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Antimicrobial Agents and Chemotherapy, May 2004, p. 1652-1663, Vol. 48, No. 5
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.5.1652-1663.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Bis-Anthracycline Antibiotics Inhibit Human Immunodeficiency Virus Type 1 Transcription

Olaf Kutsch,1* David N. Levy,2 Paula J. Bates,3 Julie Decker,2 Barry R. Kosloff,1 George M. Shaw,2,4 W. Priebe,5 and Etty N. Benveniste1

Departments of Cell Biology,1 Medicine, The University of Alabama at Birmingham,2 The Howard Hughes Medical Institute, Birmingham, Alabama,4 The Department of Medicine, University of Louisville, Louisville, Kentucky,3 The University of Texas M. D. Anderson Cancer Center, Houston, Texas5

Received 26 August 2003/ Returned for modification 2 December 2003/ Accepted 8 January 2004


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The increasing numbers of human immunodeficiency virus type 1 (HIV-1) strains that exhibit resistance to antiretroviral agents used at present require the development of new effective antiretroviral compounds. Tat transactivation was recognized early on as an attractive target for drug interference. To screen for and analyze the effects of compounds that interfere with Tat transactivation, we developed several cell-based reporter systems in which enhanced green fluorescence protein is a direct and quantitative marker of HIV-1 expression or Tat-dependent long terminal repeat activity. Using these reporter cell lines, we found that the bis-anthracycline WP631, a recently developed DNA intercalator, efficiently inhibits HIV-1 expression at subcytotoxic concentrations. WP631 also abrogated acute HIV-1 replication in peripheral blood mononuclear cells infected with various primary virus isolates. We demonstrate that WP631-mediated HIV-1 inhibition is caused by the inhibition of Tat transactivation. The data presented suggest that WP631 could serve as a lead compound for a new type of HIV-1 inhibitor.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The advent of highly active antiretroviral therapy (HAART) in 1995 profoundly increased the life expectancy of human immunodeficiency virus (HIV) type 1 (HIV-1)-infected people in developed countries (31), but despite its tremendous benefits, HAART is not able to eradicate HIV from treated patients (for reviews, see references 2 and 34). As cessation of antiretroviral therapy results in an immediate rebound of viremia, life-long treatment is required. In this scenario, the continuously increasing numbers of viral strains that are resistant to therapy pose a serious threat to the current treatment success and require the development of new anti-HIV-1 compounds (29).

A possible alternative therapeutic target is Tat transactivation. Tat transactivation is dependent on the interaction of HIV-1 Tat with the conserved RNA structure of the transactivation responsive (TAR) element, which consists of RNA with a complex three-dimensional structure which contains a stem-loop and a bulge (5, 11, 17). The TAR element is a part of the nascent viral RNA and is essential for efficient initiation of viral transcription and subsequent elongation (1, 9, 14, 23).

To screen for and analyze the effects of compounds that would target HIV-1 transcription, we established several reporter cell lines in which HIV-1 expression or Tat-mediated long terminal repeat (LTR) activity is directly and quantitatively linked to enhanced green fluorescence protein (EGFP) fluorescence. For this purpose we infected Jurkat (T-cell lymphoma) cells with an HIV-1 strain engineered to express EGFP (22). Following the initial infection, we established several cell clones that constitutively expressed EGFP and secreted infectious viral particles. Using these cell lines and two additional reporter cell lines in which EGFP expression is controlled by the HIV-1 LTR in the absence or presence of Tat, we discovered that the bis-anthracycline WP631 efficiently and specifically inhibits HIV-1 expression by interfering with Tat transactivation. WP631 was designed by using the established anticancer drug daunorubicin as a scaffold compound (4). Two daunorubicin molecules were linked by a p-xylene linker, while the individual DNA-binding capacity of each daunorubicin molecule was maintained. While daunorubicin preferentially binds to triplets of the type 5'-(A/T)CG or 5'-(A/T)GC (3), WP631 binds to a CG(A/T)(A/T)CG hexanucleotide sequence, thereby greatly increasing its DNA-binding specificity (16, 38) and DNA-binding affinity (24). Interestingly, although WP631 had been described to act as a DNA intercalator, we could not find evidence that WP631 would interact with any of the HIV-1 key promoter elements (e.g., NF-{kappa}B and Sp1) and suppress LTR activity at the DNA level. Although it is possible that WP631 binds to the double-stranded parts of the TAR elements, the inhibitory kinetics of WP631 rather suggest interaction with an unknown cellular factor, as has been described for the Tat inhibitor Ro24-7429 (15).

By using WP631 as a lead compound, it should be possible to design bis-anthracyclines by altering the major scaffold molecule, the side chains, or the linker type that specifically inhibits HIV-1 while exhibiting very low DNA-binding activities and, thus, cytotoxicities. On the other hand, bimodal agents of the WP631 type with HIV-1-inhibitory and antineoplastic properties could be ideal compounds for the treatment of AIDS-related malignancies.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents. WP631 was purchased from ALEXIS (San Diego, Calif.) and was dissolved in dimethyl sulfoxide at a stock concentration of 5 mg/ml. Daunorubicin and doxorubicin were obtained from Calbiochem (San Diego, Calif.) and were dissolved in methanol at stock concentrations of 5 mg/ml. All antibodies were purchased from BD Pharmingen (San Diego, Calif.). Ro24-7429 was kindly provided by Hoffmann-La Roche, Inc. (Nutley, N.J.).

Cell lines and cell culture. All cell lines are maintained in RPMI 1640 medium supplemented with 2 mM L-glutamine, 100 U of penicillin per ml, 100 µg of streptomycin per ml, and 10% heat-inactivated fetal bovine serum.

Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of HIV-1-seronegative volunteers after informed consent was obtained from each individual. The lymphocyte fraction was isolated by using Ficoll-Paque gradients (Pharmacia). Cells were stimulated with phytohemagglutinin (PHA-L; 5 µg/ml) and interleukin-2 (IL-2; 100 U/ml) for 4 days, and then the cells were infected with patient HIV-1 isolates at multiplicities of infection of 0.06 for HIV-1 WEAU and OVWI and 0.03 for HIV-1 CUCY. After 2 h, the inocula were washed off, the cells were seeded in 48-well plates at a density of 106 cells/ml, and WP631 was added at the indicated concentrations (0.01 to 0.3 µg/ml). For experiments in which the ability of WP631 to inhibit cell-to-cell transmission of HIV-1 was tested, PBMCs were infected 4 days after PHA-L and IL-2 stimulation and then on day 8 were mixed with syngeneic cells at a ratio of 5 x 103 infected cells to 1 x 106 uninfected cells. WP631 was added at the time of cell mixing.

