Optimized chimeras between kinase-inactive mutant Cdk9 and truncated cyclin T1 proteins efficiently inhibit Tat transactivation and human immunodeficiency virus gene expression.
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.
D016475:3T3 Cells; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D056906:Cyclin T; D042863:Cyclin-Dependent Kinase 9; D018844:Cyclin-Dependent Kinases / Q000235:genetics / Q000378:metabolism*; D016213:Cyclins / Q000235:genetics / Q000378:metabolism*; D015967:Gene Expression Regulation, Viral; D015696:Gene Products, tat / Q000378:metabolism*; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000235:genetics*; D051379:Mice; D016296:Mutagenesis; D042925:Positive Transcriptional Elongation Factor B; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation; D054322:tat Gene Products, Human Immunodeficiency Virus
C503684:CCNT1 protein, human; C503685:Ccnt1 protein, mouse; D056906:Cyclin T; D016213:Cyclins; D015696:Gene Products, tat; D011993:Recombinant Fusion Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; D042925:Positive Transcriptional Elongation Factor B; D017346:Protein Serine-Threonine Kinases; C467972:Cdk9 protein, mouse; D042863:Cyclin-Dependent Kinase 9; D018844:Cyclin-Dependent Kinases