Modulation of HIV-1 enhancer activity and virus production by cAMP.

The effect of cAMP on the transcriptional activity of the HIV-1 long terminal repeat/enhancer was investigated and compared to the effect of cAMP on virus replication. In culture cAMP repressed virus replication in vivo using different cell types. Transient transfection studies with HIV-1 enhancer-derived luciferase reporter gene constructs identified the minimal DNA sequence mediating the negative regulatory effect of cAMP on HIV-1 transcription. A single nuclear factor kappaB element from the HIV-1 enhancer mediates the repressive effect on transcription. AP-2 is not involved in cAMP repression. Stable transfection of Jurkat T cells with the co-activators CREB binding protein (CBP) and p300 completely abolished the cAMP repressive effect, supporting the hypothesis that elevation of intracellular cAMP increases phosphorylation of CREB, which then competes with phosphorylated p65 and Ets-1 for limiting amounts of CBP/p300 thereby mediating the observed repressive effect on transcription. These findings suggest an important role of cAMP on HIV-1 transcription.

Hydroxyurea inhibits the transactivation of the HIV-long-terminal repeat (LTR) promoter.

HIV-1 gene expression is regulated by the promoter/enhancer located within the U3 region of the proviral 5' LTR that contains multiple potential cis-acting regulatory sites. Here we describe that the inhibitor of the cellular ribonucleoside reductase, hydroxyurea (HU), inhibited phorbol myristate acetate- or tumour necrosis factor-alpha-induced HIV-1-LTR transactivation in both lymphoid and non-lymphoid cells in a dose-dependent manner within the first 6 h of treatment, with a 50% inhibitory concentration of 0.5 mM. This inhibition was found to be specific for the HIV-1-LTR since transactivation of either an AP-1-dependent promoter or the CD69 gene promoter was not affected by the presence of HU. Moreover, gel-shift assays in 5.1 cells showed that HU prevented the binding of the NF-kappaB to the kappaB sites located in the HIV-1-LTR region, but it did not affect the binding of both the AP-1 and the Sp-1 transcription factors. By Western blots and cell cycle analyses we detected that HU induced a rapid dephosphorylation of the pRB, the product of the retinoblastoma tumour suppressor gene, and the cell cycle arrest was evident after 24 h of treatment. Thus, HU inhibits HIV-1 promoter activity by a novel pathway that implies an inhibition of the NF-kappaB binding to the LTR promoter. The present study suggests that HU may be useful as a potential therapeutic approach for inhibition of HIV-1 replication through different pathways.

Genetic evidence that stress-activated p38 MAP kinase is necessary but not sufficient for UV activation of HIV gene expression.

We have examined the role of stress-activated p38 MAP kinase in regulating human immunodeficiency virus (HIV) gene expression in response to ultraviolet light (UV). We found that UV activated p38 in HeLa cells harboring stably integrated copies of an HIVcat plasmid to levels similar to those obtained by hyperosmotic shock. However, hyperosmotic shock resulted in one order of magnitude smaller increase in CAT activity than treatment with UV. The specific p38 inhibitor SB203580 significantly decreased (>80%) UV activation of HIV gene expression whereas PD98059, a specific MEK-1 inhibitor did not, suggesting that p38 is specifically involved in the HIV UV response and little to no contribution is provided by MEK-1 and the p42/p44 MAP kinase pathway. Whereas increased binding of NF-kappaB to an oligonucleotide spanning the HIV enhancer was observed after UV, as expected, this binding was not affected by SB203580. Furthermore, UV activation of HIV gene expression in cells having the cat reporter gene under control of an HIV promoter deleted of the enhancer (-69/+80) produced results indistinguishable from those using HIVcat/HeLa cells with an intact HIV promoter (-485/+80), suggesting that SB203580 acts through the basal transcription machinery. Northern blot analysis of steady-state RNA from HIVcat/HeLa cells revealed an almost complete inhibition of UV activation with SB203580 at the RNA level. Similarly, the UV response was almost completely obliterated at the CAT and RNA levels in HIVcat/HeLa cells stably transfected with a plasmid expressing a kinase-inactive mutant of p38 (isoform alpha), without affecting NF-kappaB activation, providing strong genetic evidence that p38, at least the alpha isoform, is necessary for UV activation of HIV gene expression and that NF-kappaB activation alone is insufficient. These results firmly establish p38 MAP kinase as a key modulator of HIV gene expression in response to UV that acts independently of NF-kappaB.

