Accumulation of transcripts coding for prion protein in human astrocytes during infection with human immunodeficiency virus.

The abnormal isoforms of the normal cellular prion protein (PrP), also termed Scrapie-associated fibril protein, are assumed to be one causative factor of spongiform encephalopathies. The mRNA of PrP contains stem-loop structures which are very similar to the human immunodeficiency virus-1 (HIV-1) cis-acting sequence TAR within the LTR; both structures contain the pentanucleotide CUGGG in the loop, and the uridine- and adenine-bulge in the stem. In this study, using purified HIV-encoded trans-activator, Tat, and HIV-1 TAR-RNA or PrP-mRNA containing the stem-loop structure, we demonstrate by use of gel-retardation and filter binding assays that Tat binds to TAR- and PrP-RNA with the dissociation constants of 2.9 or 37.0 nM, respectively, at a molar ratio of 0.7 mol of Tat to 1 mol of RNA fragment. The Tat-RNA (TAR or PrP) complexes bind to protein(s) in the nuclear matrix, isolated from human astrocytes (glial fibrillary acidic protein positive brain cells). Infection of astrocytes with HIV-1 resulted in an increased level of PrP mRNA. The data presented led us to assume that certain sequences in the PrP mRNA might be targets for proteins acting in trans.     

HIV-1 Tat protein-mediated transactivation of the HIV-1 long terminal repeat promoter is potentiated by a novel nuclear Tat-interacting protein of 110 kDa, Tip110

Human immunodeficiency virus type 1 (HIV-1) gene expression and replication is highly dependent on and modulated by interactions between viral and host cellular factors. Tat protein, encoded by one of the HIV-1 regulatory genes, tat, is essential for HIV-1 gene expression. A number of host cellular factors have been shown to interact with Tat in this process. During our attempts to determine the molecular mechanisms of Tat interaction with brain cells, we isolated a cDNA clone that encodes a novel Tat-interacting protein of 110 kDa or Tip110 from a human fetal brain cDNA library. GenBank BLAST search revealed that Tip110 was almost identical to a previously cloned KIAA0156 gene with unknown functions. In vivo binding of Tip110 with Tat was confirmed by immunoprecipitation and Western blotting, in combination with mutagenesis. The yeast three-hybrid RNA-protein interaction assay indicated no direct interaction of Tip110 with Tat transactivating response element RNA. Nevertheless, Tip110 strongly synergized with Tat on Tat-mediated chloramphenicol acetyltransferase reporter gene expression and HIV-1 virus production, whereas down-modulation of constitutive Tip110 expression inhibited HIV-1 virus production. Northern blot analysis showed that Tip110 mRNA was expressed in a variety of human tissues and cells. Moreover, digital fluorescence microscopic imaging revealed that Tip110 was expressed exclusively in the nucleus, and within a nuclear speckle structure that has recently been described for human cyclin T and CDK9, two critical components for Tat transactivation function on HIV-1 long terminal repeat promoter. Taken together, these data demonstrate that Tip110 regulates Tat transactivation activity through direct interaction, and suggest that Tip110 is an important cellular factor for HIV-1 gene expression and viral replication.     

The endocannabinoid system protects rat glioma cells against HIV-1 Tat protein-induced cytotoxicity. Mechanism and regulation

Cannabinoids modulate nitric oxide (NO) levels in cells of the central nervous system. Here we studied the effect of cannabinoid CB(1) and CB(2) receptor agonists on the release of NO and cell toxicity induced by the human immuno-deficiency virus-1 Tat protein (HIV-1 Tat) in rat glioma C6 cells. The CB(1) and CB(2) agonist WIN 55,212-2 inhibited the expression of inducible NO synthase (iNOS) and NO release caused by treatment of C6 cells with HIV-1 Tat and interferon-gamma (IFN-gamma). The effect of WIN 55,212-2 was uniquely due to CB(1) receptors, as shown by experiments carried out with selective CB(1) and CB(2) receptor agonists and antagonists. CB(1) receptor stimulation also inhibited HIV-1 Tat + IFN-gamma-induced and NO-mediated cell toxicity. Moreover, cell treatment with HIV-1 Tat + IFN-gamma induced a significant inhibition of CB(1), but not CB(2), receptor expression. This effect was mimicked by the NO donor GSNO, suggesting that the inhibition of CB(1) expression was due to HIV-1 Tat + IFN-gamma-induced NO overexpression. HIV-1 Tat + IFN-gamma treatment also induced a significant inhibition of the uptake of the endocannabinoid anandamide by C6 cells with no effect on anandamide hydrolysis. These findings show that the endocannabinoid system, through the modulation of the l-arginine/NO pathway, reduces HIV-1 Tat-induced cytotoxicity, and is itself regulated by HIV-1 Tat.     

