A Repression-derepression Mechanism Regulating the Transcription of Human Immunodeficiency Virus Type 1 In Primary T Cells

Molecular Medicine 6(5): 377–390, 2000 Molecular Medicine © 2000 The Picower Institute Press Original Articles A Repression-derepression Mechanism Regulating the Transcription of Human Immunodeficiency Virus Type 1 In Primary T Cells Athanasia Mouzaki1, Arlette Doucet2, Emmanuel Mavroidis1, Lisbeth Muster3, and Duri Rungger3 1 Experimental Hematology and Transfusion Medicine, Department of Medicine, University of Patras, Patras, Greece 2 Division of Hematology, Department of Medicine, Geneva University Hospital, Geneva, Switzerland 3 Station de Zoologie Expérimentale, University of Geneva, Chêne-Bougeries, Switzerland Accepted February 29, 2000. Abstract Background: Despite some controversy regarding the preferential infection and replication of human immunodeficiency virus type 1 (HIV-1), it appears that primary T lymphocytes, in their quiescent state, are nonpermissive for viral expression and propagation. Massive activation of viral gene expression occurs only when the host lymphocyte is activated. These observations prompted us to investigate the transcriptional regulation of HIV-1 in resting or activated T cells that were isolated from cord blood or adult peripheral blood. Materials and Methods: To this end, we employed cellular purification and phenotyping techniques, in vitro protein-DNA binding studies, functional transactivation assays using proteins isolated from cord blood or adult peripheral blood T lymphocytes, and transfection experiments in primary T cells. Results: We showed that transcription from the HIV-1 long terminal repeat is repressed in resting naive T lymphocytes; whereas, mitogenically stimulated CD4 cells form an activator that derepresses transcription. Negative and positive regulation act through a repressor-activator target sequence (RATS), which shares homology with the interleukin-2 (IL-2) purine-rich response element, through the adjacent binding site of the nuclear factor of activated T cells (NFAT), and weakly, through the B region. Conclusions: This regulation exerted by cellular transcription factors can account for several important features of HIV-1 expression in primary CD4 cells. Tight repression in resting naive T helper cells may be a main cause of viral latency and transcriptional activation accounts for massive viral production in activated T lymphocytes. Introduction tor and one or both of the co-receptors, CXCR4 or CCR5, are the main target of infection by human immunodeficiency virus type 1 (HIV-1) and disease progression is directly related to a gradual loss of CD4 lymphocytes, accompanied by an increase in the viral burden (1,2). Despite some controversy regarding the preferential infection and replication of HIV-1 (3), it Cells such as T lymphocytes and monocytederived macrophages, bearing the CD4 recepAddress correspondence and reprint requests to: D. Rungger, Station de Zoologie Expérimentale, 154, route de Malagnou, CH-1224 Chêne-Bougeries, Switzerland. Phone: 41 22 349 9925; Fax: 41 22 349 2647; E-mail: 378 Molecular Medicine, Volume 6, Number 5, May 2000 appears that in their quiescent state, T lymphocytes are non-permissive for viral expression and propagation. Massive activation of viral gene expression occurs only when the host lymphocyte is activated (4–6). However, the cellular mechanism underlying latency and productive lytic activation is still poorly understood. HIV-1 gene transcription is controlled by the interplay of viral and cellular transcription factors (TFs) interacting with sequence elements in the viral long terminal repeat (LTR), most of which interact with the core promoter and the upstream enhancer region (7–9). An ever increasing number of binding sites for cellular TFs has been identified (8–10). The activation of HIV-1 transcription in response to mitogenic stimulation is triggered by the signaling pathways of the infected host cell (11). It was anticipated that this response is primarily controlled by cellular factors. Indeed, several sequence elements present in the LTR have been shown to be functionally involved in the response to T-cell activation, namely the B region (6,12), with the cooperative interaction of Sp1 (13). The AP-1 site of the R/U5 region also participates in stimulation (14). The so-called negative regulatory element (NRE), located between nucleotides 340 and 185 bp upstream of the transcriptor initiation site (15), contains a region (position 279 to 250) that shares extensive sequence homology with the interleukin-2 (IL-2) distal purinerich response elements and is alternatively called PRRE (16), IL-2 consensus sequence (17) or IL-2 homology region (6). Though it was occasionally also named (NFAT) motif (8), NFAT does not bind to this segment, but just downstream to it between positions 255 and 216 (18; Fig. 1). In the HIV-LTR, the PRRE and NFAT sites, thus, are not superimposed as in the IL-2 promoter, but sit side by side and exhibit astonishingly little sequence similarity. The response of HIV-1 to host cell activation is strikingly similar to that of the IL-2 gene which, in the T leukemia Jurkat, is controlled by NFAT through the distal PRRE (19–22). For this reason, the HIV-1 PRRE, as well as the adjacent NFAT motif, are often considered as putative response elements to T-cell activation. In vivo footprinting patterns reveal changes in protein binding in the PRRE region and in the adjacent NFAT-binding site during T-cell activation (23). Attempts to functionally prove the involvement of PRRE and NFAT sites in HIV-1 activation gave contrasting results. In stably transfected T-cell lines, the HIV-1 LTR responds, Fig. 1. HIV-1 LTR and derived target genes. (A) Relative position of relevant promoter elements within the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR). (B) Sequence and position of oligonucleotides used as probes in the bandshift assays, and as cis-acting elements inserted into the hybrid promoter of target genes. The interleukin-2 (IL-2) consensus sequence is marked by a grey bar. (C) Map of target genes containing one or several copies of single cis-acting elements. In the Sp1 promoters, the elements are linked by an irrelevant spacer to a TATA box followed by the gene coding for chloramphenicol-acetyl-transferase (CAT) and a polyadenylation signal. In the Sp1 promoters, an Sp1 site is intercalated between the cisactive element(s) and the TATA box. NFAT, nuclear factor of activated T cells; NFB, nuclear factor kappa B. similar to the IL-2 PRRE, to a variety of T-cell activation signals that trigger calcium increase and protein kinase C signals (24). Deletion of the PRRE element from the HIV-1 LTR results in increased transcription in the Jurkat T cell line (25) and the human melanoma cell line SK23 (16); whereas, a single PRRE in the context of a hybrid promoter mediates activation, indicative of a dual, negative and positive role of the PRRE (16). A (HeLa) cell protein was found to bind to the PRR (...truncated)