Inhibition of Non Canonical HIV-1 Tat Secretion Through the Cellular Na+,K+-ATPase Blocks HIV-1 Infection.

Besides its essential role in the activation of HIV-1 gene expression, the viral Tat protein has the unusual property of trafficking in and out of cells. In contrast to Tat internalization, the mechanism involved in extracellular Tat release has so far remained elusive. Here we show that Tat secretion occurs through a Golgi-independent pathway requiring binding of Tat with three short, non-consecutive intracytoplasmic loops at the C-terminus of the cellular Na+,K+-ATPase pump alpha subunit. Ouabain, a pump inhibitor, blocked this interaction and prevented Tat secretion; virions produced in the presence of this drug were less infectious, consistent the capacity of virion-associated Tat to increase HIV-1 infectivity. Treatment of CD4+ T-cells with short peptides corresponding to the Tat-binding regions of the pump alpha subunit impaired extracellular Tat release and blocked HIV-1 replication. Thus, non canonical, extracellular Tat secretion is essential for viral infectivity.

Novel human-derived cell-penetrating peptides for specific subcellular delivery of therapeutic biomolecules.

Short peptide sequences that are able to transport molecules across the cell membrane have been developed as tools for intracellular delivery of therapeutic molecules. This work describes a novel family of cell-penetrating peptides named Vectocell peptides [also termed DPVs (Diatos peptide vectors)]. These peptides, originating from human heparin binding proteins and/or anti-DNA antibodies, once conjugated to a therapeutic molecule, can deliver the molecule to either the cytoplasm or the nucleus of mammalian cells. Vectocell peptides can drive intracellular delivery of molecules of varying molecular mass, including full-length active immunoglobulins, with efficiency often greater than that of the well-characterized cell-penetrating peptide Tat. The internalization of Vectocell peptides has been demonstrated to occur in both adherent and suspension cell lines as well as in primary cells through an energy-dependent endocytosis process, involving cell-membrane lipid rafts. This endocytosis occurs after binding of the cell-penetrating peptides to extracellular heparan sulphate proteoglycans, except for one particular peptide (DPV1047) that partially originates from an anti-DNA antibody and is internalized in a caveolar independent manner. These new therapeutic tools are currently being developed for intracellular delivery of a number of active molecules and their potentiality for in vivo transduction investigated.

Transcellular protein transduction using the Tat protein of HIV-1.

The Tat protein of HIV-1 is a powerful transactivator of gene expression. By interacting with a structured RNA sequence at the 5' end of the viral mRNA, it promotes the remodeling of chromatin and the recruitment of processive RNA polymerase complexes at the viral promoter. In addition to these transcriptional functions, a short amino acid motif, highly enriched in basic amino acids, promotes the export of the protein from the expressing cells. Once in the extracellular environment, the same basic domain of Tat binds to cell surface heparan sulfate proteoglycans; through this interaction, the protein is internalized by a variety of different cell types. Cellular internalization of Tat and Tat fusion proteins requires the integrity of cell membrane lipid rafts and mainly occurs through caveolar endocytosis. The Tat basic domain, when attached to large protein cargos, also mediates their efficient cellular internalization and can be thus utilized for transcellular protein transduction. This property has already been successfully exploited for the delivery of heterologous proteins, nanoparticles, liposomes, phage and viral vectors, and plasmid DNA. The biological significance of intercellular Tat trafficking in the context of viral infection still remains elusive.

Enhanced caveolae-mediated endocytosis by diagnostic ultrasound in vitro.

