Inhibition of HIV-1 multiplication by a modified U7 snRNA inducing Tat and Rev exon skipping.

The HIV-1 regulatory proteins Tat and Rev are encoded by multiply spliced mRNAs that differ by the use of alternative 3' splice sites at the beginning of the internal exon. If these internal exons are skipped, the expression of these genes, and hence HIV-1 multiplication, should be inhibited. We have previously developed a strategy, based on antisense derivatives of U7 small nuclear RNA, that allows us to induce the skipping of an internal exon in virtually any gene. Here, we have successfully applied this approach to induce a partial skipping of the Tat, Rev (and Nef) internal exons. Three functional U7 constructs were subcloned into a lentiviral vector. Two of them strongly reduced the efficiency of lentiviral particle production compared to vectors carrying either no U7 insert or unrelated U7 cassettes. This defect could be partly or fully compensated by coexpressing Rev from an unspliced mRNA in the producing cell line. Upon stable transduction into CEM-SS or CEM T-lymphocytes, the most efficient of these constructs inhibits HIV-1 multiplication. Although the inhibition is not complete, it is more efficient in combination with another mechanism inhibiting HIV multiplication. Therefore, this new approach targeting HIV-1 regulatory genes at the level of pre-mRNA splicing, in combination with other antiviral strategies, may be a useful new tool in the fight against HIV/AIDS.

Transcriptional potencies of inhaled glucocorticoids.

Glucocorticoids (GC) are the most effective anti-inflammatory drugs used in asthma. By a process called trans-activation, they increase the transcription of genes involved in either beneficial processes or certain side effects. Through trans-repression, they inhibit the transcription factors nuclear factor kappa B (NF-kappaB) and activator protein-1 (AP-1), thereby decreasing the expression of many genes encoding inflammatory mediators such as the cytokine RANTES. We have measured the trans-activation and trans-repression potencies of the five currently available inhaled GC using reporter gene assays. The rank order of trans-activation potencies in HeLa cells stably transfected with a GC-inducible luciferase gene was fluticasone propionate > budesonide and triamcinolone acetonide > beclomethasone dipropionate and flunisolide. For all GC except beclomethasone dipropionate, there was a highly significant correlation between their potency to trans-activate in HeLa cells and their capacity to induce the gluconeogenic enzyme tyrosine aminotransferase in hepatoma tissue culture (HTC) cells. The rank order of trans-repression potencies in A549 lung cells transiently transfected with an AP-1- or NF-kappaB-dependent luciferase gene was fluticasone propionate > budesonide > beclomethasone dipropionate, triamcinolone acetonide, and flunisolide. The same rank order was found for inhibition of RANTES release. Thus, determination of trans-repression and trans-activation potencies of GC may help to predict their capacity to produce anti-inflammatory and side effects, respectively.

A second target for the peptoid Tat/transactivation response element inhibitor CGP64222: inhibition of human immunodeficiency virus replication by blocking CXC-chemokine receptor 4-mediated virus entry.

The peptoid CGP64222 has been previously demonstrated to inhibit the human immunodeficiency virus (HIV) Tat/transactivation response element complex formation. It has previously been shown that CGP64222 selectively inhibits HIV-1 long terminal repeat-driven gene expression and HIV-1(LAV) replication in lymphocytes. Here, we show that CGP64222 inhibits the replication of a wide range of laboratory strains of HIV-1 and HIV-2 in MT-4 cells. However, CGP64222 proved inactive in MT-4 cells against HIV-1 strains that are resistant to the bicyclams. The bicyclams are known to specifically interact with CXC-chemokine receptor 4, the main coreceptor used by T-tropic HIV strains to enter the cells. Mechanism of action studies revealed that CGP64222 can inhibit the HIV replicative cycle, also through a selective interaction with the CXC-chemokine receptor 4 coreceptor.

Synthesis and anti-HIV activity of 10,11-dihydropyrrolo [1,2-b][1,2,5]benzothiadiazepine-11-acetic acid 5,5-dioxide derivatives and related compounds.

The synthesis and the in vitro anti-HIV-1 activity of novel pyrrolo annulated benzothiadiazepine acetic acids and some related derivatives are reported. The new compounds share chemical features with pyrrolo[1,2-d][1,4]benzodiazepin-6-one 1 and Ro 5-3335 pyrrylbenzodiazepinone 4, two inhibitors of HIV-replication at the level of reverse transcriptase (RT) and transcriptional transactivation by Tat, respectively. Two derivatives, namely methyl 10,11-dihydropyrrolo[1,2-b][1,2,5]benzothiadiazepine-11-acetic-5,5 -dioxide (5a) and 1,12b-dihydro-2H-azeto[2,1-d]pyrrolo[1,2-b][1,2,5]benzoth iadiazepin-2-one 8,8-dioxide (7a), were found to exhibit a significant, although not very potent, activity against human immunodeficiency virus Type 1 (HIV-1).

