMed Google Scholar, 43McElhinny J.A. MacMorran W.S. Bren G.D. Ten R.M. Israel A. Paya C.V. J. Virol. 1995; 69: 1500-1509Crossref PubMed Google Scholar, 44Asin S. Taylor J.A. Trushin S. Bren G. Paya C.V. J. Virol. 1999; 73: 3893-3903Crossref PubMed Google Scholar). The activity of activator protein-1 (AP-1), a transcription factor, consisting of a homodimer and heterodimers of members of the Jun family (c-Jun, JunB, and JunD) and heterodimers of the Jun and Fos (c-Fos, FosB, Fra1, and Fra2) families, is regulated, at least in part, by the activation of c-Jun N-terminal kinase (JNK) (45Karin M. J. Biol. Chem. 1995; 270: 16483-16486Abstract Full Text Full Text PDF PubMed Scopus (2256) Google Scholar). It has also been suggested that the activation of NF-κB is regulated by some upstream MAP kinases that also regulate JNK activation in the cells (46Lee F.S. Hagler J. Chen Z.J. Maniatis T. Cell. 1997; 88: 213-222Abstract Full Text Full Text PDF PubMed Scopus (660) Google Scholar). Induction of AP-1 in macrophages by endogenous HIV-1 Nef has been reported to be a cell type-specific response that requires both Hck and MAPK signaling (47Biggs T.E. Cooke S.J. Barton C.H. Harris M.P. Saksela K. Mann D.A. J. Mol. Biol. 1999; 290: 21-35Crossref PubMed Scopus (55) Google Scholar). Exogenous Nef protein is detected in the serum of HIV-infected subjects (48Fujii Y. Otake K. Tashiro M. Adachi A. FEBS Lett. 1996; 393: 93-96Crossref PubMed Scopus (120) Google Scholar). Both antibodies and cytotoxic T lymphocytes (CTLs) directed against Nef have been found in a large proportion of infected individuals (49Ameisen J.C. Guy B. Chamaret S. Loche M. Mouton Y. Neyrinck J.L. Khalife J. Leprevost C. Beaucaire G. Boutillon C. et al.AIDS Res. Hum. Retroviruses. 1989; 5: 279-291Crossref PubMed Scopus (62) Google Scholar, 50Bahraoui E. Yagello M. Billaud J.N. Sabatier J.M. Guy B. Muchmore E. Girard M. Gluckman J.C. AIDS Res. Hum. Retroviruses. 1990; 6: 1087-1098Crossref PubMed Scopus (31) Google Scholar). This suggests that in vivo Nef is processed and presented by antigen-presenting cells, as the result of uptake of extracellular Nef possibly released by infected apoptotic cells (51Alessandrini L. Santarcangelo A.C. Olivetta E. Ferrantelli F. d'Aloja P. Pugliese K. Pelosi E. Chelucci C. Mattia G. Peschle C. Verani P. Federico M. J. Gen. Virol. 2000; 81: 2905-2917Crossref PubMed Scopus (43) Google Scholar). Exogenous Nef protein has been shown to enter the cell by adsorptive endocytosis following nonspecific binding to the surface of CD4+ T cells, primary macrophages, and U937 promonocytic cells (51Alessandrini L. Santarcangelo A.C. Olivetta E. Ferrantelli F. d'Aloja P. Pugliese K. Pelosi E. Chelucci C. Mattia G. Peschle C. Verani P. Federico M. J. Gen. Virol. 2000; 81: 2905-2917Crossref PubMed Scopus (43) Google Scholar) and to activate the signal transducer and activator of transcription 1 (STAT-1) in human monocytes/macrophages (52Federico M. Percario Z. Olivetta E. Fiorucci G. Muratori C. Micheli A. Romeo G. Affabris E. Blood. 2001; 98: 2752-2761Crossref PubMed Scopus (87) Google Scholar). Confocal microscopy indicates that the intracellular distribution of internalized FITC-labeled recombinant Nef is identical to that of endogenously produced Nef, localizing both in an intracytoplasmic punctate pattern and at the cell margin (51Alessandrini L. Santarcangelo A.C. Olivetta E. Ferrantelli F. d'Aloja P. Pugliese K. Pelosi E. Chelucci C. Mattia G. Peschle C. Verani P. Federico M. J. Gen. Virol. 2000; 81: 2905-2917Crossref PubMed Scopus (43) Google Scholar, 53Greenberg M.E. Bronson S. Lock M. Neumann M. Pavlakis G.N. Skowronski J. EMBO J. 1997; 16: 6964-6976Crossref PubMed Scopus (199) Google Scholar). Although most of the results reported so far in regard to signaling were obtained following expression of endogenous Nef protein, exogenous Nef protein could also be involved in the modulation of cell signaling, especially in promonocytic cells and primary macrophages. Since activation of NF-κB and AP-1 results in stimulation of both HIV-1 and SIV replication (41Bachelerie F. Alcami J. Arenzana-Seisdedos F. Virelizier J.L. Nature. 1991; 350: 709-712Crossref PubMed Scopus (151) Google Scholar, 42Jacque J.M. Fernandez B. Arenzana-Seisdedos F. Thomas D. Baleux F. Virelizier J.L. Bachelerie F. J. Virol. 