Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

Hydroxyurea and Interleukin-6 Synergistically Reactivate HIV-1 Replication in a Latently Infected Promonocytic Cell Line via SP1/SP3 Transcription Factors

2006; Elsevier BV; Volume: 282; Issue: 6 Linguagem: Inglês

10.1074/jbc.m608150200

ISSN

1083-351X

Autores

Raphael M. Oguariri, Terrence W. Brann, Tomozumi Imamichi,

Tópico(s)

HIV/AIDS drug development and treatment

Resumo

The existence of viral latency limits the success of highly active antiretroviral therapy. With the therapeutic intention of reactivating latent virus to induce a cure, in this study we assessed the impact of cell synchronizers on HIV gene activation in latently infected U1 cells and investigated the molecular mechanisms responsible for such effect. Latently infected U1 cells were treated with 10 drugs including hydroxyurea (HU) and HIV-1 replication monitored using a p24 antigen capture assay. We found that HU was able to induce HIV-1 replication by 5-fold. HU has been used in the clinical treatment of HIV-1-infected patients in combination with didanosine; therefore, we investigated the impact of HU on HIV-1 activation in the presence of the proinflammatory cytokines, interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α). IL-6 or TNF-α alone induced HIV replication by 18- and ∼500-fold, respectively. Of interest, in the presence of HU, IL-6-mediated HIV-1 activation was enhanced by >90-fold, whereas TNF-α-mediated activation was inhibited by >30%. A reporter gene assay showed that HU and IL-6 synergized to activate HIV promoter activity via the Sp1 binding site. Electrophoretic mobility shift and supershift assays revealed increased binding of the Sp1 and Sp3 transcription factors to this region. Western blot analysis showed that HU and IL-6 co-stimulation resulted in increased levels of Sp1 and Sp3 proteins. In contrast, treatment with HU plus TNF-α down-regulated the expression of NF-κB. These findings suggest that Sp1/Sp3 is involved in controlling the HU/IL-6-induced reactivation of HIV-1 in latently infected cells. The existence of viral latency limits the success of highly active antiretroviral therapy. With the therapeutic intention of reactivating latent virus to induce a cure, in this study we assessed the impact of cell synchronizers on HIV gene activation in latently infected U1 cells and investigated the molecular mechanisms responsible for such effect. Latently infected U1 cells were treated with 10 drugs including hydroxyurea (HU) and HIV-1 replication monitored using a p24 antigen capture assay. We found that HU was able to induce HIV-1 replication by 5-fold. HU has been used in the clinical treatment of HIV-1-infected patients in combination with didanosine; therefore, we investigated the impact of HU on HIV-1 activation in the presence of the proinflammatory cytokines, interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α). IL-6 or TNF-α alone induced HIV replication by 18- and ∼500-fold, respectively. Of interest, in the presence of HU, IL-6-mediated HIV-1 activation was enhanced by >90-fold, whereas TNF-α-mediated activation was inhibited by >30%. A reporter gene assay showed that HU and IL-6 synergized to activate HIV promoter activity via the Sp1 binding site. Electrophoretic mobility shift and supershift assays revealed increased binding of the Sp1 and Sp3 transcription factors to this region. Western blot analysis showed that HU and IL-6 co-stimulation resulted in increased levels of Sp1 and Sp3 proteins. In contrast, treatment with HU plus TNF-α down-regulated the expression of NF-κB. These findings suggest that Sp1/Sp3 is involved in controlling the HU/IL-6-induced reactivation of HIV-1 in latently infected cells. Hydroxyurea (HU) 2The abbreviations used are: HU, hydroxyurea; ddI, didanosine; HIV, human immunodeficiency virus; HAART, highly active antiretroviral therapy; LTR, long terminal repeat; IL, interleukin; TNF, tumor necrosis factor; EMSA, electrophoretic mobility shift assays; RT, reverse transcriptase; STAT, signal transducers and activators of transcription. 