IL-7 up-regulates HIV-1 replication in naturally infected peripheral blood mononuclear cells.

We have investigated the effect of exogenous IL-7 on replication of HIV-1 in PBMCs isolated from asymptomatic, chronically infected donors. We show that IL-7 induced virus replication and increased proviral DNA levels in CD8- PBMC cultures. IL-7 also increased the levels of doubly spliced HIV-1 tat RNA in these cultures. In comparison, IL-2 induced lower levels of virus production than IL-7, but had a more pronounced effect on cell proliferation. The IL-7-mediated increase in virus replication was not inhibited by neutralizing mAbs to IL-1 beta, IL-2, IL-6, or TNF-alpha, and was only partially dependent on ligation of the T cell accessory molecule CD28. CD8+ cells inhibited the increase in viral replication following IL-7 stimulation, but did not prevent virus replication following ligation of CD3 in the presence of IL-7. The data shows that IL-7 regulates HIV-1 replication in naturally infected PBMCs.

CT-2576, an inhibitor of phospholipid signaling, suppresses constitutive and induced expression of human immunodeficiency virus.

Viruses such as human immunodeficiency virus (HIV) require cellular activation for expression. Cellular activation in lymphoid cells is associated with augmented accumulation of certain phosphatidic acid (PA) species derived from the hydrolysis of glycan phosphatidylinositol (GPI). This suggests that activation of a phospholipid pathway may play a role in initiation of viral replication. To test this hypothesis, we examined the effect of tat gene expression on the production of cellular PA species, as the Tat protein is essential for HIV expression and has been implicated in activating the expression of multiple host cellular genes. Expression of tat increased the expression of PA. We then tested whether synthetic inhibitors of PA metabolism would inhibit activation of the HIV long terminal repeat by Tat and tumor necrosis factor alpha (TNF-alpha). CT-2576 suppressed both PA generation induced by Tat and HIV long terminal repeat-directed gene expression in response to Tat or TNF-alpha at a posttranscriptional step. CT-2576 also inhibited constitutive as well as TNF-alpha- and interleukin 6-induced expression of HIV p24 antigen in chronically infected U1 cells and in peripheral blood lymphocytes acutely infected with a clinical isolate of HIV. Pharmacological inhibition of synthesis of selected PA species may therefore provide a therapeutic approach to suppression of HIV replication.

Regulation of bone sialoprotein gene transcription by steroid hormones.

During the initial formation of bone, dentine and cementum in tooth morphogenesis, fully differentiated osteoblasts, odontoblasts and cementoblasts express bone sialoprotein (BSP), a mineralized tissue-specific acidic glycoprotein that has been implicated in the nucleation of hydroxyapatite crystal growth. The expression of BSP is regulated by steroid hormones that modulate mineralized tissue formation. Thus, the transcription of the BSP gene is induced by glucocorticoids in association with osteoblast differentiation and glucocorticoids also stimulate the expression of BSP in differentiated osteoblasts. In contrast, however, vitamin D3 suppresses bone formation and abrogates the expression of BSP. Our studies, using the osteoblastic cell lines ROS 17/2.8 and UMR 106-06, have revealed that the glucocorticoid (10(-8) M dexamethasone; dex) effect on BSP mRNA involves both direct and indirect pathways. To determine the molecular basis of the direct pathway on transcriptional regulation of the BSP we have isolated and characterized the promoter regions of both the human and rat BSP genes. The promoters are characterized by a highly conserved region (BSP box) encompassing the immediate promoter region, which includes a unique inverted TATA box overlapped by a putative (DR3) vitamin D3 response element (VDRE). Possible glucocorticoid response elements are present approximately 1 kb and approximately 1.4 kb further upstream. Transient transfection analysis of chimeric constructs linked to a luciferase reporter gene have shown Dex-stimulated expression in constructs that include one or both GREs, whereas vit D3 suppresses expression in a short construct that includes the VDRE.(ABSTRACT TRUNCATED AT 250 WORDS)

Complete inactivation of target mRNA by biotinylated antisense oligodeoxynucleotide-avidin conjugates.