1996; 70: 2930-2938Crossref PubMed Google Scholar, 54Yang X. Chen Y. Gabuzda D. J. Biol. Chem. 1999; 274: 27981-27988Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 55Canonne-Hergaux F. Aunis D. Schaeffer E. J. Virol. 1995; 69: 6634-6642Crossref PubMed Google Scholar, 56Asin S. Bren G.D. Carmona E.M. Solan N.J. Paya C.V. J. Virol. 2001; 75: 11408-11416Crossref PubMed Scopus (25) Google Scholar), we investigated the role of exogenous Nef protein in this process. We observed that exogenous Nef protein activates NF-κB, AP-1, and JNK in promonocytic cells U937 and enhances HIV-1 replication in chronically infected U1 cells. Recombinant SIVmac Nef protein (AIDS Research and Reference Reagent Program, National Institutes of Health cat. 2999, kindly provided by Jose Torres) and recombinant Nef protein derived from HIV-1 strains, SF-2, BH10 (kindly provided by M. Harris, Leeds University, UK) and NL4-3 (kindly provided by U. Mahlknecht, Frankfurt University, Germany) were used to treat U937 cells and U1 cells. Anti-SIV Nef antibody was provided by the AIDS Research and Reference Reagent Program, National Institutes of Health (cat. 2659). Anti-HIV-1 Nef antibody was provided by AbCys (Paris, France). Antibody against IκBα and the double-stranded oligonucleotide having the AP-1 consensus sequence were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Phospho-IκBα (Ser32) antibody was purchased from New England BioLabs (Beverly, MA). TNF was purchased from R&D Systems. Most of the studies were performed with the promonocytic cells U937 obtained from the American Tissue Cell Culture Collection (ATCC, Manassas, VA). The promonocytic cell line U1, derived from cells surviving acute infection of the U937 cell line, contains two integrated HIV copies per cell (57Folks T.M. Justement J. Kinter A. Schnittman S. Orenstein J. Poli G. Fauci A.S. J. Immunol. 1988; 140: 1117-1122PubMed Google Scholar). U1 cells were a gift from Dr. U. Mahlknecht (University of Frankfurt, Germany). U937 and U1 cells were cultivated in RPMI 1640 supplemented with 10% fetal bovine serum. To measure NF-κB and AP-1 activation, EMSA were carried out as previously described by Manna and Aggarwal (58Manna S.K. Aggarwal B.B. J. Immunol. 1999; 162: 2095-2102PubMed Google Scholar) and by Van Lint et al. (59Van Lint C. Ghysdael J. Paras P. Burny A. Verdin E. J. Virol. 1994; 68: 2632-2648Crossref PubMed Google Scholar). Briefly, nuclear extracts prepared from cells treated with exogenous Nef protein were incubated with 32P-end-labeled 45-mer double-stranded NF-κB oligonucleotide, 5′-TTGTTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGG-3′ (bold indicates NF-κB binding sites), and the DNA-protein complex formed was resolved from free oligonucleotide on a 6% native polyacrylamide gel. A double-stranded mutated oligonucleotide 5′-TTGTTACAACTCACTTTCCGCTGCTCACTTTCCAGGGAGGCGTGG-3′, was used to examine the specificity of binding of NF-κB to the DNA. The specificity of binding was also examined by competition with the unlabeled oligonucleotide and a heterologous unlabeled oligonucleotide. To measure AP-1 activation, nuclear extracts, prepared as described above, were incubated with the 32P-end-labeled AP-1 consensus oligonucleotide 5′-CGCTTGATGACTCAGCCGGAA-3′ (bold indicates AP-1 binding site) and analyzed on a 6% native polyacrylamide gel. The specificity of the binding was examined by competition with unlabeled oligonucleotide, with a heterologous unlabeled oligonucleotide and with a consensus unlabeled oligonucleotide. The dried gels were visualized and radioactive bands quantified by a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) using ImageQuant software. Cytoplasmic extracts of U937 cells treated for different times with exogenous SIV Nef protein were used to examine IκBα degradation by Western blot procedure as described (58Manna S.K. Aggarwal B.B. J. Immunol. 1999; 162: 2095-2102PubMed Google Scholar). The c-Jun kinase (JNK) assay was performed according to the method of Manna and Aggarwal (58Manna S.K. Aggarwal B.B. J. Immunol. 