RESUMO
The human immunodeficiency virus type 1 (HIV-1) Tat protein has been reported to transactivate several cellular genes, including the potent chemotactic factor interleukin-8 (IL-8). Consistent with these in vitro assays, elevated levels of IL-8 protein are found in the serum of HIV-infected individuals. We now extend these observations by demonstrating that Tat induction of IL-8 is linked to the cell cycle. Cells that constitutively express the Tat(1-86) protein (eTat) and control cells (pCEP) were reversibly blocked at the G(1)/S border with hydroxyurea or thymidine. The cells were subsequently released, and IL-8 expression was monitored by RNase protection assays and enzyme-linked immunosorbent assay (ELISA). RNase protection assays demonstrated that IL-8 mRNA expression is transiently induced, approximately fourfold, as the Tat-expressing cells enter S phase. Consistent with the RNase protection assay, an increase in IL-8 protein was observed in the cell supernatant using an IL-8 ELISA. Similar experiments were performed following a reversible block at the G(2)/M border with nocodazole and release into G(1). Using the RNase protection assay and ELISA, little or no increase in IL-8 expression was observed during G(1). Using gel shift as well as an immobilized DNA binding assay, we demonstrate that the increase in IL-8 gene expression correlates with a specific increase in p65 NF-kappa B binding activity only in the nucleus of the Tat-expressing cells. Moreover, the CREB-binding protein coactivator is present in the complex in the Tat cell line. Finally, we demonstrate that the presence of the proteasome inhibitor MG-132 inhibits the induction of NF-kappa B binding, as well as IL-8 expression, supporting the role of NF-kappa B.
Assuntos
Ciclo Celular/fisiologia , Regulação da Expressão Gênica , Produtos do Gene tat/metabolismo , HIV-1/metabolismo , Interleucina-8/biossíntese , Interleucina-8/genética , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Produtos do Gene tat/genética , HIV-1/genética , Células HeLa , Humanos , Hidroxiureia/farmacologia , Leupeptinas/farmacologia , NF-kappa B/metabolismo , Regiões Promotoras Genéticas , Fase S , Transcrição Gênica , Transfecção , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
Cyclin-dependent kinases are required for the Tat-dependent transition from abortive to productive elongation. Further, the human immunodeficiency virus type 1 (HIV-1) Vpr protein prevents proliferation of infected cells by arresting them in the G(2) phase of the cell cycle. These findings suggest that the life cycle of the virus may be integrally related to the cell cycle. We now demonstrate by in vitro transcription analysis that Tat-dependent transcription takes place in a cell cycle-dependent manner. Remarkably, Tat activates gene expression in two distinct stages of the cell cycle. Tat-dependent long terminal repeat activation is observed in G(1). This activation is TAR dependent and requires a functional Sp1 binding site. A second phase of transactivation by Tat is observed in G(2) and is TAR independent. This later phase of transcription is enhanced by a natural cell cycle blocker of HIV-1, vpr, which arrests infected cells at the G(2)/M boundary. These studies link the HIV-1 Tat protein to cell cycle-specific biological functions.
Assuntos
Ciclo Celular , Regulação Viral da Expressão Gênica , Produtos do Gene tat/metabolismo , HIV-1/genética , Extratos Celulares , Epitopos/genética , Fase G1 , Fase G2 , Produtos do Gene tat/genética , Produtos do Gene vpr/genética , Produtos do Gene vpr/metabolismo , Repetição Terminal Longa de HIV , Células HeLa , Hemaglutininas/genética , Humanos , Fator de Transcrição Sp1/metabolismo , Transcrição Gênica , Ativação Transcricional , Produtos do Gene tat do Vírus da Imunodeficiência Humana , Produtos do Gene vpr do Vírus da Imunodeficiência HumanaRESUMO
Human immunodeficiency virus type 1 (HIV-1) Tat-mediated trans-activation requires the structural integrity of TAR RNA and the cooperative interaction of human host cell proteins. The TAR domain, minimally required for tat response, includes the Tat binding pyrimidine bulge, the TAR RNA upper stem, and the loop sequences. However, little is known about the significance of the 5'-stem structure of TAR in the regulation of viral growth. We designed viral mutations, specifically in the TAR RNA lower stem structure, and studied their effects on the kinetics of viral growth in T-lymphocyte cell lines and in activated human peripheral blood mononuclear cells. Mutations that destabilized the lower TAR stem structure inhibited viral growth to various degrees in different CD4+ T-cells. These results suggest that the structural integrity of the lower stem structure of TAR plays an important role in viral growth, presumably by binding to specific host cell proteins that stabilize Tat-TAR interactions.