Viruses and plasmids. HIV-1 NLENG1 and HIV-1 89ENG are described elsewhere (22). Primary HIV-1 isolates were isolated from HIV-1-infected patients after they had given informed consent, and viral stocks were grown in PHA-L- and IL-2-stimulated PBMC cultures. pLTRGFP was constructed by excising the cat gene from pU3R-III cat (40, 43) by using XhoI-HindIII and inserting the EGFP open reading frame excised from pEGFP-1 (Clontech) by using the same enzymes. pLZRS-YFP-Tat was constructed by replacing EGFP with enhanced yellow fluorescence protein (EYFP; pEYFP-C1; Clontech) in the original vector pLZRS-pBMN-link-EGFP (a kind gift of M. Andersson, University of Alabama at Birmingham) by using NcoI and BsrG1 and inserting HIV-1 tat (from pBabe-puro-tat [26]) by using BamHI and SalI.

Flow cytometric analysis of HIV-1 expression and cytotoxicity. JNLG cells were seeded at a density of 106 cells/ml in 24-well plates and were then treated with the respective drugs. EGFP expression was analyzed with a FACStar Plus flow cytometer (Becton Dickinson). Dead-cell exclusion was performed by addition of propidium iodide (PI) and gating on PI-negative cells. Data were analyzed with CellQuest software (Becton Dickinson). Quantification of cytotoxicity levels for each sample was based on the ratio of PI-negative cells in the live gate to the total cell counts by using forward scatter (FSC) and side scatter (SSC) analysis. In comparison to the level of cell death determined with a Neubauer chamber and by trypan blue staining, this method underestimated cell death by 5%.

Analysis of WP631-mediated HIV-1 inhibition in PBMC cultures. To analyze the effect of WP631 on HIV-1 infection in PBMCs, freshly isolated cells were stimulated and infected as described above. Supernatants for HIV-1 p24 enzyme-linked immunosorbent assays (ELISAs) were collected on day 5 postinfection. At 24 h prior to collection of the supernatants, the medium from each culture was replaced with fresh medium. For flow cytometric analysis, the cells were stained with CD3-fluorescein isothiocyanate (FITC) and CD4-phycoerythrin (PE) antibodies or CD3-FITC and CD8-PE antibodies to determine the CD4 T-cell counts in the infected cultures. Depending on the antibody combination used, the percentage of CD4+ cells was defined as the CD3+CD4+ fraction or as the CD3+CD8– fraction. In all cultures tested, staining of CD3 and CD4 cells or CD3 and CD8 cells revealed identical results with respect to those achieved from the CD4+ T-cell counts, indicating that at the time of analysis, no CD4+ T cells were undetected because they had down modulated CD4 as a consequence of the HIV-1 infection. Use of an anti-CD4 antibody that could not be blocked by HIV-1 gp120 further ensured the accuracy of the CD4+ T-cell count (clone L120; BD Science, San Diego, Calif.).

Electrophoretic mobility shift assay and TransFactor assay. Duplex HIV-1 LTR probes (5'-AGGGAGGCGTGGCCTGGGCGGGACTGGGGAGTGGCGAG-3') were constructed by first 5' labeling the C-rich strand with [{gamma}-32P]ATP by using T4 polynucleotide kinase. Unincorporated ATP was removed with a G25 column (5 Prime-3 Prime Inc., Boulder, Colo.). Next, the labeled oligonucleotide was annealed to its complementary strand by heating to 95°C and slowly cooling to room temperature in TAM buffer (40 mM Tris acetate [pH 7.0], 5 mM MgCl2). Labeled duplex (20,000 cpm, equivalent to approximately 1 nM duplex per reaction mixture) was incubated with 2.5 µg of HeLa cell nuclear extract (gel shift grade HeLa NE; Promega Corp., Madison, Wis.) and 0.2 µg of poly(dI-dC) nonspecific competitor in buffer consisting of 25 mM HEPES (pH 7.4), 12.5 mM MgCl2, 70 mM KCl, 1 mM ZnSO4, l mM dithiothreitol, 0.1% Nonidet P-40, and 10% (vol/vol) glycerol. The samples were incubated for 1 h at room temperature without competitor (a buffer containing dimethyl sulfoxide at an amount equivalent to that in the highest concentration of WP631 was added) or in the presence of various concentrations of WP631 or doxorubicin.

Mercury TransFactor kits were used to determine the ability of WP631 to inhibit binding of several transcription factors (NF-{kappa}B p50, NF-{kappa}B p65, c-rel, c-fos, CREB1, and ATF2) to their respective consensus binding sites. Experiments were performed according the instructions of the manufacturer (Clontech) by using nuclear extracts from JNLG cells either in the absence or in the presence of WP631 (0.3 µg/ml).


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
EGFP-based reporter systems for the study of compounds suppressing HIV-1 expression. To screen for and analyze the effects of potential pharmaceutical compounds that may inhibit HIV-1 expression at the transcriptional level, we established several reporter cell lines based on Jurkat (T-cell lymphoma) cells (Fig. 1A). In these cell lines EGFP fluorescence is a quantitative marker of HIV-1 LTR activity. The clonal chronically actively infected JNLG and J89G cell lines (Fig. 1B) were established by infecting Jurkat cells with HIV-1 NLENG1 and HIV-1 89ENG, respectively (22), and allow quantification of LTR activity in the context of an integrated full-length virus. As in some previously established latently infected cell lines (22), EGFP expression in these chronically infected cells is controlled by the viral LTR, and EGFP fluorescence can be used as a direct and quantitative marker of HIV-1 expression.



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  		FIG.1. Novel reporter cell lines used to study inhibition of Tat transactivation and HIV-1 transcription. To study the influence of different agents on HIV-1 transcription in the context of a full-length virus, we infected Jurkat cells with either HIV-1 NLENG1 or HIV-1 89ENG and cloned the cells that had high levels of constitutive EGFP expression and that secreted infectious viral particles (A). The resulting chronically active HIV-1-infected cell lines were termed JNLG and J89G, respectively (B). To investigate the modulation of HIV-1 LTR activity, we stably transfected Jurkat cells with the reporter construct pLTRGFP (C). JLTRGon cells, which constitutively express EGFP in the absence of Tat expression, were derived by bulk sorting (sorting gate R2) (D). JLTRGon cells allow the study of Tat-independent regulation of LTR activity. JLTRG cells hold a stably integrated pLTRGFP, but in the absence of Tat expression or virus infection, they do not express EGFP (E). Retroviral transduction of JLTRG cells with pLZRS-YFP-Tat, followed by single-cell cloning, resulted in JLTRG/Tat-Y cells, in which EGFP fluorescence is a direct indicator of HIV-1 LTR activity; and EYFP expression serves as a quantitative marker for Tat expression (F).

 
To study Tat-dependent and Tat-independent regulation of HIV-1 LTR activity, we stably transfected Jurkat cells with a reporter construct (pLTRGFP) in which the HIV-1 LTR controls EGFP expression (Fig. 1C). Cells that constitutively expressed EGFP were bulk sorted, and the resulting polyclonal cell population was termed JLTRGon (Fig. 1D). EGFP expression in these cells is probably mediated by integration of the reporter plasmid in a transcriptionally active environment, as no mutation in the integrated LTR was detected (data not shown).

Following G418 selection, EGFP-negative cells were cloned and screened for cells that would exhibit EGFP expression following infection with HIV-1 NL4-3. The resulting clonal cell line was termed JLTRG (Fig. 1E).

