293T cells were purchased from ATCC (Manassas, VA). Histone H1 was purchased from Upstate Cell Signaling Solutions (Charlottesville, VA). Anti-FLAG monoclonal antibodies, protein G and protein A agarose were purchased from Sigma (Atlanta, GA). Recombinant CDK2/cyclin E and CDK9/cyclin T1 were purchased from ProQinase (Freiburg, Germany). Antibodies against CDK9, CDK2 and cyclin T1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-CDK9 phospho-Thr186 polyclonal antibodies were a kind gift from Dr. Qiang Zhou (University of California, Berkeley). Preparation of CDK9 Ser 90 phospho-specific antibodies is described below. Horseradish peroxidase (HRP)-conjugated F(ab)2 fragment was purchased from GE Healthcare (Piscataway, NJ). All other inorganic reagents were purchased from Fisher Scientific (Fair Lawn, NJ) or Sigma (St. Louis, MO). Radioactive materials were purchased from Perkin-Elmer (Waltham MA). ARC (4-Amino-6-hydrazino-7-beta-D-Ribofuranosyl-7H-Pyrrolo (2,3-d)-pyrimidine-5-Carboxamide)  was a gift from Dr. Andrei L. Gartel (Departments of Medicine, Microbiology and Immunology, University of Illinois at Chicago).
The HIV-1 genomic vector, pNL4-3.Luc.R-E- (Courtesy of Prof. Nathaniel Landau, NYU School of Medicine, New York, NY) was obtained from the NIH AIDS Research and Reference Reagent Program. The Tat expression plasmid was a gift from Dr. Ben Berkhout (University of Amsterdam). WT HIV-1 LTR (−105 to +77) followed by the luciferase reporter gene (KB SP WT) was kindly provided by Dr. Manuel López-Cabrera (Unidad de Biología Molecular, Madrid, Spain).
293T cells were seeded in 6 well plates to achieve 50% confluence at the day of transfection. The cells were transfected with indicated plasmids using Lipofectamine and Plus reagents (Life Technologies) following manufacturer’s protocol. The efficiency of transfection was verified using a plasmid encoding green fluorescent protein. The cells were cultured for 48 hrs post-transfection and then analyzed for HIV-1 transcription or phosphorylation of CDK9.
CDK2-directed siRNA (siGENOME SMARTpool reagent M-003236-03-0005) and a control siRNA (D-001206-13-05) were purchased from Dharmacon (Dallas, TX). Control siRNA targets firefly luciferase gene. The siRNAs were transfected at final concentration of 100 nM using Lipofectamin reagent (Invitrogen) according to the manufacturer’s recommendations. The siRNAs were incubated with cells for 2 days before cells were lysed for Western blotting analysis or retransfected.
Stable CDK2-knock-down cell line
293T cells were transfected with HSH000225-1-LvH1 vectors expressing shRNA that targeted 399gcttaaggagctttaaccat418 (OS211957), 919ccaggagttacttctatgc937 (OS211958), 1010atggacggagcttgttatc1028 (OS211959) and 49aggcggcaacattgtttca67 (OS211960) sequences of CDK2 (GeneCopoeia, Rockville, MD). Stable clones were selected with 10 μg/ml puromycin. Several clones transfected with OS211959 vector showed significant decrease in CDK2 expression and one of these clones, designated as 293T-CDK KD cells, was used for further studies.
293T and 293T-CDK KD cells were co-transfected with pNL4-3 Luc plasmids and CMV-LacZ expression vector. After 48 hours the cells were collected, washed in PBS, and lysed with Steady Lite Luciferase. The samples luminescence was determined in a Luminoskan Perkin-Elmer). The lysates were then used to measure β-galactosidase activity with ONPG-based assay . Luciferase activity was normalized on the basis of the obtained β-galactosidase activity.
HIV-1 Tat activated transcription
293T cells were co-transfected with CDK9 WT and mutants and KB SP WT. Cells were also co-transfected with Tat-expressing vector. After 48 hrs the cells were collected, washed in PBS, lysed with Steady Lite Luciferase lysis buffer, and luminescence was determined in a Luminoskan. Luciferase activity was normalized to GFP expression.
VSVG-HIV-1 was added to 293T and 293T-CDK2 KD cells that were seeded in 24-well plate at ~30 confluence. After 48 hours, the cells were washed in PBS, and lysed with Steady Lite Luciferase buffer. Light emission was analyzed in a Luminoskan. To adjust for the cell number, the MTT assay was performed. Control samples were supplemented with 0.5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and incubated for 1 hr at 37°C. Media was removed, formazan crystals were solubilized in dimethyl sulfoxide and absorbance was measured at 654 nm. Luciferase activity was normalized for MTT reading at 654 nm.