1999; 162: 2095-2102PubMed Google Scholar). To examine SIV Nef-induced NF-κB-dependent gene expression, cells were transfected with the secreted alkaline phosphatase (SEAP) expression plasmid for 10 h before treatment with SIV Nef. After 24 h, cell culture conditioned-medium was harvested and analyzed (25 μl) for alkaline phosphatase activity, as described in the manufacturer's protocol (Clontech, Palo Alto, CA). SEAP activity was assayed on a 96-well fluorescence plate reader (Fluoroscan II, Lab Systems) with excitation set at 360 nm and emission at 460 nm. This reporter system was specific since TNF-induced NF-κB SEAP activity was inhibited by overexpression of the IκBα mutant, IκBα-DN, which lacks Ser-32 and Ser-36 (60Darnay B.G., Ni, J. Moore P.A. Aggarwal B.B. J. Biol. Chem. 1999; 274: 7724-7731Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar). To examine NF-κB LTR-driven gene expression by exogenous HIV Nef, 107 U937 cells were transfected with 750 ng of pLTR-Luc or 750 ng of pLTRmut-NFkB-Luc using the DEAE-dextran procedure (61Van Lint C. Amella C.A. Emiliani S. John M. Jie T. Verdin E. J. Virol. 1997; 71: 6113-6127Crossref PubMed Google Scholar). Twenty-four hours later, the cells were stimulated with different concentrations of exogenous HIV-1 Nef or with TNF. At 48-h post-transfection, luciferase activity was measured in cell lysates using a luminometer (TD-20/20; Promega, Madison, WI) as previously reported (11Mahlknecht U. Deng C., Lu, M.C. Greenough T.C. Sullivan J.L. O'Brien W.A. Herbein G. J. Immunol. 2000; 165: 6437-6446Crossref PubMed Scopus (54) Google Scholar). Values normalized to protein concentrations were expressed in fold increase over unstimulated control values. U1 cells were treated with different concentrations of exogenous HIV-1 Nef or TNF. Culture supernatants were collected every day and assessed for p24 antigen using a microELISA assay (Organon Teknika). Treatment of U937 cells with exogenous SIV Nef for 90 min revealed a dose-dependent activation of NF-κB by EMSA (Fig. 1 A), with maximum activation at ∼50–100 ng/ml, but to a lesser extent than TNF, the most potent activator of NF-κB (3.5-fold versus 5.2-fold induction). Exogenous SIV Nef activated NF-κB in a time- and dose-dependent manner reaching a peak by 90 min (Fig. 1 B). The gel shift band was specific as formation of the complex was blocked with an unlabeled oligonucleotide and was supershifted by either anti-p50 or anti-p65 antibody alone, and also by a mixture of anti-p50 and anti-p65 antibodies (Fig. 1 C), indicating that it is composed of p50 and p65 subunits. To rule out the possibility that a TNF inducer, such as lipopolysaccharide (LPS), produced the activity, we treated exogenous SIV Nef protein with 1% trypsin or boiled it at 100 °C (Fig. 1 D). Both treatments abolished Nef-induced NF-κB activity, indicating that exogenous Nef protein, but not LPS contamination, was responsible for NF-κB activation. The pretreatement of exogenous Nef protein with a neutralizing anti-Nef monoclonal antibody blocked NF-κB activation (Fig. 1 D), thereby indicating the effect was Nef-specific. The degradation of IκBα in U937 cells treated with exogenous SIV Nef protein for different periods of time was also examined using Western blot analysis. We observed that IκBα started to degrade at 15 min, degraded maximally by 30–60 min, and started to be resynthesized at 90 min (Fig. 1 E, upper panel). The presence of a slow migrating band of IκBα in samples prepared from SIV Nef-treated cells suggested the appearance of the phosphorylated form of IκBα, which is required for IκBα degradation. The induction of the phosphorylated form of IκBα by exogenous SIV Nef was detected by using an antibody directed against the phosphorylated form of IκBα, with a maximum detection by 30 min (Fig. 1 E, lower panel). Since SIV Nef and HIV-1 Nef may differ in regard to some of their biological functions (62Kirchhoff F. Munch J. Carl S. Stolte N. Matz-Rensing K. Fuchs D. Haaft P.T. Heeney J.L. Swigut T. Skowronski J. Stahl-Hennig C. J. Virol. 