Retroviral transduction of JLTRG cells with the pLZRS-YFP-Tat vector that expresses HIV-1 Tat and EYFP under the control of the same cytomegalovirus (CMV) promoter, followed by single-cell cloning, resulted in the establishment of JLTRG/Tat-Y cells (Fig. 1F). Through Tat-mediated activation of the viral LTR, JLTRG/Tat-Y cells express high levels of EGFP. As expression of HIV-1 Tat and the EYFP gene in these cells is linked through an internal ribosome entry site element, EYFP can be used as a marker for Tat expression. JLTRG/Tat-Y and JLTRGon cells thus allow quantification of the LTR activity in a Tat-dependent manner and a Tat-independent manner, respectively.

In all cell lines, regulation of HIV-1 expression or LTR activity (EGFP fluorescence) following treatment with a compound or stimulus of choice can be correlated with the regulation of cellular parameters such as expression of cell surface markers, viability, proliferation, apoptosis, and, in case the cells are treated with a fluorescent compound, drug uptake. This is demonstrated in Fig. 2A by use of the established Tat inhibitor Ro24-7429 (15) and the DNA-bis-intercalating agent WP631. On day 4 following treatment with Ro24-7429, 75% inhibition of EGFP expression was achieved with both compounds, which, in the case of Ro24-7429, is consistent with the results presented in previous reports (15). In the case of WP631, WP631-mediated HIV-1 inhibition and WP631 uptake can be measured simultaneously, as the compound is a fluorochrome. The inability of Ro24-7249 and WP631 to completely inhibit EGFP fluorescence as an indicator of HIV-1 expression suggests that part of EGFP expression in these cells is Tat independent.



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  		FIG. 2. WP631-mediated inhibition of HIV-1 expression and EGFP fluorescence in JNLG cells. (A) JNLG cells were cultivated in medium only or were treated with Ro24-7429 (10 µM) or WP631 (0.2 µg/ml). The level of EGFP fluorescence, which was used as a measure of HIV-1 expression, was determined by flow cytometric analysis 4 days posttreatment. Changes in EGFP expression are presented over a logarithmic scale. Numbers represent the EGFP mean channel fluorescence (MCF). The results are representative of those of five independent experiments. (B) JNLG cells were treated with various concentrations of WP631, and the level of EGFP fluorescence was monitored over time by flow cytometric analysis. Gray circles, the theoretical decay rate of EGFP if WP631 treatment would have resulted in an immediate and complete block of HIV-1 LTR activity. The results represent the means ± standard deviations of six independent experiments. (C) JNLG cells were treated with WP631 (0.3 µg/ml) for 4 days. At that time, viability could be determined by comparing the ratios of cells in the live gate (R1) to the total cell counts in the FSC-SCC dot plots of untreated and WP631-treated cells. Simultaneously, the levels of EGFP expression as measures of HIV-1 expression and relative drug uptake were determined. (D) On day 4 following treatment of JNLG cells with WP631 at various concentrations (0 to 0.4 µg/ml), the levels of EGFP fluorescence, as measured by flow cytometry, and p24 Gag expression, as measured by ELISA, in the supernatants of the respective cultures were determined. The results represent the means ± standard deviations of six independent experiments. (E) JNLG cells were treated with either daunorubicin (DA; 0.001 µg/ml) or WP631 (0.2 µg/ml) for 2 days (C, control). Then, cell viability and EGFP mean channel fluorescence were determined by flow cytometry. The results represent the means ± standard deviations of three independent experiments.

 
HIV-1-inhibitory effect of the bis-anthracycline WP631. Treatment of JNLG cells with the bis-anthracycline antibiotic WP631 resulted in a decrease in the level of EGFP expression. The observed inhibitory effect of WP631 was concentration dependent. WP631 at 0.05 µg/ml decreased the level of EGFP expression by 25%. WP631 at 0.2 µg/ml decreased the level of EGFP expression by 65% (Fig. 2B). Higher concentrations of WP631 did not further decrease the level of EGFP fluorescence but resulted in increasing levels of cell death.

Following treatment with a single dose of WP631, the level of EGFP fluorescence, which is used as a marker of HIV-1 expression, in JNLG cells continuously decreased for 4 to 6 days (Fig. 2B). The level of EGFP fluorescence then slowly increased again, until it finally regained full expression in the treated JNLG cells after 3 weeks, indicating that a single dose of WP631 can inhibit HIV-1 expression in cell culture over an extended period. With respect to the concentration-dependent WP631-mediated decrease in the level of EGFP expression and the kinetics of HIV-1 inhibition, similar results were obtained with J89G cells (data not shown). In both cell lines, WP631 concentrations of 0.3 µg/ml and higher started to cause cell death, as shown in the FSC-SSC dot plot shown in Fig. 2C. By using the ratio of the cells in the live gate and the total event counts, it is possible to determine the 50% cytotoxic concentration (CC50), which for WP631 was 0.4 µg/ml. The ability of EGFP to serve as a marker for HIV-1 expression is demonstrated by the tight correlation of EGFP fluorescence and the level of p24 Gag protein production (Fig. 2D). The slight difference in the slopes of the two curves can be explained by the antiproliferative properties and the cytotoxic effect of WP631, which decrease the levels of p24 Gag protein production in the supernatants of the cells treated with higher concentrations of WP631.

Interestingly, treatment of JNLG and J89G cells with daunorubicin, the parent compound of WP631, and the related compound doxorubicin, which are part of the standard treatment protocols for AIDS-related malignancies (10, 25), increased the levels of EGFP expression in the cell lines. The levels of EGFP fluorescence, which was used as an indicator of HIV-1 expression, were increased by daunorubicin and doxorubicin to 170 and 300%, respectively, of the levels observed in untreated control cells (Fig. 2E). As expected, daunorubicin and doxorubicin also exhibited very high levels of cytotoxicity in comparison to that exhibited by WP631 when JNLG cells and PBMCs were treated with the two compounds (CC50s of daunorubicin and doxorubicin for JNLG cells, 0.001 µg/ml).

WP631 inhibits HIV-1 replication in acutely infected PBMCs. PBMCs activated with PHA-L and IL-2 were infected with various primary HIV-1 isolates (HIV-1 CUCY, OVWI, and WEAU). At 2 h following infection, PBMCs were treated with WP631 at various concentrations (0.001 to 0.3 µg/ml), and at 7 days following infection, the p24 Gag protein concentrations in the culture supernatants were determined. The concentration-dependent ability of WP631 to suppress the infectivities of the primary HIV-1 isolates is depicted in Fig. 3A to C. Although the results varied slightly for the different donors and the different HIV-1 isolates tested, addition of WP631 to the infected cultures at 0.3 µg/ml reduced the level of HIV-1 p24 Gag expression by 80 to 90%.



