Transient induction of cyclin T1 during human macrophage differentiation regulates human immunodeficiency virus type 1 Tat transactivation function

Abstract

The human immunodeficiency virus type 1 (HIV-1) Tat protein is essential for viral replication and stimulates transcription of the integrated provirus by recruiting the kinase complex TAK/P-TEFb, composed of cyclin T1 (CycT1) and Cdk9, to the viral TAR RNA element. TAK/P-TEFb phosphorylates the RNA polymerase II complex and stimulates transcriptional elongation. In this report, we investigated the regulation of TAK/P-TEFb in primary human macrophages, a major target cell of HIV infection. While Cdk9 levels remained constant, CycT1 protein expression in freshly isolated monocytes was very low, increased early during macrophage differentiation, and, unexpectedly, decreased to very low levels after about 1 week in culture. The kinase activity of TAK/P-TEFb paralleled the changes in CycT1 protein expression. RNA analysis indicated that the transient induction of CycT1 protein expression involves a posttranscriptional mechanism. In transient transfection assays, the ability of Tat to transactivate the HIV long terminal repeat (LTR) in the late differentiated macrophages was greatly diminished relative to its ability to transactivate the HIV LTR in early differentiated cells, strongly suggesting that CycT1 is limiting for Tat function in late differentiated macrophages. Interestingly, lipopolysaccharide, a component of the cell wall of gram-negative bacteria, reinduced CycT1 expression late in macrophage differentiation. These results raise the possibility that regulation of CycT1 expression may be involved in establishing latent infection in macrophages and that opportunistic infection may reactivate the virus by inducing CycT1 expression.

FIG. 1.

CycT1 protein expression and TAK/P-TEFb kinase activity increase during early MDM differentiation. (A) Expression of CycT1 and Cdk9 proteins during early MDM differentiation. Human monocytes were isolated as indicated from healthy blood donors and cultured for 4 days. Cell lysates were prepared at the indicated times, and the expression levels of CycT1, Cdk9, Cdk8, Cdk7, and CycC were examined by immunoblotting. Membranes were cut in four sections according to the sizes of proteins to be detected. The membrane section for Cdk9 was stripped and reprobed with an Ab against β-actin as an internal control for loading and transfer. (B) TAK/P-TEFb enzymatic activity increases during MDM differentiation. Cell extracts were prepared at the indicated days of MDM differentiation, and TAK kinase assays were performed as described in Materials and Methods. Methods used for monocyte preparation are indicated. Arrows, phosphorylated GST-Tat-2, Cdk9, and hyper- and hypophosphorylated CTD (CTDo and CTDa, respectively).

FIG. 2.

Transient induction of CycT1 protein and kinase activity during differentiation. (A) Expression of CycT1 and Cdk9 proteins during late MDM differentiation. MDMs with extended culturing time were analyzed for the expression of CycT1 and Cdk9. Cell lysates were prepared and immunoblotting was carried out as described in Materials and Methods. (B) Kinase activity of TAK/P-TEFb decreases while CycT1 declines at late time points of differentiation. Kinase assays were carried out with cell extracts made from MDMs with a high level of CycT1 (day 6) or undetectable levels of CycT1 (day 8). The CycT1 protein levels from the same donor at these time points are shown in Fig. 7.

FIG. 3.

Cyclin T1 RNA levels during monocyte differentiation. RNA analyses were performed by an RT-PCR method as described in Materials and Methods. Methods used to isolate monocytes from donors are indicated.

FIG. 4.

Tat transactivation activity of HIV-1 LTR during MDM differentiation. MDMs from the indicated donors were cotransfected with Tat expression or vector plasmids and HIV-1 LTR-luciferase and TK-Renilla luciferase reporter plasmids. Luciferase activities were measured 24 h posttransfection and normalized to Renilla luciferase activity.

FIG. 5.

Cyclin T1 protein decline is accelerated at high culture density. PBMCs were seeded in culture dishes at the indicated densities, and monocytes were prepared by the adherence methods. After 4 or 10 days, cell extracts were prepared and CycT1, Cdk9, Cdk8, and CycC were analyzed by immunoblotting.

FIG. 6.

LPS induces CycT1 expression at late time points of differentiation. After confirming that CycT1 expression had declined, MDMs were treated with LPS at the indicated days. Cell extracts were prepared either 1 or 2 days after addition of LPS. CycT1, Cdk9, and β-actin were detected by immunoblotting. Similar results were observed for nine additional donors.

FIG. 7.

CycT1 level is maintained during differentiation of monocyte-derived DC. Isolated monocytes were cultured under conditions to induce macrophage differentiation or differentiation to immature DC (iDC) or mature DC (mDC) as described in Materials and Methods. DC were treated with TNF-α to induce maturation. On the indicated days, cell extracts were prepared and CycT1, Cdk9, and β-actin protein levels were determined by immunoblotting. Similar results were obtained with DC from three additional donors.