Biotinylation of phosphodiester oligodeoxynucleotides (PO-ODN) allows for conjugation to avidin-based transcellular delivery systems. In addition, biotinylation of PO-ODN at the 3'-terminus provides complete protection against serum 3'-exonuclease degradation. The present study was undertaken to determine if antisense 3'-biotinylated PO-ODN-avidin constructs are able to recognize and inactivate the target mRNA through RNase H-mediated degradation. A 21-mer antisense PO-ODN complementary to the tat gene encompassing nucleotides 5402-5422 of the HIV-1 genome was synthesized with biotin conjugated to the 3'-terminus (bio-tat). Gel mobility assays using [5'-32P]-labeled bio-tat ODN and avidin showed that the bio-tat ODN was fully monobiotinylated. Aliquots of [32P]-labeled sense or antisense tat RNA (337 and 351 nucleotides, respectively) were prepared from transcription plasmids and were preincubated with an excess of bio-tat ODN with or without avidin constructs and digested with RNase H. Products were resolved with sequencing gel and analyzed by autoradiography. Complete conversion to predicted RNA fragments resulting from RNase H digestion of the RNA-ODN duplex (53 and 263 nucleotides) was observed when [32P]-tat sense RNA was incubated with antisense bio-tat ODN or conjugated to avidin or an avidin-cationized human serum albumin (cHSA) complex. Conversely, no degradation of [32P]-tat-antisense RNA was observed after incubation with antisense bio-tat ODN and RNase H. In addition, the avidin-cHSA complex significantly increased (84-fold) the uptake of [32P]-internally labeled bio-tat ODN and its stability against cellular nuclease degradation in peripheral blood lymphocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

A novel transgenic mouse model for the in vivo evaluation of anti-human immunodeficiency virus type 1 drugs.

We have developed a binary transgenic mouse system that allows easy in vivo evaluation of new anti-human immunodeficiency virus type 1 (HIV-1) drugs or therapies specifically designed to target the viral transactivator protein (TAT) or long terminal repeat (LTR) functions. This approach consists of a simple genetic cross between an "activator" transgenic mouse expressing the HIV-1-tat gene exclusively to T lymphocytes and a "target" transgenic mouse bearing a silent reporter gene whose expression is under the control of the HIV-1-LTR. As expected, most of the target transgenic animals did not express the reporter gene; on the contrary, all the double-transgenic mice bearing both the activator and target transgenes strongly expressed the TAT-induced reporter gene. The choice of a secreted human alpha 1-antitrypsin variant (alpha 1-AT) as reporter gene readily permits in a single animal the quantitative determination of the plasma level of alpha 1-AT protein before and after anti-LTR or anti-TAT treatments. Such mice may be valuable as new laboratory models for the in vivo evaluation of agents with potential anti-HIV-1 activity.

Discovery and characterization of an HIV-1 Tat antagonist.

Ro 5-3335, 7-chloro-5-(2-pyrryl)-3H-1,4-benzo-diazepin-2-(H)-one, has been shown to inhibit gene expression controlled by the human immunodeficiency virus-1 (HIV-1) LTR promoter. The inhibition was specific for the viral transcriptional transactivator Tat. The compound did not inhibit the basal activity of the HIV-1 LTR or the activity of promoters not responsive to Tat. Consistent with its mode of action, Ro 5-3335 inhibited HIV-1 replication (IC50 = 0.1-1 microM) by reducing viral RNA synthesis in acutely, as well as chronically, infected cells in vitro. The compound was active against HIV-1 and HIV-2, and AZT-resistant clinical isolates.

Inhibition of human immunodeficiency virus-1 proliferation by liposome-encapsulated sense DNA to the 5' tat splice acceptor site.

A liposome formulation containing a distearoylphosphatidylethanolamine analog was developed that was endocytosed by both lymphocytes and monocytes. This formulation was used to encapsulate sense and antisense 20-mer oligodeoxynucleotides to the 5' tat splice acceptor site of human immunodeficiency virus type 1. At a DNA concentration of 140 nM, the liposome-encapsulated sense DNA inhibited p24 production by as much as 84% in human peripheral blood leukocytes infected with "wild-type" virus. This treatment also reduced the number of peripheral blood leukocytes producing intracellular viral antigen by 71%. Of interest, no reduction in either parameter was observed for the antisense-containing liposomes. The results demonstrate the promise of a new liposomal delivery vehicle to inhibit human immunodeficiency virus replication by an entrapped oligodeoxynucleotide.