2The abbreviations used are: HU, hydroxyurea; ddI, didanosine; HIV, human immunodeficiency virus; HAART, highly active antiretroviral therapy; LTR, long terminal repeat; IL, interleukin; TNF, tumor necrosis factor; EMSA, electrophoretic mobility shift assays; RT, reverse transcriptase; STAT, signal transducers and activators of transcription. or hydroxycarbamide is a ribonucleotide reductase inhibitor and has been extensively used in medical practice including treatment of chronic leukemia and sickle cell anemia (1Lori F. Lisziewicz J. J. Biol. Regul. Homeost. Agents. 2000; 14: 45-48PubMed Google Scholar). HU has been reported to possess antiretroviral activity due to its ability to deplete the intracellular dNTP pool, thereby directly inhibiting viral DNA synthesis (2Gao W.Y. Cara A. Gallo R.C. Lori F. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8925-8928Crossref PubMed Scopus (279) Google Scholar, 3Gao W.Y. Johns D.G. Mitsuya H. Mol. Pharmacol. 1994; 46: 767-772PubMed Google Scholar, 4Johns D.G. Gao W.Y. Biochem. Pharmacol. 1998; 55: 1551-1556Crossref PubMed Scopus (37) Google Scholar, 5Lori F. Kelly L.M. Foli A. Lisziewicz J. Expert Opin. Drug Saf. 2004; 3: 279-288Crossref PubMed Scopus (14) Google Scholar). It is thought that by reducing endogenous levels of deoxynucleoside triphosphates, HU may potentiate the effects of certain reverse transcriptase inhibitors (6Romanelli F. Hoven A.D. Curr. Pharm. Des. 2006; 12: 1121-1127Crossref PubMed Scopus (14) Google Scholar). However, HU alone is not potent enough to reduce initial viral load in treatment-naïve patients (7Foli A. Lori F. Maserati R. Tinelli C. Minoli L. Lisziewicz J. Antivir. Ther. 1997; 2: 31-38PubMed Google Scholar); however, in combination with didanosine (ddI) HU has been shown to induce substantial and durable reductions in human immunodeficiency virus (HIV) RNA levels (5Lori F. Kelly L.M. Foli A. Lisziewicz J. Expert Opin. Drug Saf. 2004; 3: 279-288Crossref PubMed Scopus (14) Google Scholar, 8Lori F. Malykh A.G. Foli A. Maserati R. De Antoni A. Minoli L. Padrini D. Degli Antoni A. Barchi E. Jessen H. Wainberg M.A. Gallo R.C. Lisziewicz J. AIDS. Res. Hum. Retroviruses. 1997; 13: 1403-1409Crossref PubMed Scopus (94) Google Scholar, 9Rutschmann O.T. Opravil M. Iten A. Malinverni R. Vernazza P.L. Bucher H. Bernasconi E. Perrin L.H. Yerly S. Hirschel B. Antivir. Ther. 1998; 3: 65-67PubMed Google Scholar, 10Frank I. J. Biol. Regul. Homeost. Agents. 1999; 13: 186-191PubMed Google Scholar, 11Havlir D.V. Gilbert P.B. Bennett K. Collier A.C. Hirsch M.S. Tebas P. Adams E.M. Wheat L.J. Goodwin D. Schnittman S. Holohan M.K. Richman D.D. AIDS. 2001; 15: 1379-1388Crossref PubMed Scopus (67) Google Scholar, 12Barreiro P. de Mendoza C. Camino N. Garcia-Benayas T. Blanco F. Nunez M. Gonzalez-Lahoz J. Soriano V. HIV Clin. Trials. 2003; 4: 361-371Crossref PubMed Scopus (18) Google Scholar). As a result, the HU plus ddI combination has been proposed as maintenance therapy in patients on prolonged successful highly active antiretroviral therapy (HAART) (12Barreiro P. de Mendoza C. Camino N. Garcia-Benayas T. Blanco F. Nunez M. Gonzalez-Lahoz J. Soriano V. HIV Clin. Trials. 