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  		FIG. 3. Influence of WP631 on acute HIV-1 infection in PBMCs. PBMCs from three different donors (donors MP, MS, and OL) were infected with free virus from three different primary HIV-1 patient isolates, isolates OVWI (A), WEAU (B), and CUCY (C). At 2 h following the initial infection, WP631 was added at various concentrations (0 to 0.3 µg/ml). At 5 days postinfection, virus infectivity in the absence or presence of WP631 was determined on the basis of the level of HIV-1 p24 Gag protein expression. (D) To determine the ability of WP631 to inhibit cell-to-cell viral transmission, PBMCs from three different donors (donors 46-5, 59-4, and 63-7) were infected with primary HIV-1 patient isolate CUCY. At 4 days postinfection, 5 x 103 infected cells were mixed with 1 x 106 syngeneic PBMCs, and WP631 was added at the indicated concentrations (0.03 to 0.3 µg/ml). On day 8 of the experiment, HIV-1 replication was determined by HIV-1 p24 ELISA. (E) Relative CD4 counts were determined by staining the cells for CD3 and CD4 and subjecting them to flow cytometric analysis.

 
Similar results were obtained under more stringent experimental conditions when we tested the ability of WP631 to inhibit HIV-1 replication in a cell-to-cell viral transmission assay, which generally results in higher levels of virus replication. PBMCs were infected with HIV-1 CUCY, and at 4 days postinfection they were mixed with syngeneic uninfected PBMCs and were then treated with WP631. Supernatants for the p24 protein ELISA and cells were harvested on day 8, and the cells were subsequently stained for CD3 and CD4 to determine the influence of WP631 on the CD4+ T-cell count in the infected culture. The effect of WP631 on HIV-1 replication varied slightly for the three donors (Fig. 3D). Again, in contrast to the cell lines used in the initial experiments, WP631 concentrations of up to 0.3 µg/ml had no influence on cell viability but suppressed HIV-1 replication by 80 to 90% compared to the level of replication of HIV-1-infected but untreated cells, as measured by the p24 Gag protein ELISA. WP631 also stabilized the CD4+ T-cell count, another measure of HIV-1 expression, in the infected cultures in a concentration-dependent manner (Fig. 3E).

Influence of WP631 on binding of transcription factors to the HIV-1 LTR Sp1 element. We initially investigated whether WP631 would interfere with the binding of transcription factors that are vital for HIV-1 expression after time was allowed for intercalation into the integrated viral LTR. The HIV-1 LTR contains three Sp1 sites located in close proximity to each other, and these are essential for HIV-1 expression (12). As WP631 has been described to bind with an extremely high affinity to an Sp1 consensus sequence (27), we tested whether WP631 could compete away nuclear factors binding to a 38-bp fragment containing all three Sp1 sites of the HIV-1 HxB LTR (Fig. 4). Nuclear extracts derived from HeLa cells formed a complex with the labeled LTR-Sp1 probe (lane 1). Addition of WP631 (0.5 to 8 µM, equivalent to 0.6 to 9.2 µg/ml; lanes 2 to 6) or doxorubicin (0.5 to 8 µM, equivalent to 0.3 to 4.6 µg/ml; lanes 7 to 11) inhibited the formation of the complex in a concentration-dependent manner. In these experiments, abrogation of the complex required more than 4.6 µg of WP631 per ml, which is more than 20 times the dose required for inhibition of HIV activity in JNLG cells and PBMCs. Furthermore, doxorubicin, which we demonstrated increases the level of HIV-1 expression in JNLG cells (data not shown), exhibits a similar, concentration-dependent ability to compete with the binding of transcription factors to the LTR-Sp1 probe.



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  		FIG. 4. Influence of WP631 on binding of transcription factors to the HIV-1 LTR Sp1 element. Electrophoretic mobility shift assay experiments were performed with a 38-bp oligonucleotide comprising the three Sp1 elements of the HIV-1 LTR and HeLa nuclear extracts. Samples were incubated without competitor (lane 1) or in the presence of various concentrations of WP631 (0.5 to 8 µM; lanes 2 to 6) and doxorubicin (Doxo; 0.5 to 8 µM; lanes 7 to 11).

 
Using the Mercury TransFactor system, we further investigated whether WP631 would inhibit the binding of transcription factors to NF-{kappa}B p50, NF-{kappa}B p65, c-rel, c-fos, CREB1, and ATF2 consensus binding sequences, all of which have been described to be important for HIV-1 expression (6, 8, 20, 21, 30, 33, 36, 37, 39, 41). Binding of transcription factors present in the nuclear extracts of JNLG cells to the respective consensus binding sites was analyzed in the absence or the presence of 0.3 µg of WP631 per ml. We found that nuclear extracts of JNLG cells contain high levels of NF-{kappa}B p50 and ATF2 and low levels of NF-{kappa}B p65, c-rel, c-fos, and CREB1; but binding of these transcription factors to the respective consensus sequences was not influenced by the presence of WP631 (data not shown).

Influence of WP631 on expression of immune-relevant genes. To determine the mechanism underlying the observed WP631-mediated inhibition of HIV-1, we investigated the specificity of the HIV-1-inhibitory effect of WP631. We initially analyzed whether WP631 influences the expression of several immune-relevant genes, such as major histocompatibility complex (MHC) class I, MHC class II, CD3, CD4, CD8, CD28, CCR, or CXCR4, by PBMCs or immortalized cell lines (Jurkat and THP-1 cells) but found that none of them were influenced by WP631 in any of the cell types tested (Table 1). These results suggest that WP631 most likely does not modulate antigen presentation and recognition, which would result in additional immune suppression. The inability of WP631 to regulate expression of CD4, CCR5, and CXCR4 further suggests that WP631-mediated inhibition of HIV-1 replication in PBMCs does not occur at the level of receptor modulation.


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  		TABLE 1. Regulation of expression of several cellular immune-relevant genes following treatment with WP631a

 
WP631 specifically targets Tat transactivation. As we had observed that WP631 did not alter the expression of a variety of cellular genes, we wanted to investigate whether the compound would be generally active against retroviruses. For this purpose, we established Jurkat cell lines that constitutively express EGFP under the control of the murine leukemia virus (MuLV) promoter (JEGFP) and monitored the level of EGFP expression in the respective cells over a period of 7 days following treatment with WP631 at various concentrations (0.03 to 0.3 µg/ml). In contrast to EGFP expression in JNLG and J89G cells, EGFP fluorescence in JEGFP cells was not regulated by WP631 over the entire period of the experiment. Figure 5 depicts the relative levels of EGFP expression in JNLG, J89G, and JEGFP cells 4 days following treatment with WP631. WP631 also did not influence EGFP expression in Jurkat cells controlled by the CMV promoter (data not shown). As MuLV, unlike HIV-1, is a retrovirus that expresses its genes in a transactivation-independent manner, we continued to investigate whether WP631 would specifically target Tat transactivation.



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  		FIG. 5. Effect of WP631 on LTR-controlled Tat-dependent and Tat-independent expression of EGFP. The influence of WP631 on Tat-dependent EGFP expression in the context of a full-length virus (JNLG and J89G) and in the context of a stable integrated LTR-EGFP reporter plasmid (JLTRG/Tat-Y) was compared to the influence of WP631 on EGFP expression controlled by the HIV-1 LTR in the absence of Tat (JLTRGon) and by the MuLV promoter (JEGFP). All cells were cultured in medium or were treated with WP631 at various concentrations (0.03 to 0.3 µg/ml) for 4 days and then subjected to flow cytometric analysis of EGFP fluorescence. The results represent the means ± standard deviations of three independent experiments.

