Proposed model of CDK9/cyclin T1 activation in viral transcription.

<p>CDK9's Thr186 dephosphorylation by PP1/PIP1 complex leads to the dissociation of 7SK RNA and HEXIM1 protein and the release of inactive CDK9. CDK9 is phosphorylated by a cellular kinase, which may include active CDK9/cyclin T1 on Ser175 and Thr 186 that creates inactive CDK9/cyclin T1. This inactive phosphorylated CDK9/cyclin T1 is activated by PP1/PIP2 complex. The active CDK9/cyclin T1 can be recruited by Tat or re-associated with 7SK snRNP.</p

Inhibition of PP1 prevents RNAPII CTD phosphorylation and inhibits CDK9 activity.

<p>(<b>A</b>) <b>High concentration of okadaic acid inhibits RNAPII phosphorylation </b><b><i>in vitro</i></b><b>.</b> HeLa cell nuclear extract was subjected to <i>in vitro</i> transcription without (lane 2) or with the addition of 10 nM okadaic acid (lane 3) or 1 µM okadaic acid (lane 4). Lane 1, untreated HeLa cell nuclear extract. RNAPII was resolved on 5% SDS-PAGE and analyzed with RNAPII CTD serine 2 phospho-epitope specific antibodies (Ser2). (<b>B</b>) <b>NIPP1 prevents RNAPII phosphorylation </b><b><i>in vitro</i></b>. HeLa cell nuclear extract was subjected to <i>in vitro</i> transcription without (lane 2) or with the addition of 5 µM NIPP1 (lane 3). Lane 1, untreated HeLa cell nuclear extract. RNAPII was resolved on 5% SDS-PAGE and analyzed with Ser2 phospho-epitope specific antibodies. (<b>C</b>) <b>Expression of cdNIPP1 prevents RNAPII phosphorylation in cultured cells.</b> 293T cells were transfected with vectors expressing wt cdNIPP1 (lane 2) or mutant cdNIPP1 (lane 3) or mock transfected (lane 1). At 48 hours post transfection, the cells were treated with 0.1 µM okadaic acid and pulsed with (³²P) orthophosphate for 3 hours. The cellular lysates were subjected to immunoprecipitation with 8WG16 antibodies against RNAPII CTD (lanes 1 to 3) or with non-specific mouse IgG2a (lane 4). Immunoprecipitated RNAPII was resolved on 5% SDS-PAGE and the gel was analyzed on Phosphor Imager. Separately, RNAPII was immunoprecipitated and analyzed by Western blotting (lower panel). (<b>D & E</b>) <b>Expression of cdNIPP1 inhibits enzymatic activity of CDK9.</b> Lysates of 293T cells (lane 1) or 293T cells continuously expressing cdNIPP1 (293T-cdNIPP1 cells) (lane 2) were immunoprecipitated with anti-CDK9 antibodies. Precipitated CDK9 was supplemented with γ-(³²P) ATP and purified yeast RNAPII (panel D) or GST-CTD (panel E) as substrates. GST-CTD and RNAPII were resolved on 10% and 7.5% SDS-PAGE gels and the gels were analyzed on Phosphor Imager. Immunoprecipitation of CDK9 was verified by immunoblotting (lower panel D). Also there was phosphorylation in the absence of substrate ((lower panel E) or when non-specific antibodies were used (panel E, lanes 3 and 4). Results are from a typical experiment of 2–4 performed.</p

CDK9 S175A activates HIV-1 transcription.

<p>(<b>A</b>) <b>Analysis of CDK9 mutants for the activation of HIV-1 transcription.</b> 293T cells were transfected with HIV-1 LTR-LacZ and Tat expression vectors along with the indicated WT or mutated Flag- CDK9. At 48 hours posttransfection β-galactosidase activity was analyzed using ONPG substrate. Results are averages of quadruplicates from a typical experiment of 3 performed. (<b>B</b>) <b>PP1 induction of HIV-1 transcription is reduced in the presence of CDK9 S175A mutant.</b> 293T cells were EGFP (control) or PP1-EGFP expression vector in combination with indicated WT or mutated Flag-tagged CDK9 and also with HIV-1 LTR-LacZ and Tat expression vectors. At 48 hours posttransfection β-galactosidase activity was analyzed using ONPG substrate. Results are from a typical experiment of 3 performed. (<b>C</b>) <b>Expression of CDK9, PP1 and Tat</b>. Protein expression from panels A and B was verified by immunoblotting.</p

CDK9 is phosphorylated on Ser175 residue in cultured cells.