Fibrinogen activates NF-kappa B transcription factors in mononuclear phagocytes.

Adhesion to extracellular matrices is known to modulate leukocyte activation, although the mechanisms are not fully understood. Mononuclear phagocytes are exposed to fibrinous provisional matrix throughout migration into inflammatory foci, so this study was undertaken to determine whether fibrinogen triggers activation of NF-kappa B transcription factors. U937 cells differentiated with PMA in nonadherent culture were shown to express two fibrinogen-binding integrins, predominately CD11b/CD18, and to a lesser extent, CD11c/CD18. Cells stimulated with fibrinogen (10-100 microg/ml)/Mn2+ (50 microM) for 2 h were examined by electrophoretic mobility shift assay. NF-kappa B activation, minimal in unstimulated cells, was substantially up-regulated by fibrinogen. Fibrinogen also caused activation of AP-1, but not SP1 or cAMP response element-binding protein (CREB) factors. Blocking mAbs against CD18 and CD11b abrogated fibrinogen-induced NF-kappa B activation. To determine the effects on transcriptional regulation, U937 cells were transfected with a plasmid containing the HIV-1 enhancer (bearing two NF-kappa B sites) coupled to a chloramphenicol acetyltransferase (CAT) reporter. Cells were subsequently stimulated with 1) PMA for 24 h, inducing CAT activity by 2.6-fold, 2) fibrinogen/Mn2+ for 2 h, inducing CAT activity by 3.2-fold, or 3) costimulation with fibrinogen and PMA, inducing 5.7-fold the CAT activity induced by PMA alone. We conclude that contact with fibrinogen-derived proteins may contribute to mononuclear phagocyte activation by signaling through CD11b/CD18, resulting in selective activation of transcriptional regulatory factors, including NF-kappa B.

Hexamethylene bisacetamide activates the human immunodeficiency virus type 1 provirus by an NF-kappa B-independent mechanism.

Expression of the human immunodeficiency virus type 1 (HIV-1) provirus in T lymphocytic and monocytic cells can be induced by treatment with hexamethylene bisacetamide (HMBA). The induction occurs at the transcriptional level within 1 to 3 h after the addition of the drug, and is not associated with detectable changes in the binding of transcription factors to the enhancer, TATA box or other regulatory regions of the HIV-1 long terminal repeat (LTR). Using the 5' deletion mutants of HIV-1 LTR controlling the expression of the chloramphenicol acetyltransferase gene, we found that the deletion of the kappa B enhancer did not affect HIV-1 inducibility, whereas the deletion of the Sp1 binding sites abolished transcriptional activation. However, the presence of the HIV-1 LTR Sp1 binding sites in the context of the heterologous promoter did not induce responsiveness to HMBA. We conclude that HMBA increases transcription through the secondary modification of the basal transcription complex suggesting the existence of a regulatory pathway that circumvents the requirement for the induction of NF-kappa B or other DNA-specific binding proteins.

Human thioredoxin/adult T cell leukemia-derived factor activates the enhancer binding protein of human immunodeficiency virus type 1 by thiol redox control mechanism.

Transcription from the human immunodeficiency virus type 1 (HIV-1) provirus is activated by a cellular factor, NF kappa B, recognizing the tandemly repeated 10-base-pair sequences, termed the kappa B sequence, present in the enhancer region within the viral long terminal repeat (LTR). Using electrophoretic mobility shift assay (EMSA), which demonstrates specific DNA-protein interaction in vitro, we could demonstrate that reducto-oxidative modulation of NF kappa B dramatically changes its DNA binding activity and that a cellular physiological reducing catalyst, thioredoxin (TRX) also known as adult T cell leukemia derived factor (ADF), fully restored the DNA-binding activity of the oxidized NF kappa B. We also observed that purified TRX/ADF protein could augment gene expression from HIV LTR as demonstrated by transient chloramphenicol acetyltransferase (CAT) assay. These observations confirmed the previous notion that ADF might be an inducing factor of cellular interleukin-2 receptor alpha subunit (IL-2R alpha) through the kappa B sequence that is a common central cis-regulatory element in both IL-2R alpha and HIV gene expression. These observations indicate that reducto-oxidative regulation (or redox regulation) of a cysteine residue(s) on the NF kappa B molecule might play an important role in its specific DNA interaction and that it might provide a clue to the understanding of a pathway of cellular signal transduction to NF kappa B that is independent from the known pathways involving protein phosphorylation.