2003; 4: 361-371Crossref PubMed Scopus (18) Google Scholar).HIV-1 encodes a transactivator protein, Tat (13Feinberg M.B. Jarrett R.F. Aldovini A. Gallo R.C. Wong-Staal F. Cell. 1986; 46: 807-817Abstract Full Text PDF PubMed Scopus (418) Google Scholar), that stimulates transcription elongation through interaction with a transactivation-responsive element located at the 5′ end of the nascent transcript (14Berkhout B. Silverman R.H. Jeang K.T. Cell. 1989; 59: 273-282Abstract Full Text PDF PubMed Scopus (508) Google Scholar). High level expression of HIV requires transactivation by Tat (15Arya S.K. Guo C. Josephs S.F. Wong-Staal F. Science. 1985; 229: 69-73Crossref PubMed Scopus (590) Google Scholar). Sp1 is one member of a multigene family (16Hagen G. Muller S. Beato M. Suske G. EMBO J. 1994; 13: 3843-3851Crossref PubMed Scopus (650) Google Scholar), has been shown to contribute significantly to the expression of the HIV-1 LTR (17Jones K.A. Kadonaga J.T. Luciw P.A. Tjian R. Science. 1986; 232: 755-759Crossref PubMed Scopus (444) Google Scholar), and plays a pivotal role in Tat activation of LTR-driven transcription (18Jeang K.T. Chun R. Lin N.H. Gatignol A. Glabe C.G. Fan H. J. Virol. 1993; 67: 6224-6233Crossref PubMed Google Scholar). Post-translational modification of Sp1 through glycosylation and phosphorylation (19Jackson S.P. MacDonald J.J. Lees-Miller S. Tjian R. Cell. 1990; 63: 155-165Abstract Full Text PDF PubMed Scopus (516) Google Scholar), rather than absolute increase in protein levels, has also been implicated to be important to HIV-1 gene expression (20Chun R.F. Semmes O.J. Neuveut C. Jeang K.T. J. Virol. 1998; 72: 2615-2629Crossref PubMed Google Scholar).The persistence of latent HIV-infected cellular reservoirs despite prolonged treatment with HAART represents the major hurdle to virus eradication. These latently infected cells are a permanent source for virus reactivation and lead to a rebound of the virus load after interruption of HAART (21Pierson T. McArthur J. Siliciano R.F. Annu. Rev. Immunol. 2000; 18: 665-708Crossref PubMed Scopus (456) Google Scholar, 22Quivy V. Adam E. Collette Y. Demonte D. Chariot A. Vanhulle C. Berkhout B. Castellano R. de Launoit Y. Burny A. Piette J. Bours V. Van Lint C. J. Virol. 2002; 76: 11091-11103Crossref PubMed Scopus (112) Google Scholar). The cellular reservoirs for HIV-1 infection could be macrophages (23Ho D.D. Rota T.R. Hirsch M.S. J. Clin. Investig. 1986; 77: 1712-1715Crossref PubMed Scopus (476) Google Scholar) or CD4+ T lymphocytes not fully activated, which carry the integrated provirus in a non-replicative state until the activation process is complete (24Marcello A. Retrovirology. 2006; 3: 7-15Crossref PubMed Scopus (99) Google Scholar). The reservoir that appears to be the major barrier to eradication is composed of latently infected cells that carry an integrated provirus that is transcriptionally silent (25Chun T.W. Finzi D. Margolick J. Chadwick K. Schwartz D. Siliciano R.F. Nat. Med. 1995; 1: 1284-1290Crossref PubMed Scopus (593) Google Scholar, 26Chun T.W. Carruth L. Finzi D. Shen X. DiGiuseppe J.A. Taylor H. Hermankova M. Chadwick K. Margolick J. Quinn T.C. Kuo Y.H. Brookmeyer R. Zeiger M.A. Barditch-Crovo P. Siliciano R.F. Nature. 1997; 387: 183-188Crossref PubMed Scopus (1659) Google Scholar). The extremely long half-life of these cells combined with a tight control of HIV-1 expression has been reported (24Marcello A. Retrovirology. 2006; 3: 7-15Crossref PubMed Scopus (99) Google Scholar) to make the reservoir ideally suited to maintain hidden copies of the virus, which are in turn able to trigger a novel systemic infection upon discontinuation of therapy. At the cellular level, preintegration and postintegration latency have been described in HIV-1 (27McCune J.M. Cell. 1995; 82: 183-188Abstract Full Text PDF PubMed Scopus (68) Google Scholar). The U1 promonocytic cell line is one of the most thoroughly characterized models of postintegration latency. The U1 cell line was derived from a population of U937 cells and harbors two copies of integrated proviruses (28Folks T.M. Justement J. Kinter A. Dinarello C.A. Fauci A.S. Science. 