The persistence of transcriptionally silent but replication-competent HIV-1 reservoirs in Highly Active Anti-Retroviral Therapy (HAART)-treated infected individuals, represents a major hurdle to virus eradication. Activation of HIV-1 gene expression in these cells together with an efficient HAART has been proposed as an adjuvant therapy aimed at decreasing the pool of latent viral reservoirs. Using the latently-infected U1 monocytic cell line and latently-infected J-Lat T-cell clones, we here demonstrated a strong synergistic activation of HIV-1 production by clinically used histone deacetylase inhibitors (HDACIs) combined with prostratin, a non-tumor-promoting nuclear factor (NF)- κB inducer. In J-Lat cells, we showed that this synergism was due, at least partially, to the synergistic recruitment of unresponsive cells into the expressing cell population. A combination of prostratin+HDACI synergistically activated the 5′ Long Terminal Repeat (5'LTR) from HIV-1 Major group subtypes representing the most prevalent viral genetic forms, as shown by transient transfection reporter assays. Mechanistically, HDACIs increased prostratin-induced DNA-binding activity of nuclear NF-κB and degradation of cytoplasmic NF-κB inhibitor, IκBα . Moreover, the combined treatment prostratin+HDACI caused a more pronounced nucleosomal remodeling in the U1 viral promoter region than the treatments with the compounds alone. This more pronounced remodeling correlated with a synergistic reactivation of HIV-1 transcription following the combined treatment prostratin+HDACI, as demonstrated by measuring recruitment of RNA polymerase II to the 5'LTR and both initiated and elongated transcripts. The physiological relevance of the prostratin+HDACI synergism was shown in CD8+-depleted peripheral blood mononuclear cells from HAART-treated patients with undetectable viral load. Moreover, this combined treatment reactivated viral replication in resting CD4+ T cells isolated from similar patients. Our results suggest that combinations of different kinds of proviral activators may have important implications for reducing the size of latent HIV-1 reservoirs in HAART-treated patients.

Efficient bovine leukemia virus (BLV) transcription requires the virus-encoded transactivatorBovine leukemia virus (BLV) 1 is a B-lymphotropic retrovirus associated with enzootic bovine leukosis, a disease characterized by an increased number of B-lymphocytes and, in some cases, after a long latency period, by the subsequent development of B-cell leukemia or lymphosarcoma (1). BLV is closely related structurally and biologically to the human T-lymphotropic viruses HTLV-I and HTLV-II (1). Expression of BLV is regulated at the transcriptional level by the virus-encoded transactivator Tax BLV (2, 3). The molecular mechanism by which Tax BLV activates viral transcription is not fully understood. Transactivation by Tax BLV requires three 21-bp imperfect repeats located in the U3 region of the 5Ј long terminal repeat (LTR) (2, 4). These Tax BLV -responsive elements (called TxREs) contain a core octanucleotide sequence with similarity to the cAMP-responsive element (CRE) consensus (TGACGTCA) (5). The BLV CRE-like motifs located in the middle of each TxRE have been shown to serve as binding sites for three members of the basic leucine zipper (bZIP) family of cellular transcription factors: the CRE-binding protein (CREB) and the activating transcription factors-1 and Ϫ2 (ATF-1 and ATF-2) (4, 6). Because there is no evidence for direct binding of Tax BLV to DNA, it has been proposed that Tax BLV transactivation of the BLV promoter could be mediated, as reported for the HTLV-I system, through protein-protein interactions with CREB/ATF (4, 6, 7). The formation of this promoter-bound Tax BLV -CREB/ATF complex could then serve for the recruitment of the multifunctional cellular coactivators CBP (CREB-binding protein) and p300.There is now strong evidence that both transcriptional activation and silencing are mediated through the recruitment of enzymes that control protein acetylation: the histone deacetylases (HDACs) and the histone acetyltransferases (HATs). Acetylation of specific lysine residues within the amino-terminal tails of nucleosomal histones is generally linked to chroma-

Bovine leukemia virus (BLV) expression is controlled at the transcriptional level through three Tax Bovine leukemia virus (BLV)7 infection is characterized by viral latency in the large majority of infected cells and by the absence of viremia. These features are thought to be due to the transcriptional repression of viral expression in vivo (1, 2). BLV transcription initiates at the unique promoter located in the 5Ј-long terminal repeat (5Ј-LTR) of the BLV genome. The 5Ј-LTR is composed of the U3, R, and U5 regions and transcription initiates at the U3-R junction. BLV exhibits two distinct functional transcriptional states as follows: a low basal level of transcription ensured by host cellular transcription factors, and a high level of transcription directed by the virus-encoded transcriptional activator Tax BLV (3,4). In the early stages of BLV transcription, before Tax BLV expression and transactivation, the basal transcriptional promoter activity is ensured by several cis-acting elements located in the 5Ј-LTR. In the U3 region are present the promoter CAAT and TATA boxes (5, 6), and three 21-bp enhancers, each containing an imperfectly conserved 8-bp cyclic AMP-responsive element (CRE), which binds at least three proteins: CRE-binding protein (CREB) and activating transcription factors 1 and 2 (ATF-1 and ATF-2) (7-10). Importantly, the 21-bp enhancers are also called Tax BLV -responsive elements (TxREs) because transactivation of the BLV LTR by Tax BLV requires these enhancers. It has been proposed that Tax BLV activation of transcription

LIGHT (TNFSF14) is a newly identified tumor necrosis factor superfamily member involved in the regulation of immune responses by control of activation, maturation, and survival of immune effector cells. Despite the immunological relevance of the LIGHT protein, little knowledge is available as to how light gene expression is regulated. In T-lymphocytes, most LIGHT surface expression and transcript accumulation occurs after T cell activation. In this study, we have shown that these events are blocked at the transcriptional level by cyclosporin A, an immuno-suppressive drug. Besides, we identified a role for Ca 2؉ -signaling pathways and NFAT transcription factors in T cell activation-induced LIGHT expression. To further investigate this process, we have identified, cloned, and characterized a 2.1-kilobase 5-flanking DNA genomic fragment from the human light gene. We have shown the transcriptional activity of the herein-identified minimal 5 regulatory region of human light gene parallels the endogenous expression of light in T cells. Moreover, we demonstrated that induced LIGHT promoter activity can be equally blocked by cyclosporin A treatment or dominant negative NFAT overexpression and further identified by site-directed mutagenesis and electrophoretic mobility supershift analysis of a NFAT transcription factor binding site within the human light minimal promoter. Finally, Sp1 and Ets1 binding sites were identified and shown to regulate light basal promoter activity. Thus, the present study establishes a molecular basis to further understand the mechanisms governing human light gene expression and, consequently, could potentially lead to novel therapeutic manipulations that control the signaling cascade, resulting in LIGHT production in conditions characterized by immunopathologic activation of T cells.