Expression of a protein phosphatase 1 inhibitor, cdNIPP1, increases CDK9 threonine 186 phosphorylation and inhibits HIV-1 transcription

Abstract

CDK9/cyclin T1, a key enzyme in HIV-1 transcription, is negatively regulated by 7SK RNA and the HEXIM1 protein. Dephosphorylation of CDK9 on Thr(186) by protein phosphatase 1 (PP1) in stress-induced cells or by protein phosphatase M1A in normally growing cells activates CDK9. Our previous studies showed that HIV-1 Tat protein binds to PP1 through the Tat Q(35)VCF(38) sequence, which is similar to the PP1-binding RVXF motif and that this interaction facilitates HIV-1 transcription. In the present study, we analyzed the effect of expression of the central domain of nuclear inhibitor of PP1 (cdNIPP1) in an engineered cell line and also when cdNIPP1 was expressed as part of HIV-1 pNL4-3 in place of nef. Stable expression of cdNIPP1 increased CDK9 phosphorylation on Thr(186) and the association of CDK9 with 7SK RNA. The stable expression of cdNIPP1 disrupted the interaction of Tat and PP1 and inhibited HIV-1 transcription. Expression of cdNIPP1 as a part of the HIV-1 genome inhibited HIV-1 replication. Our study provides a proof-of-concept for the future development of PP1-targeting compounds as inhibitors of HIV-1 replication.

FIGURE 1.

Effect of cdNIPP1 expression on NIPP1 distribution, CDK9 phosphorylation, and association with 7SK RNA. A, analysis of 293T-cdNIPP1 cells is shown. Western blot analysis of 293T and 293T-cdNIPP1 cells with antibodies for EGFP (to check for the expression of cdNIPP1-EGFP), CDK9, PP1γ, and α-tubulin is shown. B, expression of cdNIPP1 changes NIPP1 distribution and increases CDK9 phosphorylation on Thr¹⁸⁶. Whole cell extracts were prepared from 293T and 293T-cdNIPP1 cells and separated on glycerol gradients by ultracentrifugation. The gradients were fractionated and analyzed by Western blotting for NIPP1, PP1, CDK9, and CDK9 phosphorylated on Thr¹⁸⁶. The NIPP1 and CDK9 phosphorylated on Thr¹⁸⁶ immunoblots were scanned and quantified using PhosphorImager OptiQuant software. The pixels were adjusted to have the same total amount for the 293T and the cdNIPP1 extracts, and the normalized results are graphed (lower panels). C, expression of cdNIPP1 increases the association of CDK9 with 7SK RNA. RNA was isolated from material that co-precipitated with CDK9 from 239T or 293T-cdNIPP1 whole cell lysates. The RNA was reverse transcribed and analyzed by real time PCR. Results are presented as numbers of copies of 7SK RNA. D, PP1 removes CDK9-associated 7SK RNA. RNA was isolated from material that co-immunoprecipitated with CDK9 from 239T or 293T-cdNIPP1 cells, reverse-transcribed, and analyzed by semiquantitative PCR (26 cycles) using 7SK-specific primers. Lane 1, control using 7SK expression vector. Lane 2, control that was not reverse-transcribed. Lanes 3 and 7 are inputs. Lanes 4 and 8, 7SK RNA associated with CDK9 in 293T or 293T-cdNIPP1 whole cell lysates. Lanes 5 and 9, immunoprecipitated CDK9 was treated with purified rabbit muscular PP1C prior to the RNA extraction. Lanes 6 and 10, controls in which the immunoprecipitations were carried out with nonspecific rabbit IgG.

FIGURE 2.

Expression of cdNIPP1 inhibits HIV-1 transcription. A, expression of cdNIPP1 disrupts the interaction of Tat and PP1. 293T and 293T-cdNIPP1 cells were transfected with FLAG-Tat expression vectors. Tat and associated proteins were immunoprecipitated with anti-FLAG antibodies, resolved on SDS-PAGE, and probed with antibodies against Tat, CDK9, and PP1γ. Lane 1, inputs. Lane 2, 293T cells without FLAG-Tat expressed. Lanes 3 and 4, 293T and 293T-cdNIPP cells, respectively, transfected with FLAG-Tat. B, expression of cdNIPP1 inhibits Tat-induced HIV-1 transcription. 293T and 293T-cdNIPP1 cells were transiently transfected with HIV-1 LTR-LacZ without (lane 1) or with co-transfection of Tat expressing plasmid (lane 2); or a TAR deleted construct of HIV-1 LTR-LacZ (HIV-1 LTRΔTAR) (lane 3). At 24 h after the transfection, the cells were lysed and analyzed for β-galactosidase activity. HIV-1 LTR-expression plasmid was analyzed by real time PCR and used for normalization. C, co-expression of cdNIPP1 and PP1γ or PP1γ-D64N-EGFP (DN PP1γ-EGFP) affects basal and Tat-induced HIV-1 transcription. 293T and 293T-cdNIPP1 cells were transfected with HIV-1 LTR-LacZ (lane 1) or in combination with Tat expression vector (lanes 2–8) and the indicated amounts of PP1γ (lanes 3–5) or DN PP1γ (lanes 6–8) expression vectors and co-transfected with CMV-EGFP expression vector. At 24 h the cells were lysed and analyzed for β-galactosidase and EGFP fluorescence. D, expression of PP1γ rescues cdNIPP1-mediated inhibition of Tat-induced HIV-1 transcription. 293T cells were transfected with HIV-1 LTR-LacZ and Tat expression vectors and with WT cdNIPP1 (lanes 1 and 3) or mutant cdNIPP1 (lanes 2 and 4) expression vectors and with PP11γ expression vector (lanes 3 and 4). At 48 h after transfection, the cells were lysed and analyzed for β-galactosidase activity. E, expression of mutant NIPP1 does not affect HIV-1 transcription. 84-31 cells and 84-31 cells stably expressing mutant NIPP1 were transfected with HIV-1 LTR-LacZ without (lane 1) or with co-transfection of Tat-expressing plasmid (lane 2); or HIV-1 LTRΔTAR (lane 3). At 24 h the cells were lysed and analyzed for β-galactosidase activity.

FIGURE 3.

Expression of cdNIPP1 inhibits HIV-1 replication. A, cloning strategy. Diagram of the HIV-1 genome indicates the insertion of cdNIPP1-EGFP into nef. B, expression of cdNIPP1. EGFP-fused WT cdNIPP1 and mutant RATA cdNIPP1 were expressed in 293T cells transfected with recombinant pNL4-3 constructs or Ad8-GFP expression vector as control. C, replication of recombinant pNL4-3 cdNIPP1. Replication kinetics of WT pNL4-3 and control pNL4-3 398 and recombinant pNL4-3 expressing cdNIPP1 and mutant RATA cdNIPP1 in MT4 cells determined by RT measurement in cell media are shown. No new cells were added into the infected cultures.