1987; 238: 800-802Crossref PubMed Scopus (714) Google Scholar, 29Folks T.M. Justement J. Kinter A. Schnittman S. Orenstein J. Poli G. Fauci A.S. J. Immunol. 1988; 140: 1117-1122PubMed Google Scholar). In the absence of stimulation, U1 cells maintain a pattern of low viral mRNA expression. Several studies have shown that the relative state of latency in U1 cells is as a result of defective Tat, the HIV transactivating protein (30Cannon P. Kim S.H. Ulich C. Kim S. J. Virol. 1994; 68: 1993-1997Crossref PubMed Google Scholar, 31Emiliani S. Van Lint C. Fischle W. Paras Jr., P. Ott M. Brady J. Verdin E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6377-6381Crossref PubMed Scopus (138) Google Scholar) One rational strategy of purging the latent HIV reservoir is to understand the molecular mechanisms regulating viral latency and reactivation (32Cohen J. Science. 1998; 279: 1854-1855Crossref PubMed Scopus (38) Google Scholar).To activate latently infected HIV in vitro, proinflammatory cytokines such as granulocyte macrophage colony-stimulating factor, interleukin (IL) 3, IL-6, and tumor necrosis factor (TNF)-α have been used (28Folks T.M. Justement J. Kinter A. Dinarello C.A. Fauci A.S. Science. 1987; 238: 800-802Crossref PubMed Scopus (714) Google Scholar, 29Folks T.M. Justement J. Kinter A. Schnittman S. Orenstein J. Poli G. Fauci A.S. J. Immunol. 1988; 140: 1117-1122PubMed Google Scholar). These agents have been shown to activate HIV-1 expression in U1 cells by affecting distinct steps of the virus life cycle, including NF-κB-dependent transcription in the case of TNF-α and phorbol 12-myristate 13-acetate (33Osborn L. Kunkel S. Nabel G.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2336-2340Crossref PubMed Scopus (1354) Google Scholar, 34Duh E.J. Maury W.J. Folks T.M. Fauci A.S. Rabson A.B. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5974-5978Crossref PubMed Scopus (631) Google Scholar) or a posttranscriptional event(s) in cells stimulated with IL-6 (35Poli G. Bressler P. Kinter A. Duh E. Timmer W.C. Rabson A. Justement J.S. Stanley S. Fauci A.S. J. Exp. Med. 1990; 172: 151-158Crossref PubMed Scopus (420) Google Scholar).Our laboratory previously reported that a low concentration of actinomycin D, a known transcription inhibitor and a cell cycle synchronizer, induces HIV-1 activation in latently infected cells and HTLV-1-transformed cells (36Imamichi T. Murphy M.A. Adelsberger J.W. Yang J. Watkins C.M. Berg S.C. Baseler M.W. Lempicki R.A. Guo J. Levin J.G. Lane H.C. J. Virol. 2003; 77: 1011-1020Crossref PubMed Scopus (20) Google Scholar, 37Imamichi T. Conrads T.P. Zhou M. Liu Y. Adelsberger J.W. Veenstra T.D. Lane H.C. J. Acquired Immune Defic. Syndr. 2005; 40: 388-397Crossref PubMed Scopus (9) Google Scholar). To further understand the mechanism of HIV gene activation, we investigated the impact of another synchronizer, HU, on HIV-1 gene activation in latently infected U1 cells. We demonstrate here for the first time that HU activates HIV-1 replication in latently infected U1 cells and synergizes with IL-6 to enhance HIV-1 gene reactivation and that binding of the Sp1 and Sp3 transcription factors to the LTR promoter is critical for this synergism.EXPERIMENTAL PROCEDURESCells and Reagents—The promonocytic cell line, U1 (28Folks T.M. Justement J. Kinter A. Dinarello C.A. Fauci A.S. Science. 