 
For this purpose, we treated JLTRG/Tat-Y cells with WP631 at various concentrations (0.03 to 0.3 µg/ml). The level of Tat-dependent LTR activity in these cells is indicated by the expression of EGFP, whereas the levels of Tat expression are linked to the level of EYFP fluorescence. In the presence of WP631, EGFP expression in JLTRG/Tat-Y cells is reduced in a manner similar to that seen in JNLG cells containing full-length HIV-1, indicating that WP631-mediated HIV-1 inhibition is a result of specific interference with Tat transactivation (Fig. 5 and Fig. 6). Four days following treatment with 0.3 µg of WP631 per ml, the level of EGFP expression was reduced by 70% compared to that for untreated JLTRG/Tat-Y cells. At the same time, the level of EYFP expression under the control of the CMV promoter was not reduced (Fig. 6A and B), indicating that Tat expression in JLTRG/Tat-Y cells was not affected by WP631, which was also confirmed by Western blotting analysis (data not shown). The inhibitory effect determined from the levels of EGFP reduction in JLTRG/Tat-Y cells achieved with WP631 at this time was comparable to the inhibitory effect demonstrated by the Tat inhibitor Ro24-7429 (Fig. 2A). To determine the level of Tat-independent EGFP expression that can occur in these cells, we stimulated parental JLTRG cells (Fig. 6C) with tumor necrosis factor alpha (TNF-{alpha}; 10 ng/ml), which resulted in a 1-log shift in the level of the EGFP fluorescence intensity (Fig. 6D). The level of EGFP fluorescence observed in JLTRG cells following stimulation with TNF-{alpha} is equivalent to the level of Tat-independent EGFP expression seen in JLTRGon cells (Fig. 1). Hence, it is expected that a specific Tat inhibitor could not reduce the level of EGFP expression to levels lower than this, as long as the treated cells remain activated due to the previous presence of Tat. Indeed, EGFP expression in cells of the JLTRGon cell line, a Jurkat cell-based cell line in which the cells exhibit constitutive levels of EGFP expression under the control of the HIV-1 LTR in the absence of HIV-1 Tat, was not inhibited by WP631 (Fig. 5). This result indicates that WP631 inhibits only Tat-mediated LTR activity and does not interfere with HIV-1 expression by binding to elements on the integrated HIV-1 LTR.



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  		FIG. 6. WP631-mediated inhibition of Tat transactivation. JLTRG/Tat-Y cells were cultured in medium (A) or were treated for 4 days with 0.3 µg of WP631 per ml (B). EGFP expression was determined as a measure of Tat-mediated HIV-1 LTR activity, and changes are indicated as mean channel fluorescence intensity (MCF; arbitrary units). EYFP expression serves as an indicator of HIV-1 Tat expression and is presented as Y-MCF. To determine the level of possible Tat-independent LTR activity in these experiments, parental cell line JLTRG was cultured in medium (C) or was stimulated with TNF-{alpha} (10 ng/ml) (D). The results are representative of those of three independent experiments.

 
The idea that WP631 does not interfere with HIV-1 expression at the DNA level is further strengthened by the absence of putative WP631 binding sites of the CG(A/T)(A/T)CG type in the 5' LTR regions of integrated HIV-1 NL4-3 and 89.6 (Table 2). Only some related hexanucleotide sequences (GCT/TGC, GCG/GCG, GCT/AGC, GCG/CGC) that could serve as potential WP631 binding sites were identified, but none of these overlapped with LTR elements that have been identified to be important for HIV-1 expression (e.g., NF-{kappa}B, Sp1, and AP-1 elements).


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  		TABLE 2. Frequency of putative WP631-binding motifs in the HIV-1 NL4-3 and 89.6 LTR and genes

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The increasing number of HIV-1 strains that are resistant to commonly used retroviral inhibitors requires the development of new drugs, possibly drugs that target viral structures other than the viral protease or reverse transcriptase (29). A possible alternative strategy that can be used to suppress HIV-1 expression would be the inhibition of Tat transactivation that is essential for the initiation of viral transcription and elongation (9, 14, 23). In the present study, we demonstrate that at subcytotoxic concentrations the bis-anthracycline antibiotic WP631 (4, 16, 38) is capable of suppressing HIV-1 expression in infected cell lines and PBMCs by specifically inhibiting Tat transactivation.

To screen for and study the effects of compounds that interfere with HIV-1 transcription, we established several reporter cell lines that allow the direct monitoring of HIV-1 LTR activity. JNLG and J89G cells are EGFP-based reporter cell lines that allow the study of the modulation of active HIV-1 expression in the context of a full-length virus (22). JLTRG/Tat and JLTRG/Tat-Y cells are stable T-cell lines that allow the study of modulation of Tat-dependent LTR activity by using EGFP as a direct and quantitative readout, whereas the use of JLTRGon cells allows us to study regulation of Tat-independent LTR activity.

In JNLG and J89G cells, WP631 down regulated HIV-1 expression by 70%, as measured by EGFP fluorescence levels and p24 Gag ELISA, without any apparent cytotoxicity. The level of HIV-1 inhibition achieved in these cells at this time point was comparable to that seen with the established Tat inhibitor Ro24-7249 (15). As EGFP expression in these cells is controlled by the integrated viral LTR, these results suggest that WP631 inhibits HIV-1 expression by suppressing LTR activity or interfering with viral RNA transcription. The observed long-lasting inhibition of HIV-1 expression indicates that WP631 binding to the LTR or to other structures involved in HIV-1 transcription is of extremely high affinity. Alternatively, WP631 treatment could result in alterations of the HIV-1 LTR, such as histone acetylation or DNA methylation (13, 19, 28, 35, 42, 45). WP631 not only inhibited chronic HIV-1 expression in JNLG and J89G cells, but it also efficiently abrogated acute HIV-1 infection in PBMC cultures infected with various primary HIV-1 isolates. When WP631 was applied at subcytotoxic concentrations, HIV-1 replication in infected PBMC cultures was inhibited by 80 to 90%, and the inhibition was independent of the viral strain and the donor. Notably, the cytotoxicity that was observed for the T-cell reporter lines with higher WP631 concentrations was reduced in the primary T-cell cultures, probably due to the slow proliferation rates of these cells.

