Induction of TAK (cyclin T1/P-TEFb) in purified resting CD4(+) T lymphocytes by combination of cytokines

Abstract

Combinations of cytokines are known to reactivate transcription and replication of latent human immunodeficiency virus type 1 (HIV-1) proviruses in resting CD4(+) T lymphocytes isolated from infected individuals. Transcription of the HIV-1 provirus by RNA polymerase II is strongly stimulated by the viral Tat protein. Tat function is mediated by a cellular protein kinase known as TAK (cyclin T1/P-TEFb) that is composed of Cdk9 and cyclin T1. We have found that treatment of peripheral blood lymphocytes and purified resting CD4(+) T lymphocytes with the combination of interleukin-2 (IL-2), IL-6, and tumor necrosis factor alpha resulted in an increase in Cdk9 and cyclin T1 protein levels and an increase in TAK enzymatic activity. The cytokine induction of TAK in resting CD4(+) T lymphocytes did not appear to require proliferation of lymphocytes. These results suggest that induction of TAK by cytokines secreted in the microenvironment of lymphoid tissue may be involved in the reactivation of HIV-1 in CD4(+) T lymphocytes harboring a latent provirus.

FIG. 1

CD25 and CD69 expression in cytokine-treated PBLs. PBLs purified from donor 1 were cultured for 72 h and analyzed by flow cytometry.

FIG. 2

Cdk9 and cyclin T1 protein levels and TAK kinase activity are increased in PBLs following treatment with a combination of cytokines. PBLs purified from two healthy donors were cultured in media as indicated for 72 h; CD25 and CD69 expression of donor 1 was analyzed in Fig. 1. (A) Equal amounts of protein from cell extracts were analyzed for Cdk9, cyclin T1, and Cdk7 by immunoblotting. (B) TAK kinase assays were performed with recombinant CTD as a substrate, and products were analyzed on an SDS–9% polyacrylamide gel. CTDo is the hyperphosphorylated form of CTD; CTDa is the underphosphorylated form of CTD.

FIG. 3

CD25 and CD69 expression in cytokine-treated CD4⁺ T lymphocytes. Resting CD4⁺ T lymphocytes purified from donors 3 and 4 were cultured for 72 h as indicated and analyzed by flow cytometry.

FIG. 4

Cdk9 and cyclin T1 protein levels and TAK activity are increased in resting CD4⁺ T lymphocytes following treatment with a combination of cytokines. Purified resting CD4⁺ T cells from two healthy donors were cultured in media as indicated for 72 h. Expression of CD25 and CD69 is shown in Fig. 3. (A) Equal amounts of protein from cell extracts were analyzed for Cdk9, cyclin T1, and Cdk7 by immunoblotting. (B) TAK kinase assays were performed with recombinant CTD as a substrate, and products were analyzed on an SDS–9% polyacrylamide gel. CTDo is the hyperphosphorylated form of CTD; CTDa is the underphosphorylated form of CTD.