Optimized chimeras between kinase-inactive mutant Cdk9 and truncated cyclin T1 proteins efficiently inhibit Tat transactivation and human immunodeficiency virus gene expression

Abstract

The human cyclin T1 (hCycT1) protein from the positive transcription elongation factor b (P-TEFb) binds the transactivator Tat and the transactivation response (TAR) RNA stem loop from human immunodeficiency virus type 1 (HIV). This complex activates the elongation of viral transcription. To create effective inhibitors of Tat and thus HIV replication, we constructed mutant hCycT1 proteins that are defective in binding its kinase partner, Cdk9, or TAR. Although these mutant hCycT1 proteins did not increase Tat transactivation in murine cells, their dominant-negative effects were small in human cells. Higher inhibitory effects were obtained when hCycT1 was fused with the mutant Cdk9 protein. Since the autophosphorylation of the C terminus of Cdk9 is required for the formation of the stable complex between P-TEFb, Tat, and TAR, these serines and threonines were changed to glutamate in a kinase-inactive Cdk9 protein. This chimera inhibited Tat transactivation and HIV gene expression in human cells. Therefore, this dominant-negative kinase-inactive mutant Cdk9.hCycT1 chimera could be used for antiviral gene therapy.

FIG. 1.

Mutant hCycT1 proteins defective in binding to Cdk9 weakly inhibit Tat transactivation. (A) Schematic presentation of the mutant proteins used in this study. The mutation sites (K93D and E137K) are indicated. TRM, Tat-TAR recognition motif. (B) The mutant hCycT1 proteins do not bind to Cdk9 in vivo. Myc epitope-tagged wild-type hCycT280 protein and mutant hCycT1 proteins were coexpressed with HA epitope-tagged Cdk9 protein in COS cells. Forty-eight hours after transfection, Myc epitope-tagged hCycT1 proteins were immunoprecipitated with the anti-Myc antibody from cell lysates. Bound Cdk9 was visualized by Western blotting with the anti-HA antibody (upper panel). The expression of hCycT1 proteins in the cell lysates was detected by Western blotting with the anti-Myc antibody (lower panel). (C) Mutant hCycT1 proteins do not support Tat transactivation in murine cells. Increasing amounts (0.3 to 0.6 μg) of hCycT280 and its mutant counterparts were coexpressed with Tat (0.1 μg) and pHIVCAT (0.1 μg) in NIH 3T3 cells. Forty-eight hours after the transfection, CAT activities in the cell lysates were measured. The results are presented as fold activation over the CAT activity obtained with pHIVCAT and 0.6 μg of the empty plasmid vector (lane 1). (D) The dominant-negative activity of mutant hCycT280(K93D, E137K) protein in human cells. Increasing amounts (0.2 to 0.8 μg) of hCycT280(K93D, E137K) were coexpressed with Tat (5 ng) in HeLa cells. Forty-eight hours after transfection, CAT activities in the cell lysates were measured. Results are shown as fold activation over the value obtained without Tat. All experiments were performed in triplicate and repeated at least two independent times, and the standard errors of the mean were less than 20%.

FIG. 2.

Mutant hCycT1 proteins defective for TAR binding are more potent inhibitors of Tat. (A) Schematic presentation of the mutant proteins used in this study. The mutation sites (R251A and R254A) are indicated. TRM, Tat-TAR recognition motif. (B) Mutant hCycT1 proteins do not support Tat transactivation in murine cells. Increasing amounts (0.3 to 0.6 μg) of hCycT280 and its mutant counterparts were coexpressed with Tat (0.1 μg) and pHIVCAT (0.1 μg) in NIH 3T3 cells. Forty-eight hours after transfection, CAT activities in the cell lysates were measured as for Fig. 1. (C) Mutant hCycT1 proteins can compete with the wild-type hCycT1 protein for Tat transactivation in murine cells. Increasing amounts (0.3 μg to 0.6 μg) of hCycT280 and its mutant counterparts were coexpressed with hCycT1 (0.1 μg), Tat (0.1 μg), and pHIVSCAT (0.1 μg) in NIH 3T3 cells. Forty-eight hours after transfection, CAT activities in the cell lysates were measured. (D) The dominant-negative activity of the mutant hCycT280(R251A,R254A) protein in human cells. Increasing amounts (0.2 μg to 0.8 μg) of hCycT(R251A,R254A) were coexpressed with Tat (5 ng) and pHIVCAT in HeLa cells. Forty-eight hours after transfection, CAT activities in the cell lysates were measured. Results are shown as fold activation over the value obtained without pTat. Variability between experiments was the same as in Fig. 1.

FIG. 3.

C terminus of Cdk9 is required for Tat transactivation. (A) Cdk9.hCycT280.Tat chimera and its C-terminal truncation mutant counterpart (0.5 μg) were coexpressed with pHIVCAT in NIH 3T3 cells. Forty-eight hours after transfection, CAT activities in the cell lysates were measured. (B) Expression of the fusion proteins in the cell was measured by Western blotting (WB) with the anti-Myc antibody. Variability between experiments was as in Fig. 1.

FIG. 4.

Kinase-inactive mutant Cdk9(D167N,361-5E).hCycT280 chimera inhibits Tat transactivation. Increasing amounts (0.2 μg to 0.8 μg) of Cdk9.hCycT280.Tat chimera and its mutant counterparts (illustrated on the left of the graph) were coexpressed with Tat (5 ng) and pHIVCAT in HeLa cells. Forty-eight hours after transfection, CAT activities in the cell lysates were measured. Results are shown as fold activation over the value obtained without Tat. Variability between experiments was as in Fig. 1.

FIG. 5.

Kinase-inactive mutant Cdk9(D167N,361-5E).hCycT280 chimera blocks HIV-1 gene expression. Increasing amounts (0.3 μg to 0.8 μg) of the kinase-negative mutant Cdk9(D167N,361-5E).hCycT280 and Cdk9(D167N,361-5E).hCycT280.Tat chimeras were coexpressed with 0.4 μg of HIV-1 provirus (pNL4-3) in 293T cells. Forty-eight hours after transfection, cell culture supernatants were collected, and the viral reverse transcriptase (RT) activity was measured as described in Materials and Methods.

FIG. 6.

Working hypothesis for the fusion proteins. (A) Wild-type Cdk9 protein (red rectangle) is phosphorylated in its C terminus (indicated by P) and capable of stabilizing the interaction between hCycT1 (green rectangle), Tat (purple rectangle), and TAR (red line), which is required for the hyperphosphorylation of the C-terminal domain of RNA polymerase II (RNAPIIo, red oval) and the elongation of transcription in vivo. (B) Mutant Cdk9(328).hCycT280 chimera that interacts with Tat but not with TAR can squelch Tat transactivation. (C) The kinase-inactive mutant Cdk9(D167N,361-5E).hCycT280 chimera can bind TAR in the presence of Tat as a transcriptionally inactive complex. This complex effectively forms a protective shield on TAR, which blocks the recruitment of the functional P-TEFb complex. RNA polymerase II remains unphosphorylated (RNAPIIa, gray oval).