As WP613 was published to be a major-groove DNA intercalator, we suspected that HIV-1 inhibition is caused by the specific binding of WP631 to a key regulatory site within the viral LTR. As previous work (27) suggested that WP631 interferes with Sp1 binding, we initially focused on whether WP631 could inhibit the binding of transcription factors to the three Sp1 sites that are located in the HIV-1 LTR and that have been reported to be essential for HIV-1 expression (12). Interference of WP631 with the binding of transcription factors could thus explain the observed down regulation of HIV-1 activity, but inhibition of transcription factor binding to the HIV-1 LTR Sp1 element required WP631 concentrations that greatly exceeded (20-fold higher) the concentration needed to inhibit HIV-1 infection in JNLG cells or PBMCs. These results indicate that the anti-HIV-1 effect of WP631 most likely is unrelated to the ability of WP631 to compete away transcription factor binding to the HIV-1 LTR Sp1 sequence in this system. The different abilities of WP631 to inhibit Sp1 binding in different systems might be explained by the high DNA sequence-binding specificity described for WP631 (4, 24), which would allow intercalation in the Sp1 consensus sequence used (5'-GAA TTC GGG GCG GGG CGA ATT-3') (27) but not in the HIV-1 Sp1 element (5'-AGG GAG GCG TGG CCT GGG CGG GAC TGG GGA GTG GCG AG-3').

We also could find no evidence that WP631 interferes with the binding of several other cellular transcription factors, such as NF-{kappa}B p50, NF-{kappa}B p65, c-rel, c-fos, ATF2, and CREB1, that have been described to be essential for HIV-1 expression.

Sequence analysis further revealed that the HIV-1 LTR does not contain any binding sequences for WP631 [5'-(A/T)CG or 5'-(A/T)GC]. Some related sequences that could theoretically serve as putative WP631 binding sequences were identified, but none of them overlapped with key regulatory elements important for HIV-1 transcription.

Overall, these findings make it unlikely that the observed specific HIV-1-inhibitory effect of WP631 is achieved through the DNA-intercalating abilities of the compound.

Further indirect evidence for this hypothesis is provided by the finding that a variety of cellular genes which are regulated through these transcription factors are not affected by the presence of WP631. No regulation of immune-relevant genes, such as MHC class I, MHC class II, CD3, CD4, CD8, CXCR4, or CCR5, could be detected. The inability of WP631 to interfere with the expression of the primary HIV-1 receptor CD4 or the coreceptors CXCR4 and CCR5 also indicates that the observed inhibition of HIV-1 replication in PBMCs is not related to down regulation of these HIV-1 receptors.

Whereas the observed WP631-mediated HIV-1 inhibition in JNLG cells and PBMCs could theoretically be explained by effects that target structures outside the viral LTR, the ability of WP631 to inhibit EGFP expression in JLTRG/Tat-Y cells narrows the effector side of WP631 to the HIV-1 LTR, the Tat protein, and cellular proteins interacting with Tat. With no evidence that WP631 interferes with LTR activity at the DNA level, the most likely target is Tat transactivation. Indeed, whereas Tat-dependent LTR activity in JLTRG/Tat-Y cells could be inhibited with the same efficiency as HIV-1 expression in JNLG cells, WP631 could not inhibit the basal level of HIV-1 LTR-mediated but Tat-independent EGFP expression in JLTRGon cells. Also, the EGFP expression directed by the MuLV promoter, a retroviral promoter that efficiently expresses its genes in the absence of transactivational activity, was not influenced by WP631 concentrations that efficiently inhibited HIV-1 expression.

HIV-1 Tat binds to the TAR element, which is part of each viral RNA. The TAR element forms a stem-loop RNA structure that holds stretches of double-stranded RNA (dsRNA) downstream from the site of initiation of transcription in the 5' LTR. In vitro and in vivo studies have demonstrated that correct folding of both the 5' bulge from positions +23 to +25 in the TAR element and the central loop from positions 131 to 136 in TAR are required for Tat transactivation (7, 18). Efficient activation by Tat is dependent on the cellular protein cyclin T1, which binds to the activation domain of Tat and the central loop in the TAR element (46). Cyclin T1 forms a complex with cyclin-dependent kinase 9. This complex is termed the positive transcription elongation factor b (P-TEFb) (32). P-TEFb, in turn, hyperphosphorylates the C-terminal domain of RNA polymerase II, which is required for Tat transactivation (47).

Interference of WP631 with the binding of either of these factors to the TAR element would thus result in decreased levels of HIV-1 expression. With no evidence that WP631 or the parent compound, daunorubicin, specifically binds to any proteins involved in transcription, we hypothesize that WP631 interferes with HIV-1 expression by binding to the TAR element.

As DNA usually forms B-form helices, whereas dsRNA forms A-form helices, it is unlikely that WP631 would intercalate into the dsRNA stretches of the TAR element, as it has been described to bind to DNA (4, 16, 24). Nevertheless, as the TAR element alters between different conformations with similar free energies, it is tempting to speculate that a complex molecule such as WP631 could also bind in alternative ways other than intercalation. Binding of WP631 to the TAR element would in turn prevent efficient binding of Tat, cyclin T1, or P-TEFb. Alternatively, WP631 may interfere with the binding of RNA polymerase II. In either case, direct binding of WP631 to the TAR element or WP631-mediated inhibition of RNA polymerase II, the inhibitory onset kinetics of WP631 reflected by the decrease in the level of EGFP expression should be much faster (Fig. 2B), as binding of WP631 should result in an immediate and complete block of viral transcription. In contrast, the inhibitory onset kinetic is comparable to the one seen for the established Tat inhibitor Ro24-7249 (data not shown). This inhibitor does not directly interfere with the Tat protein or binding of Tat to the TAR element but, rather, seems to inhibit an unknown cellular factor. We thus hypothesize that WP631 interferes with a cellular transcription factor important for Tat transactivation. This hypothesis is further strengthened by the finding that WP631 has a long-lasting inhibitory effect that could not be explained by the binding of WP631 to the TAR element.

To our knowledge, this is the first description of a DNA bis-intercalator that specifically interferes with Tat transactivation. The bis-anthracycline WP631 at this time can only serve as a lead compound, as the therapeutic index (CC50/IC50) is too low. Key to the further development of WP631-like compounds as specific HIV-1 inhibitors will be to decrease the intrinsic DNA-binding abilities, which are responsible for the observed cytotoxicity, without altering the HIV-1-inhibitory properties. The tremendous differences between the parental anthracycline antibiotics daunorubicin and doxorubicin and the bis-anthracycline antibiotic WP631 with respect to their cytotoxicities and their abilities to inhibit HIV-1 expression indicate that both effects can be modulated independently.

In this context the structural similarity to temacrazine, which has been described to inhibit HIV-1, is noteworthy (44). Temacrazine is a bis-triazoloacridone analog that has, on the basis of its DNA-intercalating abilities, shown some promise in the treatment of colon cancer. As both agents, WP631 and temacrazine, have been described to exert their anti-HIV-1 properties at subcytotoxic levels, it is likely that the structural properties of the compounds are more important than their DNA-binding abilities for their anti-HIV-1 effects.

Although WP631, because of potential toxicities, is not suitable as a specific HIV-1 inhibitor at present, its ability to exert simultaneous antineoplastic and HIV-1-inhibitory effects at high concentrations predestines WP631 to be a compound that should be considered for the treatment of AIDS-related malignancies. Antineoplastic compounds that control HIV-1 infection would allow the interruption of HAART during cancer treatment without the fear of the evolvement of drug-resistant virus. Thus, compounds such as WP631 should reduce the added toxicities of two simultaneous chemotherapies, which should improve the overall outcomes of treatment for AIDS-related malignancies.


