Fig. 4

Biogenesis of P-TEFb in primary T cells and proposed mechanism for the recruitment of Tat:P-TEFb to the HIV-1 provirus. A P-TEFb is expressed in resting memory CD4⁺ T cells at vanishingly low levels due to posttranscriptional mechanisms that limit CycT1 expression. This causes the CDK9 subunit to be sequestered in the cytoplasm by the kinase-specific chaperone complex Hsp90/Cdc37. TCR co-stimulation induces CycT1 protein synthesis, leading to the heterodimeric assembly of P-TEFb that is stabilized by CDK9 phosphorylation at Thr186. Upon assembly, P-TEFb then enters the nucleus where it is incorporated into the 7SK snRNP complex. BRD4 and Tat can physically engage with 7SK snRNP but compete with one another to dissociate P-TEFb from the complex. The signal-dependent modification pSer175 CDK9 preferentially enhances the binding of Tat to P-TEFb. Ser175 on the activation loop of CDK9 is an essential contact point for BRD4; mutation of Ser175 to a phosphomimetic residue or an alanine produces severe defects in the association of P-TEFb with BRD4. B Microscopic imaging of the assembly of P-TEFb in memory CD4⁺ T cells following TCR activation. Cells were stimulated or not with soluble anti-CD3 and anti-CD28 antibodies. After immunostaining, images were captured by deconvolution microscopy at 60x. Scale bar: 10 μm. CDK9 is sequestered in the cytoplasm in the unstimulated cells, and CycT1 is present in very low amounts. After stimulation, CDK9 and synthesized CycT1 are present in the nucleus as a complex (P-TEFb), presumably sequestered by 7SK snRNP. C Combined immunofluorescence and RNA FISH detection of CycT1 and 7SK snRNA in memory CD4⁺ T cells. Cells were stimulated or not with anti-CD3 and anti-CD28 Dynabeads at a 1:1 bead-to-cell ratio. Images were captured by deconvolution microscopy at 100x. Scale bar: 1 μm. Cellular stimulation results in a doughnut-shaped redistribution of 7SK snRNA within the nucleus that tightly colocalizes with induced CycT1