<p>(<b>A</b>) <b>MS/MS analysis of recombinant CDK9.</b> Recombinant CDK9/cyclin T1 was resolved on 10% SDS-PAGE. CDK9 was identified by Coomassie staining, in-gel digested with trypsin, and the eluted peptides were subjected to MS analysis on Thermo LTQ Orbitrap XL mass spectrometer as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018985#s4" target="_blank">Materials and Methods</a>. The SEQUEST search results are shown. Green, peptides identified with high probability by MS/MS analysis. Red, peptides identified with less probability. Black, peptides that were not detected. (<b>B</b>) <b>purification of (³²P)-labeled CDK9 for the peptide fingerprint analysis.</b> FLAG-tagged CDK9 was expressing in 293T cells and metabolically labeled in the presence of okadaic acid. CDK9 was immunoprecipitated, resolved on 10% SDS-PAGE and stained with colloidal Coomassie (upper panel), or exposed to Phosphor imager screen lower panel. Lane 1, mock-transfected cells. Lane 2, WT CDK9. Lane 3, CDK9 S175A mutant. (<b>C</b>) <b>Tryptic phosphopeptide mapping.</b> (³²P)-labeled CDK9 was trypsinized and resolved on Hunter thin layer peptide mapping electrophoresis system as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018985#s4" target="_blank">Materials and Methods</a>. WT CDK9 (upper panel) and CDK9 S175A (lower panel) are shown. Spots labeled as 1–3 were scraped and further analyzed by MS analysis. The results are representative from 2 experiments. (<b>D</b>) <b>Base peak chromatography of Spot 1.</b> Raw base peak chromatography data showing ion with mass 318.69 that matches to AFSLAK (M+2H)²⁺ peptide. Results are representative from 4 experiments. <b>E</b>. <b>MS/MS spectrum of derived from Spot 3.</b> The spectrum gives positive identification of GSQITQQSTNQSR peptide. Results are from a typical experiment of 3 performed.</p

PP1 does not dephosphorylate CDK9 S175A mutant.

<p>293T cells were transfected with vectors expressing Flag-tagged CDK9 WT (lanes 1, 2 and 5) or CDK9 S175A mutant (lanes 3 and 4) and treated at 48 hours posttransfection with 100 nM okadaic acid and (³²P) orthophosphate. CDK9 was immunoprecipitated from cellular lysates with anti-Flag antibodies and subjected to dephosphorylation by PP1 as indicated. Lane 5, immunoprecipitation with non-specific mouse IgG. The reactions were resolved on 10% SDS-PAGE and analyzed on Phosphor Imager and by immunoblotting with anti-CDK9 antibodies. On a lower panel quantitation of (³²P) phosphorylation of CDK9 is shown. Results are from a typical experiment of 2 performed.</p

PP1 dephosphorylates T-loop derived CDK9 peptide phosphorylated on Ser 175.

<p>(<b>A</b>) <b>Phosphorylation of CDK9's T-loop derived peptides.</b> CDK7 or CDK9 T-loop derived peptides were phosphorylated by recombinant CDK2/cyclin E or CDK9/cyclin T1, resolved on 15% SDS Tric-Tricine gel and analyzed by Phosphor Imager. Lanes 1 and 2, phosphorylation of CDK7 derived T-loop peptides by CDK2/cyclin E. Lanes 4–7, phosphoryaltion of WT and mutant CDK9's T-loop-derived peptides by recombinant CDK9/cyclin T1. (<b>B</b>) <b>Dephosphorylation of CDK9's T-loop derived peptides by PP1.</b> CDK9-derived T loop WT peptides (lanes 1–3) or T186A mutant peptides (lanes 4–6) were phosphorylated by recombinant CDK9/cyclin T1, then CDK9 activity was blocked by 10 µM ARC and the peptides were incubated with the indicated amount of PP1. The peptides were resolved on 15% Tris-Tricine gel and analyzed by Phosphor Imager (upper panel) and also showed stained with Coomassie Blue (lower panel). Results are from a typical experiment of 3 performed.</p

Dephosphorylation of <i>in vivo</i> labeled CDK9 by PP1.

<p>(<b>A</b>) <b>PP1 dephosphorylates CDK9 (³²P)-phosphorylated in cultured cells.</b> 293T cells transfected with Flag-CDK9 vector were pulsed with (³²P) orthophosphate in the presence of 100 nM okadaic acid. Flag-CDK9 was immunoprecipitated with anti-Flag antibodies (lane 2) and subjected to dephosphorylation with PP1 (lanes 3 and 4), PP2A (lanes 5 and 6) or cdc25A (lanes 7 and 8). Lane 1, immunoprecipitation with non-specific mouse IgG. The reactions were resolved on 10% SDS-PAGE and analyzed on Phosphor Imager. (<b>B</b>) <b>Comparison of phosphatase activities of PP1 and PP2A.</b> Recombinant PP1 (0.1 U) and purified from human red blood cells PP2A (0.1 U) were assayed with the generic KR-pT-IRR substrate or phospho-Rb peptide and the reactions were stopped at indicated time points by the addition of malachite green solution. The amount of malachite green was quantified by the absorbance and recalculated into the phosphate concentration using phosphate standard curve. (<b>C</b>) <b>Dephosphorylation of CDK9's Thr186 by PP1.</b> 293T cells were transfected with vectors expressing Flag-CDK9 WT (lanes 1–4) or Flag-CDK9 T186A (lane 5) and treated at 48 hours posttransfection with 100 nM okadaic acid. CDK9 was immunoprecipitated from cellular lysates with anti-Flag antibodies, subjected to dephosphorylation with PP1 (lane 2) or PP2A (lane 4) and analyzed by immunoblotting with phospho-specific CDK9 Thr186 or anti-CDK9 antibodies. (<b>D</b>) <b>Comparison of dephosphorylation of (³²P) CDK9 and Thr186-phosphorylated.</b> Combined results from three independent experiments shown as percent of CDK9 dephosphorylation. *P<0.05. Results are from a typical experiment of 4 performed.</p