The modulation of cellular endothelial permeability is a desirable goal for targeted delivery of labels and therapeutic macromolecules; the underlying mechanisms, however, remains poorly understood. Here, we hypothesize that a higher endothelial permeability may result as an outcome of selective enhancement of caveolar endocytosis by ultrasound (US), in the frequency and intensity range of current clinical diagnostic use. To assess the role of free radicals in this phenomenon, we exposed confluent human endothelial cells to pulsed diagnostic US for 30 min, with a mechanical index (MI) of 0.5 and 1.2, using a 1.6-MHz cardiac US scan, and endothelial cells not exposed to US were used as control. Here we show that pulsed diagnostic US with a MI of 1.2 (high mechanical index ultrasound [HMIUS]) were able to selectively enhance endothelial caveolar internalization of recombinant glutathione-S-transferase (GST)-Tat11-EGFP fusion protein (26 +/- 1 vs. 11.6 +/- 1 A.U, p < 0.001 vs. control), without disruption of plasma membrane integrity. Moreover, pulsed diagnostic US with a MI of 0.5 (low mechanical index ultrasound) did not increase caveolar endocytosis compared with control (11.4 +/- 1.2 vs. 11.6 +/- 1). Free-radical generation inhibitors, such as catalase and superoxide dismutase, reduced the HMIUS-induced caveolar internalization by a 49.29% factor; finally, HMIUS-induced caveolar endocytosis was found to be associated with a significant increase in the phosphorylation of tyr-14-caveolin1, ser1177-eNOS and Thr202/Tyr204-ERK(1/2) compared with control. These findings show how HMIUS irradiation of human endothelial cells cause a selective enhancement of caveolar-dependent permeability, partially mediated by free radicals generation, inducing a marked increase of phosphorylation of caveolar-related proteins. Thus, the use of diagnostic US could potentially be used as an adjuvant to drive caveolar traffic of extracellular peptides by using a higher level of US energy.

Acetylation of HIV-1 integrase by p300 regulates viral integration.

Integration of HIV-1 into the human genome, which is catalyzed by the viral protein integrase (IN), preferentially occurs near transcriptionally active genes. Here we show that p300, a cellular acetyltransferase that regulates chromatin conformation through the acetylation of histones, also acetylates IN and controls its activity. We have found that p300 directly binds IN both in vitro and in the cells, as also specifically demonstrated by fluorescence resonance energy transfer technique analysis. This interaction results in the acetylation of three specific lysines (K264, K266, K273) in the carboxy-terminus of IN, a region that is required for DNA binding. Acetylation increases IN affinity to DNA, and promotes the DNA strand transfer activity of the protein. In the context of the viral replication cycle, point mutations in the IN acetylation sites abolish virus replication by specifically impairing its integration capacity. This is the first demonstration that HIV-1 IN activity is specifically regulated by post-translational modification.

Cell membrane lipid rafts mediate caveolar endocytosis of HIV-1 Tat fusion proteins.

The transactivator protein of human immunodeficiency virus type 1 Tat has the unique property of mediating the delivery of large protein cargoes into the cells when present in the extracellular milieu. Here we show that Tat fusion proteins are internalized by the cells through a temperature-dependent endocytic pathway that originates from cell membrane lipid rafts and follows caveolar endocytosis. These conclusions are supported by the study of the slow kinetics of the internalization of Tat endosomes, by their resistance to nonionic detergents, the colocalization of internalized Tat with markers of caveolar endocytosis, and the impairment of the internalization process by drugs that disrupt lipid rafts or disturb caveolar trafficking. These results are of interest for all those who exploit Tat as a vehicle for transcellular protein delivery.

Caveolae-mediated internalization of extracellular HIV-1 tat fusion proteins visualized in real time.

The Tat protein from HIV-1, when fused with heterologous proteins or peptides, can traverse cell membranes. This ability has generated great interest due to potential therapeutic applications. However, the relevant cellular pathway and its dynamics have not been elucidated yet. Here we unravel the intracellular fate of exogenously added Tat fused with green fluorescent protein (GFP) in live HeLa and CHO cells, from the early interaction with the plasma membrane up to the long-term accumulation in the perinuclear region. We demonstrate that the internalization process of full-length Tat and of heterologous proteins fused to the transduction domain of Tat exploits a caveolar-mediated pathway and is inhibited at 4 degrees C. Remarkably, a slow linear movement toward the nucleus of individual GFP-tagged Tat-filled caveolae with an average velocity of 3 micro m/h was observed. No fluorescence was observed in the nucleus, possibly suggesting that Tat fusion protein unfolding is required for nuclear translocation. In addition, early sensitivity to cytochalasin-D treatment indicates the essential role of the actin cytoskeleton in the displacement of Tat vesicles toward the nucleus. Our results imply that HIV-1 Tat mediates the internalization of protein cargos in a slow and temperature-dependent manner by exploiting the caveolar pathway.