   ACKNOWLEDGMENTS
 
This work was supported in parts by NIH grant MH63650 and amFAR research grant 02797-RG (to E.N.B.) and The Welch Foundation, Houston, Tex. (to W.P.).


   FOOTNOTES
 
* Corresponding author. Present address: Division of Hematology/Oncology, University of Alabama at Birmingham, KAUL 840, 720 20th St. South, Birmingham, AL 35294. Phone: (205) 934-1547. Fax: (205) 934-1580. E-mail: okutsch{at}uab.edu. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Berkhout, B., R. H. Silverman, and K. T. Jeang. 1989. Tat trans-activates the human immunodeficiency virus through a nascent RNA target. Cell 59:273-282.[CrossRef][Medline]
  2. Blankson, J. N., D. Persaud, and R. F. Siliciano. 2002. The challenge of viral reservoirs in HIV-1 infection. Annu. Rev. Med. 53:557-593.[CrossRef][Medline]
  3. Chaires, J. B., J. E. Herrera, and M. J. Waring. 1990. Preferential binding of daunomycin to 5'ATCG and 5'ATGC sequences revealed by footprinting titration experiments. Biochemistry 29:6145-6153.[CrossRef][Medline]
  4. Chaires, J. B., F. Leng, T. Przewloka, I. Fokt, Y. H. Ling, R. Perez-Soler, and W. Priebe. 1997. Structure-based design of a new bisintercalating anthracycline antibiotic. J. Med. Chem. 40:261-266.[CrossRef][Medline]
  5. Cullen, B. R. 1994. RNA-sequence-mediated gene regulation in HIV-1. Infect. Agents Dis. 3:68-76.[Medline]
  6. Doerre, S., P. Sista, S. C. Sun, D. W. Ballard, and W. C. Greene. 1993. The c-rel protooncogene product represses NF-{kappa}B p65-mediated transcriptional activation of the long terminal repeat of type 1 human immunodeficiency virus. Proc. Natl. Acad. Sci. USA 90:1023-1027.[Abstract/Free Full Text]
  7. Frankel, A. D., and J. A. Young. 1998. HIV-1: fifteen proteins and an RNA. Annu. Rev. Biochem. 67:1-25.[CrossRef][Medline]
  8. Franza, B. R., Jr., F. J. Rauscher III, S. F. Josephs, and T. Curran. 1988. The Fos complex and Fos-related antigens recognize sequence elements that contain AP-1 binding sites. Science 239:1150-1153.[Abstract/Free Full Text]
  9. Fujinaga, K., T. P. Cujec, J. Peng, J. Garriga, D. H. Price, X. Grana, and B. M. Peterlin. 1998. The ability of positive transcription elongation factor B to transactivate human immunodeficiency virus transcription depends on a functional kinase domain, cyclin T1, and Tat. J. Virol. 72:7154-7159.[Abstract/Free Full Text]
  10. Gates, A. E., and L. D. Kaplan. 2002. AIDS malignancies in the era of highly active antiretroviral therapy. Oncology 16:657-665.[Medline]
  11. Grate, D., and C. Wilson. 1997. Role REVersal: understanding how RRE RNA binds its peptide ligand. Structure 5:7-11.[Medline]
  12. Harrich, D., J. Garcia, F. Wu, R. Mitsuyasu, J. Gonazalez, and R. Gaynor. 1989. Role of SP1-binding domains in in vivo transcriptional regulation of the human immunodeficiency virus type 1 long terminal repeat. J. Virol. 63:2585-2591.[Abstract/Free Full Text]
  13. He, G., and D. M. Margolis. 2002. Counterregulation of chromatin deacetylation and histone deacetylase occupancy at the integrated promoter of human immunodeficiency virus type 1 (HIV-1) by the HIV-1 repressor YY1 and HIV-1 activator Tat. Mol. Cell. Biol. 22:2965-2973.[Abstract/Free Full Text]
  14. Herrmann, C. H., and A. P. Rice. 1995. Lentivirus Tat proteins specifically associate with a cellular protein kinase, TAK, that hyperphosphorylates the carboxyl-terminal domain of the large subunit of RNA polymerase II: candidate for a Tat cofactor. J. Virol. 69:1612-1620.[Abstract]
  15. Hsu, M. C., U. Dhingra, J. V. Earley, M. Holly, D. Keith, C. M. Nalin, A. R. Richou, A. D. Schutt, S. Y. Tam, M. J. Potash, et al. 1993. Inhibition of type 1 human immunodeficiency virus replication by a Tat antagonist to which the virus remains sensitive after prolonged exposure in vitro. Proc. Natl. Acad. Sci. USA 90:6395-6399.[Abstract/Free Full Text]
  16. Hu, G. G., X. Shui, F. Leng, W. Priebe, J. B. Chaires, and L. D. Williams. 1997. Structure of a DNA-bisdaunomycin complex. Biochemistry 36:5940-5946.[CrossRef][Medline]
  17. Jeang, K. T., H. Xiao, and E. A. Rich. 1999. Multifaceted activities of the HIV-1 transactivator of transcription, Tat. J. Biol. Chem. 274:28837-28840.[Free Full Text]
  18. Jones, K. A., and B. M. Peterlin. 1994. Control of RNA initiation and elongation at the HIV-1 promoter. Annu. Rev. Biochem. 63:717-743.[CrossRef][Medline]
  19. Jordan, A., P. Defechereux, and E. Verdin. 2001. The site of HIV-1 integration in the human genome determines basal transcriptional activity and response to Tat transactivation. EMBO J. 20:1726-1738.[CrossRef][Medline]
  20. Kaufman, P. A., J. B. Weinberg, and W. C. Greene. 1992. Nuclear expression of the 50- and 65-kD Rel-related subunits of nuclear factor-kappa B is differentially regulated in human monocytic cells. J. Clin. Investig. 90:121-129.
  21. Kretzschmar, M., M. Meisterernst, C. Scheidereit, G. Li, and R. G. Roeder. 1992. Transcriptional regulation of the HIV-1 promoter by NF-{kappa}B in vitro. Genes Dev. 6:761-774.[Abstract/Free Full Text]
  22. Kutsch, O., E. N. Benveniste, G. M. Shaw, and D. N. Levy. 2002. Direct and quantitative single-cell analysis of human immunodeficiency virus type 1 reactivation from latency. J. Virol. 76:8776-8786.[Abstract/Free Full Text]
  23. Laspia, M. F., A. P. Rice, and M. B. Mathews. 1989. HIV-1 Tat protein increases transcriptional initiation and stabilizes elongation. Cell 59:283-292.[CrossRef][Medline]
  24. Leng, F., W. Priebe, and J. B. Chaires. 1998. Ultratight DNA binding of a new bisintercalating anthracycline antibiotic. Biochemistry 37:1743-1753.[CrossRef][Medline]
  25. Levine, A. M., L. Seneviratne, and A. Tulpule. 2001. Incidence and management of AIDS-related lymphoma. Oncology 15:629-639.[Medline]
  26. Levy, D. N., L. S. Fernandes, W. V. Williams, and D. B. Weiner. 1993. Induction of cell differentiation by human immunodeficiency virus 1 vpr. Cell 72:541-550.[CrossRef][Medline]