1987; 238: 800-802Crossref PubMed Scopus (714) Google Scholar), was obtained from the AIDS Research and Reference Reagent Program, NIAID, National Institutes of Health (Rockville, MD). U1 cells were maintained in RPMI 1640 (Invitrogen) supplemented with 10% fetal bovine serum (HyClone Laboratories, Logan UT), 10 mm l-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin (Quality Biologics Inc., Gaithersburg, MD). HU was purchased from Calbiochem. Recombinant IL-6 and recombinant TNF-α were purchased from R&D systems (Minneapolis, MN).Plasmid Constructions—The 5′ LTR region of HIV was subcloned upstream of luciferase reporter gene as follows. MluLTR5 (5′-ACGCGTTGGAAGGGCTAATTTGCT) and HindLTR3 (5′-AAGCTGCTAGAGATTTTCCACACT) primers were used for amplification of full-length of the 5′ LTR from a cloned proviral pNL4.3 (obtained from M. Martin through the AIDS Research and Reference Reagent Program) (38Adachi A. Gendelman H.E. Koenig S. Folks T. Willey R. Rabson A. Martin M.A. J. Virol. 1986; 59: 284-291Crossref PubMed Google Scholar). The amplified product was purified and cloned into the TOPO TA cloning pCR2.1 vector (Invitrogen). The resultant clone, pCRLTR, was then digested with MluI and HindIII, and a 0.6-kbp fragment containing the LTR region was subcloned into the corresponding sites of similarly digested pGL3-Basic vector (Promega, Madison, WI). The resultant clone is named p461. To construct a clone (designated as p470) lacking the sequence upstream of the NF-κB binding site from the full-length of 5′ LTR, pCRLTR was digested with MluI and then an inverted PCR was performed using Pfu Turbo DNA polymerase (Stratagene, La Jolla, CA) with a primer set of MluR (5′-TACGCGTAGCCGAATTCCAGCACAC-3′) and NF-κB(5′-GGGACTTTCCGCTGGGGACTT-3′). PCR product was purified, treated with T4 kinase (Promega), and then self-ligated using PanVera Takara ligation kit II (Madison, WI). The intended clone was digested with MluI and HindIII, and the 180-bp fragment was subcloned into the pGL3-Basic vector. Using the same procedure, clones lacking the upstream sequence from Sp1 site (p5006) or TATA box (p5007) were constructed from pCRLTR using primer sets of the MulR and SP1F (5′-GGGCGGGACTGGGGAGTGGCGAG-3′) or MulR and TATAF (5′-CATATAAGCTGCTTTTTGCCTG-3′). To construct a Tat expression plasmid, the full-length of Tat gene including intron region in pNL4.3 was PCR amplified using the primers FTAT/F (5′-ATGGAGCCAGTAGATCCTAG-3′(sense)) and FTAT/R (5′-CTATTCCTTCGGGCCTGTCGGG-3′ (antisense)). A 2585-bp PCR product obtained was cloned into pcDNA4/HisMax TOPO vector (Invitrogen). To delete the intron region in the PCR product (216–2539 nucleotides), an inverted PCR was performed using Pfu Turbo DNA polymerase (Stratagene) with the primer set Tat/R (5′-TGCTTTGATAGAGAAGCTTGATG) and Tat/F (5′-ACCACCTCCCAATCCCGAG-3′). The PCR product was purified and treated with T4 kinase (Promega) and then self-ligated using the PanVera Takara ligation kit II (Madison, WI). The resultant clone named p494 was verified by Western blot to confirm the expression of Tat protein. All the constructs were confirmed by nucleotide sequencing.HIV Replication Assays—Latently infected U1 cells were seeded at 2.0 × 105/ml. All conditions were performed in 96-well microplates for 4 days at 37 °C. On day 4, cell-free supernatants were collected, and virus replication was measured using a p24 antigen capture assay (Beckman-Coulter, Miami, FL). Each culture was performed in quadruplicate. Results are the means of three independent experiments. For cytokine assays, U1 cells were seeded at 2.0 × 105/ml and stimulated with HU (200 μm), IL-6 (15 ng/ml), TNF-α (5 ng/ml), or a combination of HU and IL-6 or HU and TNF-α. Replication assays were carried out as above. HIV-1 replication kinetics was performed as previously described (36Imamichi T. Murphy M.A. Adelsberger J.W. Yang J. Watkins C.M. Berg S.C. Baseler M.W. Lempicki R.A. Guo J. Levin J.G. Lane H.C. J. Virol. 