Small Molecules Targeted to a Non-Catalytic ‘‘RVxF’’ Binding Site of Protein Phosphatase-1 Inhibit HIV-1

HIV-1 Tat protein recruits host cell factors including CDK9/cyclin T1 to HIV-1 TAR RNA and thereby induces HIV-1 transcription. An interaction with host Ser/Thr protein phosphatase-1 (PP1) is critical for this function of Tat. PP1 binds to a Tat sequence, Q35VCF38, which resembles the PP1-binding ‘‘RVxF’’ motif present on PP1-binding regulatory subunits. We showed that expression of PP1 binding peptide, a central domain of Nuclear Inhibitor of PP1, disrupted the interaction of HIV-1 Tat with PP1 and inhibited HIV-1 transcription and replication. Here, we report small molecule compounds that target the ‘‘RVxF’’-binding cavity of PP1 to disrupt the interaction of PP1 with Tat and inhibit HIV-1 replication. Using the crystal structure of PP1, we virtually screened 300,000 compounds and identified 262 small molecules that were predicted to bind the ‘‘RVxF’’-accommodating cavity of PP1. These compounds were then assayed for inhibition of HIV-1 transcription in CEM T cells. One of the compounds, 1H4, inhibited HIV-1 transcription and replication at non-cytotoxic concentrations. 1H4 prevented PP1-mediated dephosphorylation of a substrate peptide containing an RVxF sequence in vitro. 1H4 also disrupted the association of PP1 with Tat in cultured cells without having an effect on the interaction of PP1 with the cellular regulators, NIPP1 and PNUTS, or on the cellular proteome. Finally, 1H4 prevented the translocation of PP1 to the nucleus. Taken together, our study shows that HIV- inhibition can be achieved through using small molecules to target a non-catalytic site of PP1. This proof-of-principle study can serve as a starting point for the development of novel antiviral drugs that target the interface of HIV-1 viral proteins with their host partners

1H4 has no effect on PP1 enzymatic activity.

<p>A. 1H4 has no effect on the kinetics of KT(pT)IRR peptide dephosphorylation by PP1α. Recombinant PP1α (0.005 Units) was assayed with KT(pT)IRR peptide (3 µM) in the absence or presence of 1H4, and the reaction was stopped at indicated time points by the addition of malachite green solution. The amount of released phosphate was quantified by the absorbance and phosphate concentration was recalculated using standards. Initial velocity was calculated by linear regression in Prism. B and C. 1H4 has no effect on Km and V_MAX of KT(pT)IRR peptide dephosphorylation by PP1α. Initial rates of KT(pT)IRR peptide dephosphorylation by PP1α were assayed at the indicated concentrations of the substrate in the absence or presence of 300 µM 1H4. The amount of released phosphate was quantified with malachite green. The V_MAX and Km were calculated by non-linear regression analysis for Michaelis-Menten equation in Prism. The data were transformed to Lineweaver-Burk representation shown in panel C.</p

1H4 compound disrupts the Tat-mediated translocation of PP1 into the nucleus.

<p>HeLa cells were transfected with PP1α-EGFP (PP1α) (A), PP1α-EGFP and WT Flag-Tat (B, D and E) or PP1α-EGFP and Flag-Tat ³⁵QACA³⁸ mutant (C) and treated with 10 µM 1H4 (D) or control 1G3 compound (E) for 18 hours. The cells were photographed on Olympus IX51 using a blue filter for EGFP fluorescence or phase contrast with 600X magnification. F, 293T cells were transfected with PP1α-EGFP or PP1α-EGFP and WT Tat or Tat QACA mutant expression vectors. At 24 hrs posttransfection cells were lysed in low salt buffer and cytoplasmic extract was separated from the nuclear material by centrifugation. Fluorescence was measured in the nuclear and cytoplasmic fractions using Perkin-Elmer Luminoscan.</p