Phosphatase PPM1A negatively regulates P-TEFb function in resting CD4+ T cells and inhibits HIV-1 gene expression

Abstract

BackgroundProcessive elongation of the integrated HIV-1 provirus is dependent on recruitment of P-TEFb by the viral Tat protein to the viral TAR RNA element. P-TEFb kinase activity requires phosphorylation of Thr186 in the T-loop of the CDK9 subunit. In resting CD4+T cells, low levels of T-loop phosphorylated CDK9 are found, which increase significantly upon activation. This suggests that the phosphorylation status of the T-loop is actively regulated through the concerted actions of cellular proteins such as Ser/Thr phosphatases. We investigated the role of phosphatase PPM1A in regulating CDK9 T-loop phosphorylation and its effect on HIV-1 proviral transcription.ResultsWe found that overexpression of PPM1A inhibits HIV-1 gene expression during viral infection and this required PPM1A catalytic function. Using an artificial CDK tethering system, we further found that PPM1A inhibits CDK9, but not CDK8 mediated activation of the HIV-1 LTR. SiRNA depletion of PPM1A in resting CD4+T cells increased the level of CDK9 T-loop phosphorylation and enhanced HIV-1 gene expression. We also observed that PPM1A protein levels are relatively high in resting CD4+T cells and are not up-regulated upon T cell activation.ConclusionsOur results establish a functional link between HIV-1 replication and modulation of CDK9 T-loop phosphorylation by PPM1A. PPM1A represses HIV-1 gene expression by inhibiting CDK9 T-loop phosphorylation, thus reducing the amount of active P-TEFb available for recruitment to the viral LTR. We also infer that PPM1A enzymatic activity in resting and activated CD4+ T cells are likely regulated by as yet undefined factors.

Cyclin-dependent kinase 7 (CDK7)-mediated phosphorylation of the CDK9 activation loop promotes P-TEFb assembly with Tat and proviral HIV reactivation

“…CDK9 phosphorylation at the conserved T-loop residue Thr186 is inducible in quiescent CD4 + T lymphocytes, but is found to be constitutive in actively replicating T cell lines and expanding, fully activated, T cells (34)(35)(36)62,63). Consistent with molecular dynamics simulations, cell-based mutagenesis experiments demonstrated that the assembly of P-TEFb in T cells is critically dependent on the stabilization of the CDK9/CycT1 interface by phosphorylation of CDK9 at Thr186.…”

“…However, the data on their role in HIV transcription has been somewhat contradictory. Rice and colleagues (62) found that the siRNA knockdown of PPM1A expression in HIV-infected resting CD4 + T cells increased the level of CDK9 Thr186 phosphorylation in the absence of T-cell stimulation, and this was coupled with enhanced HIV gene expression. By contrast, Ammosova et al (76) reported that the stable expression of a peptide-based inhibitor of PP1, cdNIPP1, promoted CDK9 Thr186 phosphorylation.…”

“…Several phosphatases, namely, PP1, PPM1A and PPM1G, have been reported to target pThr186 (43,44,62,75,76). However, the data on their role in HIV transcription has been somewhat contradictory.…”

Cyclin T1 and CDK9 T-Loop Phosphorylation Are Downregulated during Establishment of HIV-1 Latency in Primary Resting Memory CD4+T Cells

“…ϩ T cells (29). It is probable that PPM1A is involved in dephosphorylating the T loop in naïve cells and quiescent memory CD4 ϩ T cells.…”

“…While the total levels of CDK9 remain relatively constant through resting versus activated states, the levels of T-loop-phosphorylated CDK9 (pCDK9) are very low in resting CD4 ϩ T cells due to the action of the phosphatase PPM1A (29,30). However, activation of these cells induces phosphorylation of the CDK9 T loop by an activating kinase that has recently been shown to be CDK7 (31).…”