Development of a dual reporter screening assay for distinguishing the inhibition of HIV Tat-mediated transcription from off-target effects.

Human immunodeficiency virus (HIV) encodes a transcription trans-activator (Tat) with an essential role in the transcriptional elongation of viral RNA based on the viral promoter long terminal repeat (LTR). Tat-mediated transcription is conserved and can be distinguished from host transcription, so it is a therapeutic target for combating HIV replication. Traditional screening assays for Tat-mediated transcriptional inhibitors are based on the biochemical properties of Tat and transactivation-responsive RNA. We developed an inducible system based on two lentiviral expression cassettes for doxycycline (Dox)-inducible Tat and Renilla luciferase (R-Luc) using TZM-bl cells harboring LTR-driven firefly luciferase (F-Luc). The cells simultaneously expressed both Tat-induced F-Luc and R-Luc, so it was possible to recognize off-target effects in the presence of Dox. The system was validated with known inhibitors: CYC202 obtained high sensitivity and specificity, whereas 6Bio and DRB had off-target effects. The MTT-based cytotoxicity test indicated the resistance of the system even at concentrations with off-target effects. The specificity of the system was confirmed using antiretroviral drugs. Our dual reporter system can simply detect Tat inhibitory effects, as well as precisely discriminate between the inhibitory and off-target effects of inhibitors, and may be useful for the development of a therapeutic anti-HIV drug.

Probing interaction of a fluorescent ligand with HIV TAR RNA.

Trans-activator of Transcription (Tat) antagonists could block the interaction between Tat protein and its target, trans-activation responsive region (TAR) RNA, to inhibit Tat function and prevent human immunodeficiency virus type 1 (HIV-1) replication. For the first time, a small fluorescence ligand, ICR 191, was found to interact with TAR RNA at the Tat binding site and compete with Tat. It was also observed that the fluorescence of ICR 191 could be quenched when binding to TAR RNA and recovered when discharged via competition with Tat peptide or a well-known Tat inhibitor, neomycin B. The binding parameters of ICR 191 to TAR RNA were determined through theoretical calculations. Mass spectrometry, circular dichroism and molecular docking were used to further confirm the interaction of ICR 191 with TAR RNA. Inspired by these discoveries, a primary fluorescence model for the discovery of Tat antagonists was built using ICR 191 as a fluorescence indicator and the feasibility of this model was evaluated. This ligand-RNA interaction could provide a new strategy for research aimed at discovering Tat antagonists.

Extracts from Acacia catechu suppress HIV-1 replication by inhibiting the activities of the viral protease and Tat.

BACKGROUND: Acacia catechu (Mimosa family) stem bark extracts have been used traditionally as a dietary supplement as well as a folk medicine given its reported anti-inflammatory, immunomodulatory, hepatoprotective, antioxidant, anti-microbial and anti-tumor activities. The present study was undertaken to evaluate the anti-HIV-1 activity of the extracts from stem bark of A. catechu. METHODS: The aqueous and 50% ethanolic extracts of A. catechu stem bark were prepared and 50% ethanolic extract was further fractioned by successively partitioning with petroleum ether, chloroform and n-butanol. All the extracts and fractions were evaluated for cytotoxicity and anti-HIV-1 activity using different in vitro assays. The active n-butanol fraction was evaluated for its inhibition against HIV-1 reverse transcriptase, integrase, protease, pro-viral genome integration and viral Tat protein mediated transactivation. The effect of n-butanol fraction on the induction of pro-inflammatory cytokines secretion in Vk2/E6E7 cells and transepithelial resistance in Caco-2 and HEC-1A cells was investigated. RESULTS: The aqueous and 50% ethanolic extracts of A. catechu showed IC50 values of 1.8 ± 0.18 µg/ml and 3.6 ± 0.31 µg/ml, respectively in cell-free virus based assay using TZM-bl cells and HIV-1NL4.3 (X-4 tropic). In the above assay, n-butanol fraction exhibited anti-HIV-1 activity with an IC50 of 1.7 ± 0.12 µg/ml. The n-butanol fraction showed a dose-dependent inhibition against HIV-1NL4.3 infection of the peripheral blood lymphocytes and against HIV-1BaL(R-5-tropic) as well as two different primary viral isolates of HIV-1 infection of TZM-bl cells. The n-butanol fraction demonstrates a potent inhibitory activity against the viral protease (IC50 = 12.9 µg/ml), but not reverse transcriptase or integrase. Further, in Alu-PCR no effect on viral integration was observed. The n-butanol fraction interfered with the Tat-mediated Long Terminal Repeat transactivation in TZM-bl cells, mRNA quantitation (qRT-PCR) and electrophoretic mobility shift assay (EMSA). The n-butanol fraction did not cause an enhanced secretion of pro-inflammatory cytokines in Vk2/E6E7 cells. Additionally, no adverse effects were observed to the monolayer formed by the Caco-2 and HEC-1A epithelial cells. CONCLUSIONS: The results presented here show a potential anti-HIV-1 activity of A. catechu mediated by the inhibition of the functions of the viral protein and Tat.