1999; 73: 8371-8383Crossref PubMed Google Scholar), we tested recombinant HIV-1 Nef proteins derived from three HIV-1 isolates, NL4–3, SF2, and BH10, in regard to NF-κB activation in U937 cells. We observed that exogenous SF2 Nef protein activated NF-κB in a time-dependent manner (Fig. 2 A). The gel-shift band was specific as formation of the complex was blocked with an unlabeled NF-κB oligonucleotide, but not with a mutated NF-κB oligonucleotide or with a heterologous oligonucleotide (Fig. 2 B). The gel shift band was supershifted by either anti-p50 or anti-p65 antibody alone (Fig. 2 C), indicating that it is composed of p50 and p65 subunits. Nef-induced NF-κB activation was also observed following treatment of U937 cells with exogenous HIV-1 BH10 and NL4–3 Nef proteins (Fig. 2 D), indicating that NF-κB activation mediated by exogenous Nef is viral isolate-independent. Most agents that activate NF-κB also activate the transcription factor AP-1 (45Karin M. J. Biol. Chem. 1995; 270: 16483-16486Abstract Full Text Full Text PDF PubMed Scopus (2256) Google Scholar). Therefore, we investigated the ability of exogenous SIV Nef protein to activate AP-1 in U937 cells. Exogenous SIV Nef protein activated AP-1 in a dose-dependent manner, but to a lesser extent than TNF (3.5-fold versus 5-fold induction) (Fig. 3 A). AP-1 activation by exogenous SIV Nef was time-dependent, with optimum activation occurring at ∼90 min (Fig. 3 B). Supershift analysis with specific antibodies against c-Fos and c-Jun indicated that AP-1 activation induced by exogenous SIV Nef consisted of Fos and Jun (Fig. 3 C). Lack of supershift by unrelated antibodies and disappearance of the AP-1 band by competition with unlabeled oligonucleotide indicate that the interaction was specific (Fig. 3 C). Activation of JNK is another early event initiated by many stress stimuli and is required for AP-1 activation (45Karin M. J. Biol. Chem. 1995; 270: 16483-16486Abstract Full Text Full Text PDF PubMed Scopus (2256) Google Scholar). Treatment of U937 cells with exogenous SIV Nef protein led to an increase in JNK activity in a time- (Fig. 3 D) and dose-dependent manner (Fig. 3 E). The overall level of JNK activation triggered by exogenous SIV Nef protein was less than that observed following TNF treatment (3-fold versus 6-fold) (Fig. 3 E). Since SIV Nef and HIV-1 Nef could differ in regard to their biological functions (62Kirchhoff F. Munch J. Carl S. Stolte N. Matz-Rensing K. Fuchs D. Haaft P.T. Heeney J.L. Swigut T. Skowronski J. Stahl-Hennig C. J. Virol. 1999; 73: 8371-8383Crossref PubMed Google Scholar), we tested recombinant HIV-1 Nef proteins derived from three HIV-1 isolates, NL4–3, SF2, and BH10 in regard to AP-1 activation in U937 cells. Exogenous BH10 Nef protein activated AP-1 in a time-dependent (Fig. 4 A) and dose-dependent manner (data not shown). The gel shift band was specific, as formation of the complex was blocked with an unlabeled AP-1 oligonucleotide and with an unlabeled consensus AP-1 oligonucleotide, but not with a heterologous NF-κB oligonucleotide (Fig. 4 B). The gel shift band was supershifted by either anti-c-Fos or anti-c-Jun antibody alone, but also by a mixture of anti-c-Fos and anti-c-Jun antibodies (Fig. 4 C), indicating that it is composed of c-Fos and c-Jun subunits. AP-1 activation followed treatment of U937 cells with exogenous HIV-1 Nef proteins derived from NL4–3, BH10, and SF2 isolates (Fig. 4 D), indicating that AP-1 activation mediated by exogenous HIV-1 Nef was viral isolate-independent. We assessed whether NF-κB activation triggers gene expression in U937 cells treated with exogenous Nef proteins. We examined the effect of exogenous SIV Nef on NF-κB-driven SEAP gene expression in U937 cells. SIV-Nef enhanced SEAP gene expression in a dose-dependent manner, comparable with TNF (Fig. 5 A). SEAP gene expression was NF" @default.
• W2067335145 created "2016-06-24" @default.
• W2067335145 creator A5031631094 @default.
• W2067335145 creator A5042244979 @default.
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• W2067335145 creator A5062743460 @default.
• W2067335145 date "2003-01-01" @default.
• W2067335145 modified "2023-09-30" @default.
• W2067335145 title "Exogenous Nef Protein Activates NF-κB, AP-1, and c-Jun N-Terminal Kinase and Stimulates HIV Transcription in Promonocytic Cells" @default.
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• W2067335145 doi "https://doi.org/10.1074/jbc.m209622200" @default.
• W2067335145 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12419805" @default.

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