Differential role of long terminal repeat control elements for the regulation of basal and Tat-mediated transcription of the human immunodeficiency virus in stimulated and unstimulated primary human macrophages

Abstract

Primary human macrophages induced to differentiate through contact with autologous activated nonadherent cells were used to investigate the transcriptional mechanisms involved in reactivation of human immunodeficiency virus (HIV) replication. Through transient transfection experiments with an HIV long terminal repeat (LTR)-chloramphenicol acetyltransferase reporter construct, we show that macrophage differentiation results in a 20-fold upregulation of basal LTR activity. To identify sequence elements responsive to the differentiation process, point mutations introduced into the LTR were tested in differentiated and undifferentiated macrophages. Several elements were identified as positive regulators of basal transcription. TATA, Spl, and NF-KB binding sites were the most influential. The low-affinity site for LBP-1 (UBP-1) functioned as a negative regulator of LTR activity in undifferentiated macrophages, but this influence was lost upon differentiation. When tat was cotransfected into the expression system, the requirement for LTR elements identified as important for positive regulation of basal transcription remained in undifferentiated macrophages. Interestingly, however, the mutations in positive control elements which debilitated activity in undifferentiated macrophages had no effect on LTR activity in differentiated macrophages. Thus, it appears that while HIV-LTR activity is highly dependent on cellular transcription factors in undifferentiated cells, in differentiated macrophages the viral protein Tat confers pliability on the LTR and facilitates autonomy from absolute cellular control mechanisms. In vivo, release from either positive or negative regulation via cellular proteins may facilitate reactivation of HIV in macrophages.

Constitutive expression of p50 homodimer in freshly isolated human monocytes decreases with in vitro and in vivo differentiation: a possible mechanism influencing human immunodeficiency virus replication in monocytes and mature macrophages

“…The single NF-B site of simian immunodeficiency virus SIV macEm is essential for efficient replication of SIV macEm in macaque AMs but not in macaque PBLs (4). Although the NF-B sites of HIV-1 are a critical activator of transcription in monocytic cell lines when assessed by an HIV LTR-CAT expression vector (10,21), their importance for replication of HIV in differentiated macrophages is unknown. However, constitutive expression of p50/p65 and p50/ RelB would clearly favor productive HIV replication in these cells.…”

Multiorgan Nuclear Factor Kappa B Activation in a Transgenic Mouse Model of Systemic Inflammation

“…The proximal HIV-LTR is a NF-B-responsive promoter (6)(7)(8), containing a TATA box, an enhancer region between Ϫ 82 and Ϫ 103 with two NF-B motifs, and three Sp1 boxes from Ϫ 46 to Ϫ 78. In primary cell culture, NF-B activation is absolutely required for transcriptional activity of the HIV-LTR (9,10). In the HLL mice, we show that luciferase production and intracellular accumulation are dependent on NF-B-activated gene transcription; therefore, HLL mice provide a useful in vivo reporter-based assay system in which to analyze NF-B enhancer activity in response to a variety of inflammatory signals.…”

Prostaglandin I2 analogs inhibit Th1 and Th2 effector cytokine production by CD4 T cells

“…The proximal HIV-LTR contains two NF-B motifs and is a well-characterized NF-Bresponsive promoter [21]. In primary culture, NF-B activation is absolutely required for transcriptional activity of the proximal HIV-LTR [22,23]. Therefore, luciferase expression in HLL cells is used as a surrogate marker for NF-B activity.…”