Background

During HIV-1 replication, the host polymerase (Pol II) is recruited to the viral promoter within the long terminal repeat (LTR) and initiates transcription [1]. Pol II initiates transcription, but elongation of most of the transcripts is blocked by negative elongation factors [2, 3]. The HIV-1 transcription transactivator Tat binds to the bulge of the HIV-1 RNA stem loop termed TAR that is found in all nascent HIV-1 messages and recruits positive transcription elongation factor b (P-TEFb) to the LTR [reviewed in [4, 5]]. P-TEFb phosphorylates both the carboxyl-terminal domain (CTD) of Pol II [54] was used through out this study. p256 contains the V3 region from a patient isolate inserted into HIV-1pNL4-3 backbone [54]. 293T cells were seeded at 5 × 105 cells per well in a six-well tray a day before transfection. Cells were transfected with 7 μg of p256 proviral DNA expressing plasmid using the calcium phosphate procedure to generate HIV-1p256 viral stocks [53]. Virus-containing supernatants were collected at 24, 48, 72 and 96 hours post-transfection. Virus production was measured by titering the virus-containing, cell-free supernatants on HeLa37 cells using single-hit infectivity assays described below.

HIV single-hit infectivity assay

Short-term, single hit infectivity studies were performed as previously described [53]. HeLa37 cells were plated in a 48-well tray and triplicate wells were infected with a dual-tropic HIV-1p256 and serial dilutions of P-TEFb inhibitor for 40 hours. The cells were fixed with 75% acetone/25% H2O and immunostained for HIV-1 antigens using human anti-HIV serum (a gift from Dr. Jack Stapleton, Univ. of Iowa) and HRP-conjugated goat anti-human IgG followed by staining with 3-amino-9-ethylcarbazole (AEC). The HIV-1 antigen-positive cells were counted. Experiments were repeated at least three times with each drug concentration in triplicate. Results are represented as the means and standard errors of the mean of the percent of control values (the number of HIV-1 positive cells in the presence of P-TEFb inhibitors/the number of HIV-1 positive cells in untreated wells).

Primary cell isolation, maintenance and infection with HIV

Human monocyte derived macrophages (MDMs) and peripheral blood lymphocytes (PBLs) cells were isolated from 350 ml of peripheral blood from healthy, HIV negative donors. Peripheral blood mononuclear cells (PBMCs) were isolated as previously described [53]. Briefly, PBMCs were separated by centrifugation in lymphocyte separation medium (ICN Biomedicals, Solon, Ohio). The separated PBMCs were placed on gelatin and fibronectin-coated flasks in order to separate monocytes from mononuclear cells. Adherent monocytes were lifted with EDTA, washed and plated at a density of 1 × 106 per well in 48-well trays for infectivity and cytotoxicity studies. Monocytes were differentiated for 5 days in DMEM with 10% FCS, 10% human serum and 1% penicillin/streptomycin in order to generate monocyte-derived macrophages prior to HIV infections and drug treatment. PBLs were treated with 5 μg/ml of phytohaemagglutinin (PHA) for 72 hours prior to HIV infection and drug treatment. PHA-treated PBLs were plated at a density of 1 × 106 per well in 48-well trays and maintained in RPMI 1640 with 10% FCS, 1% Penicillin/Streptomycin and 10 units/ml of recombinant IL-2. Viral infection was performed in MDMs and PBLs by adding 10,000 RT units of HIV-1p256 stock per 1 × 106 cells. During long term studies in primary cells, supernatants were collected at 4, 8, 12 and 16 days post-infection, frozen at -80°C until analyzed and media was refreshed. Inhibition of HIV replication by flavopiridol in PBLs was determined in 3 independent donors and each flavopirdol concentration was tested in triplicate. Inhibition of HIV replication by flavopiridol in MDMs was determined by pooling data from 3 independent donors. A minimum of 3 data points for each flavopiridol concentration was taken into account when generating the IC50 curve for MDMs.

Cell viability assays

The impact of the P-TEFb inhibitors on cell viability was measured by ATPlite (Perkin Elmer). These cytotoxicity studies were performed as recommended by manufacturer utilizing a substrate solution that emits light in a manner proportional to the ATP present in each sample. Cells were plated in a 48-well format. Cells were treated with serial dilutions of the P-TEFb inhibitors and maintained for the indicated period of time. Mammalian cell lysis buffer was added to lyse the cells, followed by addition of the substrate solution. The amount of light produced in each well was measured in a TopCountR Microplate Scintillation and Luminescence Counter (Packard Instruments). Cytotoxicity experiments in HeLa37 cells were repeated at least three times with triplicates of each drug concentration. The results are represented as the means and standard errors of the mean of the percent of control values (the ATPLite values in the presence of P-TEFb inhibitors/the ATPLite values of untreated wells). Cytotoxicity studies in PBLs were performed in three independent donors and each flavopiridol concentration was tested in triplicate. The LD50 of flavopiridol in MDMs was determined by pooling data from 2 independent donors.