  27. Martin, B., A. Vaquero, W. Priebe, and J. Portugal. 1999. Bisanthracycline WP631 inhibits basal and Sp1-activated transcription initiation in vitro. Nucleic Acids Res. 27:3402-3409.[Abstract/Free Full Text]
  28. Marzio, G., and M. Giacca. 1999. Chromatin control of HIV-1 gene expression. Genetica 106:125-130.[CrossRef][Medline]
  29. Menendez-Arias, L. 2002. Targeting HIV: antiretroviral therapy and development of drug resistance. Trends Pharmacol. Sci. 23:381-388.[CrossRef][Medline]
  30. Muchardt, C., J. S. Seeler, A. Nirula, D. L. Shurland, and R. B. Gaynor. 1992. Regulation of human immunodeficiency virus enhancer function by PRDII-BF1 and c-rel gene products. J. Virol. 66:244-250.[Abstract/Free Full Text]
  31. Palella, F. J., K. M. Delaney, A. C. Moorman, M. O. Loveless, J. Fuhrer, G. A. Satten, D. J. Aschman, S. D. Holmberg, et al. 1998. Declining morbidity and mortality among patients with advanced human immunodeficiency virus infection. N. Engl. J. Med. 338:853-860.[Abstract/Free Full Text]
  32. Peng, J., Y. Zhu, J. T. Milton, and D. H. Price. 1998. Identification of multiple cyclin subunits of human P-TEFb. Genes Dev. 12:755-762.[Abstract/Free Full Text]
  33. Phares, W., B. R. Franza, Jr., and W. Herr. 1992. The kappa B enhancer motifs in human immunodeficiency virus type 1 and simian virus 40 recognize different binding activities in human Jurkat and H9 T cells: evidence for NF-{kappa}B-independent activation of the kappa B motif. J. Virol. 66:7490-7498.[Abstract/Free Full Text]
  34. Pierson, T., J. McArthur, and R. F. Siliciano. 2000. Reservoirs for HIV-1: mechanisms for viral persistence in the presence of antiviral immune responses and antiretroviral therapy. Annu. Rev. Immunol. 18:665-708.[CrossRef][Medline]
  35. Quivy, V., E. Adam, Y. Collette, D. Demonte, A. Chariot, C. Vanhulle, B. Berkhout, R. Castellano, Y. de Launoit, A. Burny, J. Piette, V. Bours, and C. Van Lint. 2002. Synergistic activation of human immunodeficiency virus type 1 promoter activity by NF-{kappa}B and inhibitors of deacetylases: potential perspectives for the development of therapeutic strategies. J. Virol. 76:11091-11103.[Abstract/Free Full Text]
  36. Rabbi, M. F., M. Saifuddin, D. S. Gu, M. F. Kagnoff, and K. A. Roebuck. 1997. U5 region of the human immunodeficiency virus type 1 long terminal repeat contains TRE-like cAMP-responsive elements that bind both AP-1 and CREB/ATF proteins. Virology 233:235-245.[CrossRef][Medline]
  37. Raziuddin, Mikovits, J. A., I. Calvert, S. Ghosh, H. F. Kung, and F. W. Ruscetti. 1991. Negative regulation of human immunodeficiency virus type 1 expression in monocytes: role of the 65-kDa plus 50-kDa NF-{kappa}B dimer. Proc. Natl. Acad. Sci. USA 88:9426-9430.[Abstract/Free Full Text]
  38. Robinson, H., W. Priebe, J. B. Chaires, and A. H. Wang. 1997. Binding of two novel bisdaunorubicins to DNA studied by NMR spectroscopy. Biochemistry 36:8663-8670.[CrossRef][Medline]
  39. Roebuck, K. A., D. A. Brenner, and M. F. Kagnoff. 1993. Identification of c-fos-responsive elements downstream of TAR in the long terminal repeat of human immunodeficiency virus type-1. J. Clin. Investig. 92:1336-1348.
  40. Rosen, C. A., J. G. Sodroski, K. Campbell, and W. A. Haseltine. 1986. Construction of recombinant murine retroviruses that express the human T-cell leukemia virus type II and human T-cell lymphotropic virus type III trans-activator genes. J. Virol. 57:379-384.[Abstract/Free Full Text]
  41. Ross, H. L., M. R. Nonnemacher, T. H. Hogan, S. J. Quiterio, A. Henderson, J. J. McAllister, F. C. Krebs, and B. Wigdahl. 2001. Interaction between CCAAT/enhancer binding protein and cyclic AMP response element binding protein 1 regulates human immunodeficiency virus type 1 transcription in cells of the monocyte/macrophage lineage. J. Virol. 75:1842-1856.[Abstract/Free Full Text]
  42. Sheridan, P. L., T. P. Mayall, E. Verdin, and K. A. Jones. 1997. Histone acetyltransferases regulate HIV-1 enhancer activity in vitro. Genes Dev. 11:3327-3340.[Abstract/Free Full Text]
  43. Sodroski, J., C. Rosen, F. Wong-Staal, S. Z. Salahuddin, M. Popovic, S. Arya, R. C. Gallo, and W. A. Haseltine. 1985. trans-Acting transcriptional regulation of human T-cell leukemia virus type III long terminal repeat. Science 227:171-173.[Abstract/Free Full Text]
  44. Turpin, J. A., R. W. Buckheit, Jr., D. Derse, M. Hollingshead, K. Williamson, C. Palamone, M. C. Osterling, S. A. Hill, L. Graham, C. A. Schaeffer, M. Bu, M. Huang, W. M. Cholody, C. J. Michejda, and W. G. Rice. 1998. Inhibition of acute-, latent-, and chronic-phase human immunodeficiency virus type 1 (HIV-1) replication by a bistriazoloacridone analog that selectively inhibits HIV-1 transcription. Antimicrob. Agents Chemother. 42:487-494.[Abstract/Free Full Text]
  45. Van Lint, C. 2000. Role of chromatin in HIV-1 transcriptional regulation. Adv. Pharmacol. 48:121-160.
  46. Wei, P., M. E. Garber, S. M. Fang, W. H. Fischer, and K. A. Jones. 1998. A novel CDK9-associated C-type cyclin interacts directly with HIV-1 Tat and mediates its high-affinity, loop-specific binding to TAR RNA. Cell 92:451-462.[CrossRef][Medline]
  47. Yang, X., M. O. Gold, D. N. Tang, D. E. Lewis, E. Aguilar-Cordova, A. P. Rice, and C. H. Herrmann. 1997. TAK, an HIV Tat-associated kinase, is a member of the cyclin-dependent family of protein kinases and is induced by activation of peripheral blood lymphocytes and differentiation of promonocytic cell lines. Proc. Natl. Acad. Sci. USA 94:12331-12336.[Abstract/Free Full Text]

Antimicrobial Agents and Chemotherapy, May 2004, p. 1652-1663, Vol. 48, No. 5
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.5.1652-1663.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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