A nuclear translational block imposed by the HIV-1 U3 region is relieved by the Tat-TAR interaction

Replication of HIV-1 depends on the viral Tat protein, which functions via a target sequence, TAR, present in the proviral long terminal repeat (LTR) and at the 5' end of viral mRNAs. We have shown that Tat potentiates the expression of TAR-containing RNAs, but only when Tat and the TAR-containing RNA are present in the nucleus. We now show that a small change in the TAR loop abolishes nuclear potentiation by Tat. Furthermore, the HIV-1 U3 region induces expression incompetence in mRNA synthesized by this promoter. RNAs of identical structure are, however, translated efficiently when produced from the CMV-IE promoter. The Tat-TAR system appears, therefore, to rescue the expression potential of HIV-1 LTR-directed RNA.     

HIV-1 Tat protein-induced VCAM-1 expression in human pulmonary artery endothelial cells and its signaling

Expression of cell adhesion molecule in endothelial cells upon activation by human immunodeficiency virus (HIV) infection is associated with the development of atherosclerotic vasculopathy. We postulated that induction of vascular cell adhesion molecule-1 (VCAM-1) by HIV-1 Tat protein in endothelial cells might represent an early event that could culminate in inflammatory cell recruitment and vascular injury. We determined the role of HIV-1 Tat protein in VCAM-1 expression in human pulmonary artery endothelial cells (HPAEC). HIV-1 Tat protein treatment significantly increased cell-surface expression of VCAM-1 in HPAEC. Consistently, mRNA expression of VCAM-1 was also increased by HIV-1 Tat protein as measured by RT-PCR. HIV-1 Tat protein-induced VCAM-1 expression was abolished by the NF-kappaB inhibitor pyrrolidine dithiocarbamate (PDTC) and the p38 MAPK inhibitor SB-203580. Furthermore, HIV-1 Tat protein enhanced DNA binding activity of NF-kappaB, facilitated nuclear translocation of NF-kappaB subunit p65, and increased production of reactive oxygen species (ROS). Similarly to VCAM-1 expression, HIV-1 Tat protein-induced NF-kappaB activation and ROS generation were abrogated by PDTC and SB-203580. These data indicate that HIV-1 Tat protein is able to induce VCAM-1 expression in HPAEC, which may represent a pivotal early molecular event in HIV-induced vascular/pulmonary injury. These data also suggest that the molecular mechanism underlying the HIV-1 Tat protein-induced VCAM-1 expression may involve ROS generation, p38 MAPK activation, and NF-kappaB translocation, which are the characteristics of pulmonary endothelial cell activation.     

Cellular uptake of cationic gadolinium-DOTA peptide conjugates with and without N-terminal myristoylation

Cellular and nuclear uptake of dual labelled conjugates could be of great value for chemotherapy and cancer diagnostics. Therefore we designed conjugates in which gadolinium (Gd)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), a contrast agent for magnetic resonance imaging and fluorescein isothiocyanate (FITC), a fluorescence marker were coupled to membrane translocation sequences (MTS). The MTSs we employed were the third helix of the Antennapedia homeodomain, the HIV-1 Tat peptide and the N-myristoylated HIV-1 Tat peptide. We used confocal laser scanning microscopy, fluorescence activated cell sorting, magnetic resonance imaging (MRI) and viability tests to examine the cellular and nuclear uptake of these conjugates into U373 glioma cells, as well as their cytotoxic effects. We found that the Antennapedia conjugate was taken up by no more than 20% of the cells. The HIV-1 Tat conjugate showed even lower uptake into less than 3% of cells. Interestingly, N-myristoylation of the HIV-1 Tat conjugate drastically improved its cellular uptake. Up to 70% of cells showed cellular and nuclear uptake of the N-myristoylated HIV-1 Tat conjugate. Conjugate cytotoxicity appears to correlate with cellular uptake.     

Extracellular HIV-1 Tat induces human beta-defensin-2 production via NF-kappaB/AP-1 dependent pathways in human B cells

Defensins, a family of antimicrobial peptides, are one of the first lines of host defense. Human beta-defensins (hBD) such as hBD-2 and -3 have anti-HIV activity. Previous studies have shown that HIV-1 virion can induce the expression of hBD, although the exact components of HIV-1 virion that are responsible for hBD expression have not yet been elucidated. In this study, we examined the effect of HIV-1 Tat on the expression of hBD in B cells. Stimulation of B cells with HIV-1 Tat protein significantly increased the mRNA and protein levels of hBD-2. HIV-1 Tat also induced the activation of a reporter gene for hBD-2 in a dose-dependent manner in B cells. Pretreatment of B cells with a JNK inhibitor suppressed HIV-1 Tat-induced hBD-2 expression. Pretreatment of B cells with AP-1 inhibitors or NF-κB inhibitors led to a decrease in HIV-1 Tat-induced protein and mRNA expression of hBD-2. Taken together, our results indicate that HIV-1 Tat can up-regulate the expression of hBD-2 via JNK-NF-κB/AP-1-dependent pathways in human B cells.