EGCG inhibits Tat-induced LTR transactivation: role of Nrf2, AKT, AMPK signaling pathway.

AIMS: Transcription is a crucial step for human immunodeficiency virus 1 (HIV-1) gene expression in infected host cells. The HIV-1 Tat activates the nuclear factor-kappa B (NF-κB) signaling transduction pathway, which is necessary for viral replication. Epigallocatechin-3-gallate (EGCG) has antioxidant, anti-inflammatory, and anti-viral properties. In this study, we investigated the effects of EGCG on Tat-induced HIV-1 transactivation and potential mechanisms by which EGCG inhibited activation of NF-κB pathway. MAIN METHODS: HeLa-CD4-long terminal repeat (LTR)-ß-gal (MAGI) cells were transfected with Tat plasmid. Tat-induced HIV-1 LTR transactivation was determined by MAGI cell assay. The reactive oxygen species (ROS) levels and glutathione (GSH) levels were measured. In addition, the protein expressions were assayed by western blotting. KEY FINDINGS: Tat caused a significant decrease in the intracellular glutathione (GSH) levels, a mild increase in the expression of nuclear levels of NF-E2-related factor-2 (Nrf2), a significant increase in the levels of NF-κB (phosphorylation of p65 and IKK) and a significant increase in ROS production. EGCG supplementation significantly improved the changes associated with Tat-induced oxidative stress by increasing nuclear levels of Nrf2, decreasing levels of NF-κB and ROS production. EGCG reversed Tat-mediated AKT activation and AMPK inhibition in MAGI cells. EGCG inhibited Tat-induced LTR transactivation in a dose-dependent manner. SIGNIFICANCE: The results suggest that Nrf2 signaling pathway may be the primary target for prevention of Tat-induced HIV-1 transactivation by EGCG, and EGCG also reduce NF-κB activation by inhibiting AKT signaling pathway and activating AMPK signaling pathway.

In vitro anti-HIV-1 properties of ethnobotanically selected South African plants used in the treatment of sexually transmitted diseases.

ETHNOPHARMACOLOGICAL RELEVANCE: [corrected The plants selected in this study are used traditionally in the treatment of sexually transmitted diseases and traditional healers interviewed claimed these plants can also help AIDS patients. AIM: To evaluating the in vitro anti-HIV properties of selected plants in various bioassays. MATERIALS AND METHODS: The extracts were evaluated for their inhibition against alpha-glycohydrolase, reverse transcriptase and viral proteins (NF-kappaB and Tat) which play a significant role in the HIV life cycle. RESULTS: Terminalia sericea extract (IC(50)=92mg/ml) exhibited a considerable alpha-glucosidase inhibitory activity which was better than acarbose (IC(50)=131mg/ml) under our assay conditions. In the reverse transcriptase assay, T. sericea also showed good inhibitory activity (IC(50)=43mg/ml), which was higher than that of the reference drug, Adriamycin (IC(50)=100mg/ml). The ethyl acetate extract of Elaeodendron transvaalense exhibited the most potent inhibitory activity in both the NF-kappaB and Tat assays with inhibitory activity of 76% and 75% respectively at a concentration of 15mg/ml. The acetone and chloroform extracts of E. transvaalense and Zanthoxylum davyi also showed good activity in the NF-kappaB and Tat assays.