Analysis of CDK2 mRNA expression
Total RNA was extracted from cultured 293T and 293T-CDK KD cells using TRIzol reagent according to the manufacturer’s protocol (Invitrogen Corp.). Total RNA (100 ng) was reverse transcribed to cDNA using Superscript™ RT-PCR kit (Invitrogen, Carlsbad, CA), hexamers and oligo-dT were used as primers. Real-time PCR analysis was conducted on Roche LightCycler 480 detection system (Roche Diagnostics) with SYBR Green. The cDNA was amplified in 45 cycles of denaturation at 95°C for 10 seconds, annealing at 60°C for 10 seconds, and extension at 72°C for 10 seconds and primers for β-Actin, CDK2, Cyclin A and Cyclin E. Primer sequences for β-Actin, forward-AGGCTCAGAGCAAGAGAG, reverse-TACATGGCTGGTGTGTTGA, amplicon size 229; CDK2 forward- TTTGCTGAGATGGTGACTCG, reverse- CTTCATCCAGGGGAGGTACA, amplicon size 196 bp; Cyclin A, forward-GAAACTGCAGCTCGTAGGAA, reverse-ACTTTCAGAAGCAAGTGTTCCA, amplicon size 150bp; Cyclin E, forward –AGCACTTTCTTGAGCAACACC, reverse-CGCCATATACCGGTCAAAGA, amplicon size 161bp. Mean Cp values for β-Actin, CDK2, Cyclin A and cyclin E were determined and expression levels determined using ΔΔCp analysis using β-Actin as reference. Unpaired t-test was used to test statistical significance.
Cell cycle analysis of 293T-CDK KD cells
Approximately 1 million cells were fixed in 70% ethanol at -20°C for 2 hours and stained with Propidium Iodide (10mg/ml) containing RNAse (1mg/ml) for 30 minutes. The data were acquired in BD FACSCalibur (BD Biosciences, Jose, California) and analysis was done using FlowJo software. Unpaired t-test was used to test statistical significance.
Separation of large and small P-TEFb complexes by differential salt extraction
293T cells, 293T –CDK2 KD cells that stably express CDK2 shRNA (OS211959) or 293T cells transiently transfected with a combination of Flag-tagged CDK9 (WT or S90A mutant) and cyclin T1 were cultured in DMEM containing 10% fetal bovine serum. Cells were resuspended in Buffer A (10 mM HEPES (pH 7.9), 10 mM KCl, 10 mM MgCl2, 1 mM EDTA, 250 μM sucrose, 1 mM DTT, 0.5% NP-40 and protease inhibitors) added at 500 μl/107 cells. The mixture was incubated on ice for 10 min and centrifuged at 1000 x g for 5 min to pellet the nucleus. The supernatant was removed and saved as the large complex extract (LC). The remaining pellet was resuspended in Buffer B (20 mM HEPES-KOH (pH 7.9), 450 mM NaCl, 1.5 mM MgCl2, 0.5mM EDTA, 1 mM DTT and protease inhibitors) added at 500 μl/107 cells. The mixture was incubated on ice for 10 min and centrifuged at 10,000 x g for 1 hr. The supernatant was saved as the small complex extract (SC) (11). The LC and SC were resolved by SDS-PAGE and transferred to a PVDF membrane (Millipore, Allen, TX) for Western blotting. The membrane was probed with anti-CDK9 antibodies.
293T cells were lysed in whole cell lysis buffer (50 mM Tris–HCl, pH 7.5, 0.5 M NaCl, 1% NP-40, 0.1% SDS) supplemented with protease cocktail. CDK9 was precipitated as indicated with either anti-CDK9 antibodies or anti-Flag antibodies in the case of overexpression of Flag-CDK9 as we previously described . Briefly, 400 μg of lysate and 800 ng of antibodies combined with 50 μl of 50% slurry of protein A/G agarose were incubated for 2 hrs at 4°C in a TNN Buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl and 1% NP-40). The agarose beads were precipitated and washed with TNN buffer, resolved on 10% Tris-Glycine SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membranes and immunoblotted with appropriate antibodies.
RT-PCR to detect 7SK RNA
RNA was isolated from complexes that co-immunoprecipitated with anti-CDK9 antibodies or nonspecific IgG as controls using TRIzol reagent according the Invitrogen’s protocol. RNA was also extracted from total cell extract or small and large complex extracts prepared as described above. Total RNA was reverse transcribed using SuperScript II kit (Invitrogen) with the 7SK reverse primer. The following primer sequences were used: forward primer 3′-GGATGTGAGGCGATCTGGCTG-5′; reverse primer 3′-TAAAGAAAGGCAGACTGCCAC-5′. Theses primers were used in a PCR reaction that followed the RT reaction. Real-time PCR analysis was conducted on Roche LightCycler 480 detection system (Roche Diagnostics) with SYBR Green. The cDNA was amplified in 45 cycles of denaturation at 95°C for 10 seconds, annealing at 60°C for 10 seconds, and extension at 72°C for 10 seconds. To quantify the amount of 7SK RNA, serial dilutions of 7SK RNA expressing vector  was used to determine the copy number. For ΔΔ Cp analysis of 7SK RNA in small and large complex extracts, RNA was reverse transcribed with hexamers and U6 RNA was used as reference. In semi-quantitative PCR, 7SK RNA was amplified for 30 cycles, resolved on 2% agarose gel and photographed.