P-TEFb kinase assays

Kinase reactions were carried out with recombinant, purified P-TEFb (Cdk9/cyclin T1) [6] and either DSIF subunit Spt5 or Pol II CTD as the substrate as previously described [55]. Kinase reactions contained 34 mM KCl, 20 mM HEPES pH 7.6, 7 mM MgCl2, 15 μM ATP, 1.3 μCi of [γ-32P]-ATP (Amersham) and 1 μg BSA. The reactions were incubated for 20 minutes at 30°C and stopped by addition of SDS-PAGE loading buffer. Reactions were resolved on a 7.5% SDS-PAGE gel. The dried gel was subjected to autoradiography. Quantitation was performed using an InstantImager (Packard) and data was normalized to the DMSO control. The data was fitted to a dose-response curve using TableCurve (Jandel Scientific) in order to determine the IC50.

Glycerol gradient fractionation of cell lysates

HeLa cells were grown in 100 ml of DMEM with 10% FCS to a density of 4 × 105 cells/ml in spinner flasks. The cells were treated for 1 hour with no P-TEFb inhibitor or serial dilutions of DRB ranging from 0.1 to 10 μM. Cell lysates were prepared in Buffer A (10 mM KCl, 10 mM MgCl2, 10 mM HEPES, 1 mM EDTA, 1 mM DTT, 0.1% PMSF and EDTA-free complete protease inhibitor cocktail (Roche)) containing 150 mM NaCl and 0.5% NP-40. The lysates were clarified by centrifugation at 20,000 g for 10 minutes at 4°C. The supernatant was layered on top of a 5–45% glycerol gradient containing 150 mM NaCl. Gradients were spun at 190,000 g for 16 hours using a SW-55Ti rotor. The fractions were analyzed for the presence of P-TEFb complexes by immunoblotting with anti-cyclin T1 and anti-Cdk9 antibodies (Santa Cruz). Following incubation with the appropriate HRP-conjugated secondary antibodies, the blots were developed using SuperSignal DuraWest (Pierce). The western blots were imaged using a cooled CCD camera (UVP) and the amount of P-TEFb in the large and free form was quantitated using LabWorks 4.0 software.

Separation of large and free forms of P-TEFb by differential salt extraction

HeLa37 and Jurkat cells were treated with serial dilutions of DRB, flavopiridol or seliciclib concentrations for 1 hour. The cytosolic extracts were prepared by resuspending the cells in 80 μl of Buffer A (10 mM KCl, 10 mM MgCl2, 10 mM HEPES, 1 mM EDTA, 1 mM DTT, 0.1% PMSF and EDTA-free complete protease inhibitor cocktail (Roche)) with 0.5% NP-40 for 10 minutes on ice. The nuclei were spun down at 5,000 g for 5 minutes and the supernatant was saved as the cytosolic extract (CE). The nuclei were washed once with 200 μl of Buffer A with 0.5% NP-40 and re-pelleted. The nuclei were resuspended in 80 μl of Buffer B (450 mM NaCl, 1.5 mM MgCl2, 20 mM HEPES, 0.5 mM EDTA, 1 mM DTT, 0.1% PMSF and EDTA-free complete protease inhibitor cocktail (Roche)) and incubated on ice for 10 minutes. The lysates were clarified by centrifugation at 20,000 g for 10 minutes. The supernatant was saved as the nuclear extract (NE). Western blotting was performed with one fifth of the samples and the fraction of Cdk9 and cyclin T1 in the cytosolic and nuclear extracts was determined by imaging the chemiluminescent signal using a cooled CCD camera (UVP). The signal was quantitated using LabWorks 4.0 software and the data fit to a logistic dose response curve using TableCurve (Jandel Scientific) to determine the IC50 for loss of the large, low salt extractable form of P-TEFb.

Reverse transcriptase assays

Reverse transcriptase (RT) assays were performed on supernatants from HIV-1p256 infected cells as previously described [53]. Briefly, cell-free supernatant from infected cells were added to a mix containing 50 mM Tris (pH 7.8), 75 mM KCl, 2 mM DTT, 5 mM MgCl2, 0.05% NP-40, 5 μg poly(A), 4 μg poly(d) (T12-18) and 10 μCi/ml 32P-TTP. The mixture was incubated at 37°C for 3.5 hours and then blotted onto DE81 paper. The DE81 paper was washed 4 times with 3× SSPE and the amount of radioactivity that was incorporated into negative strand DNA was quantified with an InstantImager (Packard Instruments).

Statistical analysis

All HIV infectivity and cytotoxicity data are represented as the percent of control values to allow comparisons of separate experiments. The mean of the values obtained in the infectivity studies were determined by averaging the individual experimental data points for each drug concentration. Error bars on graphs represent the calculated standard error for each drug dilution. Determination of IC50 and LD50 values was performed using TableCurve (Jandel Scientific).