Creation of a bi-directional protein transduction system for suppression of HIV-1 expression by p27SJ.

p27SJ is a novel protein from a callus culture of St. John's wort that modulates transcription of the HIV-1 promoter in several mammalian cells [Darbinian-Sarkissian, N., Darbinyan, A., Otte, J., Radhakrishnan, S., Sawaya, B.E., Arzumanyan, A., Chipitsyna, G., Popov, Y., Rappaport, J., Amini, S., Khalili, K., 2006. p27(SJ), a novel protein from St. John's wort, that suppresses expression of HIV-1 genome. Gene Ther. 13, 288-295]. Here, we armed p27SJ with signals from Ig-kappa light chain that allow its efficient excretion from the cells, and from HIV-1 Tat that facilitates its uptake by other cells for its utilization by a protein transduction method. We demonstrate that treatment of cells containing the HIV-1 LTR with conditioned media from cells expressing the armed p27SJ ((exc)p27SJ(upt)) results in suppression of the viral activation by the C/EBPbeta transcription factor. Once imported into the cells, (exc)p27SJ(upt) impacts the nuclear localization of C/EBPbeta and by retaining the protein in the cytoplasm affects its DNA binding and hence transcriptional activity. The armed p27SJ also inhibits Tat-induced activation of the LTR and decreases the level of viral replication in promonocytic cells including U-937 and T-lymphocytic cells. Our observations introduce a new bi-directional protein transduction system with a broad spectrum of applications for manufacturing therapeutic peptides by a specific group of cells called donor, and delivery to the target cells named recipient. Furthermore, our results support the utility of soluble p27SJ in suppressing transcription and replication of HIV-1 by interfering with the function of cellular proteins such as C/EBPbeta and viral activators including Tat.

Protection against human immunodeficiency virus type 1 Tat neurotoxicity by Ginkgo biloba extract EGb 761 involving glial fibrillary acidic protein.

Human immunodeficiency virus (HIV)-1 Tat protein is an important pathogenic factor in HIV-associated neuropathogenesis. Despite recent progress, the molecular mechanisms underlying Tat neurotoxicity are still not completely understood. However, few therapeutics have been developed to specifically target HIV infection in the brain. Recent development of an inducible brain-specific Tat transgenic mouse model has made it possible to define the mechanisms of Tat neurotoxicity and evaluate anti-neuroAIDS therapeutic candidates in the context of a whole organism. Herein, we demonstrate that administration of EGb 761, a standardized formulation of Ginkgo biloba extract, markedly protected Tat transgenic mice from Tat-induced developmental retardation, inflammation, death, astrocytosis, and neuron loss. EGb 761 directly down-regulated glial fibrillary acidic protein (GFAP) expression at both protein and mRNA levels. This down-regulation was, at least in part, attributable to direct effects of EGb 761 on the interactions of the AP1 and NF-kappaB transcription factors with the GFAP promoter. Most strikingly, Tat-induced neuropathological phenotypes including macrophage/microglia activation, central nervous system infiltration of T lymphocytes, and oxidative stress were significantly alleviated in GFAP-null/Tat transgenic mice. Taken together, these results provide the first evidence to support the potential for clinical use of EGb 761 to treat HIV-associated neurological diseases. Moreover, these findings suggest for the first time that GFAP activation is directly involved in Tat neurotoxicity, supporting the notion that astrocyte activation or astrocytosis may directly contribute to HIV-associated neurological disorders.