2003; 77: 1011-1020Crossref PubMed Scopus (20) Google Scholar). Briefly, U1 cells were cultured at 2.0 × 105/ml in the presence or absence of stimulation. Culture supernatants were collected every day for 4 days, and HIV-1 replication was monitored using the p24 antigen capture assay.Cell Cycle Analysis—U1 cells were treated with 200 μm HU at 37 °C for 24, 48, 72, or 96 h and washed with phosphate-buffered saline. Cell cycle analysis was performed as previously described (36Imamichi T. Murphy M.A. Adelsberger J.W. Yang J. Watkins C.M. Berg S.C. Baseler M.W. Lempicki R.A. Guo J. Levin J.G. Lane H.C. J. Virol. 2003; 77: 1011-1020Crossref PubMed Scopus (20) Google Scholar). Briefly, cells were fixed by the addition of 70% ethanol. The cell pellets were washed with phosphate-buffered saline and treated with DNase-free RNase (Roche Applied Science) at 37 °C for 15 min. Propidium iodide was added to the cell suspension, which was then incubated on ice for at least 30 min. The stained cells were analyzed for red fluorescence (FL3) on a Coulter XL flow cytometer (Beckman-Coulter) with doublet discrimination achieved with an amorphous gate based on linear and peak FL3 signal, and the distribution of cells in the G1, S, and G2/M phases of the cell cycle was calculated from the resulting DNA histogram with Multicycle AV software, based on a zero order polynomial S-phase model (Phoenix Flow Systems, San Diego, CA).Enzyme-linked Immunosorbent Assay—Levels of IL-6 and TNF-α production in culture supernatants were quantified using enzyme-linked immunosorbent assay kits for human IL-6 and human TNF-α (R&D systems), respectively, according to the manufacturer's instructions.Northern Blot Analysis—Total cellular RNA was extracted at 48 or 72 h from treated and untreated cells using the RNeasy kit (Qiagen). A total of 30 μg of RNA was loaded per lane and separated on a 1.2% agarose, formaldehyde gel and transferred to a Nytran SuperCharge nylon membrane using the TurboBlotter System (Schleicher & Schuell). After transfer, RNA was cross-linked onto the membrane by UV irradiation. Gene-specific DNA fragments were confirmed by DNA sequencing and labeled with [α-32P]dCTP (Amersham Biosciences) using the Prime-It RmT Random Primer labeling kit (Stratagene). The membrane was prehybridized for 30 min and then hybridized with the probe in the QuikHyb hybridization solution (Stratagene) at 56 °C overnight. After hybridization, the membrane was washed at 56 °C for 20 min under low stringency condition (2 × SSC (1 × SSC = 0.15 m NaCl and 0.015 m sodium citrate), 0.1% SDS) and high stringency conditions (0.1 × SSC, 0.1% SDS). Then the membrane was exposed to Eastman Kodak Co. BioMax MS film at –80 °C for 1–4 h. Ethidium bromide-stained 28 S and 18 S RNAs were used as internal controls for RNA loading. Quantification of the intensity of the bands was done using FujiFilm FLA 5100 phosphorimaging.Transient Transfection Assays—U1 cells were transiently transfected using Transit Transfection Reagent, FuGENE 6 (Roche Applied Science) with the HIV-1 LTR luciferase reporter constructs. Transfected cells were cultured in the presence or absence of 200 μm HU, 15 ng/ml IL-6, or HU plus IL-6 for 48 h. For co-transfection experiments cells were transiently transfected with Tat plasmid (p494) and either p461, p5006, or p5007 reporter constructs and cultured in the presence or absence of HU plus IL-6 for 48 h. Cells were washed, lysed, and assayed for luciferase activity using the Luciferase Assay System (Promega). Luciferase activity was normalized by total cellular protein measured with the BCA protein assay kit (Pierce).Electrophoretic Mobility Shift Assays (EMSAs)—Nuclear extracts were prepared from U1 cells treated or untreated with HU (200 μm) IL-6 (15 ng/ml), TNF-α (5 ng/ml), or a combination of HU plus IL-6 or HU plus TNF-α according to the manufacturer's instructions (Active Motive, Carlsbad, CA), and protein