CDK9 kinase assay
Kinase assay was performed at 30°C for 30 min in a kinase assay buffer (50 mM HEPES-KOH, pH 7.9, 10 mM MgCl2, 6 mM EGTA, 2.5 mM DTT) containing 100 ng of GST-CTD as substrate, 200 μM cold ATP and 5 μCi of (γ-32P) ATP. Kinase reactions were stopped with SDS-loading buffer and resolved on 10% PAGE. The dried gel was exposed to Phosphor Imager screen.
CDK2 phosphorylation assay
293T and 293T-59 cells were lysed in whole cell lysis buffer (50 mM Tris–HCl, pH 7.5, 0.5 M NaCl, 1% NP-40, 0.1% SDS) supplemented with protease cocktail. CDK2 was precipitated with anti-CDK2 antibodies as described above. Kinase assay was performed at 30°C for 20 min in the kinase assay buffer containing 2 μg Histone H1 as substrate, 200 μM cold ATP and 5 μCi of (γ-32P) ATP. Kinase reactions were stopped with SDS-loading buffer and resolved on 10% PAGE. The dried gel was exposed to Phosphor Imager screen.
Phosphorylation of CDK9-derived peptides
We designed synthetic peptides containing CDK9 residues Thr29 (21KLAKIGQGTFGEVFK35), Ser90 (88KASPYNRCKGSIYL101); and Ser175 and Thr186 (172RAFSLAKNSQPNRYTNRVV190). The peptides were phosphorylated by recombinant CDK2/cyclin E in a 10 μl reaction with 100 μM ATP (1μCi of γ-(32P) ATP) in the kinase assay buffer using 4 μg of a peptide per reaction for 20 min at 30°C. The reaction was stopped by the addition of 4X SDS-PAGE loading buffer and resolved on a 15% Tris-Tricine gel. The gel was stained with Coomassie blue for 10 min, destained for 1 hr, dried, and exposed to Phosphor Imager screen.
Constructing CDK9 S90 mutants
QuikChange XL Site-Directed Mutagenesis Kit (Stratagene) was used to generate mutants of CDK9 with the substitutions in the sites of CDK2 phosphorylation determined previously. Primers for S90A substitution were GATTTGTCGAACCAAAGCTGCCCCCTATAACCGCTGC (forward) and GCAGCGGTTATAGGGGGCAGCTTTGGTTCGACAAATC (reverse); and for S90D substitution - GATTTGTCGAACCAAAGCTGACCCCTATAACCGCTGC (forward) and GCAGCGGTTATAGGGGTCAGCTTTGGTTCGACAAATC (reverse). CDK9 S90D was created to mimic phosphorylation. PCR reactions were run for 18 cycles with the extension time of 8 min to allow the synthesis of the whole plasmid sequence. PCR products were digested with Dpn I to degrade the original template. The PCR products were transformed into XL-Gold cells. Colonies were then picked, and mini preparations were isolated using High Pure Plasmid Isolation kit (Roche Applied Sciences). The obtained clones were sequenced. CDK9 mutants were expressed in 293T cells, immunoprecipitated with anti-FLAG or anti-Cyclin T1 antibodies and tested for phosphorylation of GST-CTD as described above.
CDK9 Ser 90 phospho-specific antibodies
CDK9-derived 86 RTKASPYNR94 peptide without or with Ser 90 phosphorylated was synthesized with an additional Cys residue at the N-terminus for coupling to Keyhole limpet hemocyanin (KLH) or BSA. The KLH-coupled phospho-peptide was injected in the rabbit. The polyclonal serum was double affinity- purified on the BSA-coupled phospho- and non-phosphopeptide linked to CNBr-activated Sepharose-4B (GE Healthcare).
The 8 amino acid residues of CDK9, Ala89-Lys96, that were missing in the CDK9 crystal structure , were modeled with ICM-Pro software package [55, 56], version 3.6-1i (Molsoft LLC, 2010). First, missing residues were built with the ICM homology modeling tool, using CDK9_HUMAN sequence (Swiss-Prot accession number P50750) and coordinates of PDB entry 3MIA  as template. Next, conformation sampling of residues Thr87-Ser98 was performed by ICM ShakeLoop tool using ICM loop database and a set of low-energy backbone conformations was obtained. Finally, local energy minimization of side chains by biased probability Monte Carlo procedure provided conformation with the lowest energy.