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


RESULTS
We 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.


CONCLUSIONS
Our 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.
Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas 77030, USA.
Sona
S
Budhiraja
Rajesh
R
Ramakrishnan
Andrew P
AP
Rice
D015496:CD4-Positive T-Lymphocytes / Q000378:metabolism / Q000821:virology*; D056850:Cyclin-Dependent Kinase 8 / Q000235:genetics / Q000378:metabolism; D042863:Cyclin-Dependent Kinase 9 / Q000235:genetics / Q000378:metabolism; D015967:Gene Expression Regulation, Viral; D055785:Gene Knockdown Techniques; D057809:HEK293 Cells; D015658:HIV Infections / Q000821:virology; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000235:genetics* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D010749:Phosphoprotein Phosphatases / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D042925:Positive Transcriptional Elongation Factor B / Q000235:genetics / Q000378:metabolism*; D000071636:Protein Phosphatase 2C; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D034741:RNA, Small Interfering; D042925:Positive Transcriptional Elongation Factor B; C095686:CDK8 protein, human; C467969:CDK9 protein, human; D056850:Cyclin-Dependent Kinase 8; D042863:Cyclin-Dependent Kinase 9; C000607456:PPM1A protein, human; D010749:Phosphoprotein Phosphatases; D000071636:Protein Phosphatase 2C