concentrations were determined by the BCA assay. Sense and corresponding antisense synthetic oligonucleotides were annealed to form double-stranded oligonucleotides. Sp1 consensus and HIV-specific Sp1 oligonucleotides were end-labeled with [γ-32P]ATP (Amersham Biosciences) using T4 polynucleotide kinase (Promega). A total of 10 μg of nuclear extract was first incubated at room temperature for 10 min in binding buffer (Promega) in the absence of the probe. A 50-fold excess of cold probe or AP2 oligonucleotides was added to the reaction mixture as specific and nonspecific competitor DNAs, respectively. 20,000 cpm of probe was then added to the mixture and incubated at room temperature for 20 min. For the supershift assay, polyclonal antibodies against Sp1, Sp3, STAT3, and C/EBP (Santa Cruz Biotechnology, Santa Cruz, CA) were added to the reaction mixture and incubated for 30 min on ice before the addition of radiolabeled probe. Samples were subjected to electrophoresis on native 6% polyacrylamide gels (Invitrogen). The gel was dried and exposed to x-ray film for 20 min at –80 °C.Western Immunoblot Assays—U1 cells were stimulated with HU (200 μm), IL-6 (15 ng/ml), TNF-α (5 ng/ml), or a combination of HU plus IL-6 or HU plus TNF-α. Cells were cultured at 2.0 × 105 cells/ml for 48 h at 37 °C. Nuclear extracts were obtained as described above, and protein amount were measured by the BCA assay. 30 μg of nuclear protein was loaded onto a 10% SDS-polyacrylamide gel and subsequently transferred onto a 0.45 μm nitrocellulose membrane. Membranes were probed with anti-Sp1, Sp3, p50 NF-κB polyclonal antibodies (Santa Cruz Biotechnology) or anti-TFII-I polyclonal antibody (Cell Signaling Technology Inc., Danvers, MA) as an internal control for nuclear transcription factors. Primary antibody was detected with horseradish peroxidase-conjugated anti-goat or anti-rabbit IgG (eBioscience, San Diego, CA). Signals were detected with ECL Plus Western blotting detection system (Amersham Biosciences).Reverse Transcriptase (RT)-PCR—U1 cells were seeded at 2.0 × 105/ml, treated or untreated with 200 μm HU and 15 ng/ml IL-6, and cultured at 37 °C. Total cellular RNAs were isolated at 24 and 48 h using RNeasy kit (Qiagen). After DNase treatment, the RNA was reverse-transcribed using the Superscript First-Strand Synthesis system for RT-PCR according to the manufacturer's instructions (Invitrogen). The cDNA was amplified by PCR using the Expand High Fidelity PCR System (Roche Applied Science). Sp1 gene-specific primers used were sense, 5′-CTG GTC ATA CTG TGG GAA ACG C-3′, and antisense, 5′-TGT TGG CAA GAC GGG CAA TG-3′. Sp3 gene-specific primers used were sense, 5′-GGA AAA AGA CTT CGG AGG GTA GC-3′, and antisense, 5′-GCA AGG TGG TCA CTT CTC ATA AAG C-3′. β-Actin gene-specific primers were used as the internal control. PCR conditions were 94 °C for 2 min, 30 cycles (30 s at 94 °C, 30 s at 55 °C, and 45 s at 72 °C), then 7 min at 72 °C. PCR-generated fragments were verified on 1.2% agarose gel. The PCR products were confirmed by DNA sequence.Real-time Quantitative PCR—As above, U1 cells were mock-treated or treated with HU and IL-6, and total RNAs were extracted after 24 and 48 h. RNAs were DNase-treated and reverse-transcribed using the TaqMan reverse transcription reagents (Applied Biosystems, Foster City, CA). Real-time quantitative PCR was performed using iCycler iQ real-time PCR detection system (Bio-Rad) employing a TaqMan Universal PCR Master Mix (Applied Biosystems). The oligonucleotide primers used for the detection of Sp1, Sp3, or glyceraldehyde-3-phosphate dehydrogenase were obtained from TaqMan gene amplification assays (Applied Biosystems). In each instance the amount of real-time quantitative PCR product for the genes of interest (Sp1 and Sp3) was normalized to the amount of the reference gene glyceraldehyde-3-phosphate dehydrogenase in the same sample.RESULTSHU Activates HIV-1 Replication in the Chronically Infected Promonocytic Cell Line, U1—U1 cells, an HIV-1 latently infected cell line, harbors two integrated provirus DNA copies (28Folks T.M. Justement J. Kinter A. Dinarello C.A. Fauci A.S. Science. 