Induction of dormant HIV-1 by sodium butyrate: involvement of the TATA box in the activation of the HIV-1 promoter.

Reactivation of latent HIV-1 is believed to play a major role in the pathogenesis of AIDS. Here we show that sodium butyrate (NaB), which can cause gene induction or cell differentiation, reactivates dormant HIV-1 in vitro in chronically infected cells of T-lymphoid and monocytoid origin. The effect of NaB on HIV-1 expression in T-lymphoid cells was apparent 3 h after addition of drug and peaked at 24 h. During this time the proportion of HIV-1 antigen expressing cells increased from less than 0.5 to greater than 90%, and virus production increased by three orders of magnitude. The virus released by the NaB-induced cells was infectious. The extent and kinetics of NaB effects were similar to effects of phorbol 12-myristate 13-acetate in T cells, but not monocytes. Transient expression assays using an indicator gene under the control of the HIV-1 long terminal repeat revealed that mutations which altered the nucleotide sequence in the TATA box significantly reduced the NaB effect. These data show that NaB is a potent inducer of dormant HIV-1 and suggest that the TATA motif is required for this activity.

HIV-1 infection and expression in human colonic cells: infection and expression in CD4+ and CD4- cell lines.

Three human colonic epithelial cell lines, SW620, HT29, and T84, were characterized with respect to HIV-1 infection and gene expression. SW620 and HT29, but not T84, could be infected with HIV-1. CD4 messenger RNA and its protein product were identified in SW620 cells but not in HT29 or T84 cells. Anti-CD4 antibody blocked infection of SW620 cells but had no effect on infection of HT29 cells. In SW620 and HT29 cells transfected with the HIV-1 long terminal repeat (LTR) linked to the chloramphenicol acetyl transferase (CAT) reporter gene, an intact HIV-1 enhancer element was required for stimulation of CAT activity by tumor necrosis factor alpha (TNF alpha) and phorbol ester. T84 was not able to mediate a TNF alpha or phorbol ester response. These studies provide further evidence that HIV-1 can infect cells by mechanisms other than those mediated by the CD4 receptor and describe complementary models for analyzing HIV-1 infection and expression in colonic epithelial cells.

Stimulation of a human T-cell clone with anti-CD3 or tumor necrosis factor induces NF-kappa B translocation but not human immunodeficiency virus 1 enhancer-dependent transcription.

The expression of transiently transfected expression vectors under the control of the long terminal repeat (LTR) of the human immunodeficiency virus (HIV) or its enhancer sequence and the translocation of the HIV enhancer-binding protein NF-kappa B were analyzed in two human T-cell clones stimulated through their T-cell receptor complex or by tumor necrosis factor or phorbol 12-myristate 13-acetate. We found a dissociation of NF-kappa B translocation from transactivation of either the HIV LTR or the HIV enhancer. Interleukin 2 induced proliferation but not NF-kappa B translocation or LTR transactivation. Phorbol ester or specific antigen recognition induced HIV LTR transactivation, whereas stimulation with tumor necrosis factor or antibody to CD3 did not. The two latter signals were nevertheless able to induce NF-kappa B translocation with a pattern in the band-shift assay indistinguishable from that observed using phorbol ester. Our finding that induction of NF-kappa B by tumor necrosis factor or antibody to CD3 is not sufficient to induce HIV enhancer-dependent transcription in cloned T cells contrasts with results obtained in most lymphoblastoid T-cell lines and indicates that normal T lymphocytes differ from tumoral T cells in terms of requirements for HIV LTR activation. Furthermore, our results suggest that events linked to T-cell activation, in addition to NF-kappa B translocation per se, induce functional interactions of the NF-kappa B complex with the HIV enhancer.