1987; 238: 800-802Crossref PubMed Scopus (714) Google Scholar, 29Folks T.M. Justement J. Kinter A. Schnittman S. Orenstein J. Poli G. Fauci A.S. J. Immunol. 1988; 140: 1117-1122PubMed Google Scholar). In the absence of stimulation with certain stimulants, culturing of U1 cells leads to little virus expression. To investigate the molecular process that leads to HIV-1 reactivation, U1 cells were treated with 10 known drugs (actinomycin D, N-acetyl-Leu-Leu-norleucinal, butyrolactone, trichostatin A, paclitaxel, dexamethasone, acyclovir, tunicamycin, rapamycin, or HU) for 7 days. HIV-1 replication was monitored by measuring p24 antigen in the cell culture supernatant. Of the drugs tested, only actinomycin D and HU induced HIV-1 activation by 5–7-fold (Fig. 1A). Because HU has been used in clinical treatment of HIV-infected patients in combination with ddI, a nucleotide reverse transcriptase inhibitor, we further investigated in detail the molecular mechanisms of HU-induced HIV-1 reactivation in U1 cells. U1 cells were mock-treated or treated with increasing concentrations of HU (0. 12.5, 25, 50, 100, 200, 400, and 800 μm) and assayed for p24 antigen in culture supernatant after 4 days of culture. Results presented in Fig. 1B showed that HU induced HIV reactivation in dose-dependent manner. At 200 μm, HU enhanced the reactivation by 7-fold compared with control. Concentration of HU greater than 800 μm did not induce the reactivation (data not shown). This was due to cytotoxicity. Because the highest increase in replication was observed at 200 μm HU, this concentration was, therefore, used in all stimulations with HU in the present study. It is known that HU is able to induce cell cycle arrest. To determine whether HU induced an arrest in U1 cells, cell cycle analysis was performed. U1 cells were treated or untreated with HU (200 μm), cultured for 24, 48, 72, or 96 h, and subjected to the analysis. Our data showed an overall increase in the S and G2/M phases of the cell cycle and no arrest at the G1 phase (Fig. 2).FIGURE 2HU did not induce cell cycle arrest in U1 cells. U1 cells were cultured for 24 h (A), 48 h (B), 72 h (C), or 96 h (D) in the absence or presence of 200 μm HU. The cell cycle analysis was studied by propidium iodide staining and evaluated by flow cytometry. The left peaks constitute cells in G0/G1, the right peaks constitute cells in G2/M, and cells in S phase are between the G0/G1 and G2/M peaks. The percentages of cells in the G0/G1, S, and G2/M cell cycle phases were calculated from the resulting DNA histogram by using Multicycle AV software (Phoenix Flow Systems).View Large Image Figure ViewerDownload Hi-res image Download (PPT)HU Enhances IL-6-mediated HIV-1 Activation, Suppresses TNF-α—Several proinflammatory cytokines, for example IL-6 and TNF-α, induce HIV-1 activation in U1 cells (28Folks T.M. Justement J. Kinter A. Dinarello C.A. Fauci A.S. Science. 1987; 238: 800-802Crossref PubMed Scopus (714) Google Scholar, 33Osborn L. Kunkel S. Nabel G.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2336-2340Crossref PubMed Scopus (1354) Google Scholar, 34Duh E.J. Maury W.J. Folks T.M. Fauci A.S. Rabson A.B. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5974-5978Crossref PubMed Scopus (631) Google Scholar, 35Poli G. Bressler P. Kinter A. Duh E. Timmer W.C. Rabson A. Justement J.S. Stanley S. Fauci A.S. J. Exp. Med. 1990; 172:

Referência(s)