The HIV-1 Tat protein is secreted by infected cells. Extracellular Tat can affect bystander uninfected T cells and induce numerous biological responses such as apoptosis and cytokine secretion. Tat is likely involved in several immune disorders during AIDS. Nevertheless, it is not known whether Tat triggers cell responses directly upon binding to signaling receptors at the plasma membrane or after delivery to the cytosol. The pathway that enables Tat to reach the cytosol is also unclear. Here we visualized Tat within T-cell-coated pits and endosomes. Moreover, inhibitors of clathrin/AP-2-mediated uptake such as chlorpromazine, activated RhoA, or dominant-negative mutants of Eps15, intersectin, dynamin, or rab5 impaired Tat delivery to the cytosol by preventing its endocytosis. Molecules neutralizing low endosomal pH or Hsp90 inhibitors abolished Tat entry at a later stage by blocking its endosomal translocation, as directly shown using a cell-free translocation assay. Finally, endosomal pH neutralization prevented Tat from inducing T-cell responses such as NF-B activation, apoptosis, and interleukin secretion, indicating that cytosolic delivery is required for Tat signaling. Hence, Tat enters T cells essentially like diphtheria toxin, using clathrin-mediated endocytosis before low-pH-induced and Hsp90-assisted endosomal translocation. Cell responses are then induced from the cytosol. INTRODUCTIONTat is a strong trans-activator that enables productive transcription from the HIV-1 long terminal repeat (LTR) and is required for HIV-1 replication (Rubartelli et al., 1998;Watson and Edwards, 1999;Noonan and Albini, 2000). Albeit devoid of signal sequence, it is released by infected cells and nanomolar Tat concentrations were measured in the sera of HIV-1-infected patients (Xiao et al., 2000). Exogenous Tat can affect monocytes, endothelial cells and neurons, but one of its main targets is the T cell (Rubartelli et al., 1998;Watson and Edwards, 1999;Noonan and Albini, 2000). Indeed, Tat induces IL-2 and IL-8 hypersecretion by T cells (Ott et al., 1997(Ott et al., , 1998 and can also trigger their apoptosis (Li et al., 1995;Chen et al., 2002). Circulating Tat is thus thought to be involved in AIDS development (Rubartelli et al., 1998;Watson and Edwards, 1999). Consistently, evaluations of Tat-containing vaccines have yielded encouraging results (Voss et al., 2003).Tat has the capacity to enter the cytosol from the outside medium, like several bacterial toxins such as diphtheria and cholera toxin catalytic subunits (Falnes and Sandvig, 2000). This property was demonstrated in pioneer studies by showing that extracellular Tat could trans-activate reporter genes placed under the control of HIV-1 LTR Mann and Frankel, 1991). This finding was later confirmed using several Tat fusion proteins and different readouts for monitoring Tat cytosolic delivery (Fawell et al., 1994). Nevertheless, contrary to bacterial toxins, the overall pathway enabling extracellular Tat to access the cytosol remains elusive, although endocytosis s...

Human immunodeficiency virus type 1 (HIV‐1) transcription relies on its transactivating Tat protein. Although devoid of a signal sequence, Tat is released by infected cells and secreted Tat can affect uninfected cells, thereby contributing to HIV‐1 pathogenesis. The mechanism and the efficiency of Tat export remained to be documented. Here, we show that, in HIV‐1‐infected primary CD4+ T‐cells that are the main targets of the virus, Tat accumulates at the plasma membrane because of its specific binding to phosphatidylinositol‐4,5‐bisphosphate (PI(4,5)P2). This interaction is driven by a specific motif of the Tat basic domain that recognizes a single PI(4,5)P2 molecule and is stabilized by membrane insertion of Tat tryptophan side chain. This original recognition mechanism enables binding to membrane‐embedded PI(4,5)P2 only, but with an unusually high affinity that allows Tat to perturb the PI(4,5)P2‐mediated recruitment of cellular proteins. Tat–PI(4,5)P2 interaction is strictly required for Tat secretion, a process that is very efficient, as ∼2/3 of Tat are exported by HIV‐1‐infected cells during their lifespan. The function of extracellular Tat in HIV‐1 infection might thus be more significant than earlier thought.

Trioxaquines are dual molecules that contain a trioxane motif linked to an aminoquinoline entity. Among the different compounds of this series, trioxaquine cis-15 (DU1302 c), prepared from alpha-terpinene, a cheap natural product, showed efficient antimalarial activity in vitro on both sensitive and resistant strains of Plasmodium falciparum (IC(50)=5-19 nM). A stereochemical description of this stable, nontoxic, and non-genotoxic antimalarial agent is detailed. Mice infected with P. vinckei were successfully treated with cis-15 in a four-day suppressive test. The doses required to decrease parasitemia by 50 % (ED(50)) were 5 and 18 mg kg(-1) d(-1) after intraperitoneal and oral administration, respectively. Parasitemia clearance was complete without recrudescence at an intraperitoneal dose of 20 mg kg(-1) d(-1).

We previously showed that quaternary ammonium salts have potent antimalarial activities against the blood stage of drug-resistant Plasmodium falciparum. In the present study, 13 compounds of this series were comparatively assessed in murine in vivo malarial models. Mice infected with Plasmodium berghei were successfully treated with 11 quaternary ammonium salts in a 4-day suppressive test with a once-daily intraperitoneal administration. The dose required to decrease parasitemia by 50% (ED 50 ) ranged from 0.04 to 4.5 mg/kg of body weight. For six mono-and three bis-quaternary ammonium salts, the therapeutic indices (i.e., 50% lethal dose and ED 50 ) were higher than 5, and at best, around 20 to 30 for five of them (E6, E8, F4, G5, and G25), which is comparable to that of chloroquine under the same conditions. Plasmodium chabaudi was significantly more susceptible to G5, G15, and G25 compounds than P. berghei. Similar therapeutic indices were obtained, regardless of the administration mode or initial parasitemia (up to 11.2%). Parasitemia clearance was complete without recrudescence. Subcutaneously administered radioactive compounds had a short elimination half-life in mice (3.5 h) with low bioavailability (17.3%), which was likely due to the permanent cationic charge of the molecule. The high in vivo therapeutic index in the P. chabaudi-infected mouse model and the absence of recrudescence highlight the enormous potential of these quaternary ammonium salts for clinical malarial treatment.In the absence of vaccines, and due to the widespread resistance to antimalarials in current use, new chemotherapies are urgently needed to help in the prevention and control of malaria. The most promising strategy is to strive to discover new chemically diverse entities directed towards novel biological targets. Potential chemotherapeutic targets in the malaria parasite can be broadly classified into three categories: those involved in processes occurring in the digestive vacuole, enzymes involved in macromolecular and metabolite synthesis, and those responsible for membrane processes and signaling. The potential of putative targets to be validated will be tapped to develop effective and safe drugs (14,17,29).We previously characterized phospholipid (PL) metabolism as an attractive target for new malaria chemotherapy due to its vital importance to the parasite. PL metabolism is absent from normal mature human erythrocytes (19), but the erythrocyte PL content increases by as much as 500% after infection, specifically due to the metabolic machinery of the parasite (10, 18). Phosphatidylcholine is the major PL of infected erythrocytes, representing about 45% of the total PL. In this pathway, choline transport which regulates the supply of polar head precursors to the parasite is a regulatory rate-limiting step (3,22,23). PL polar head analogs are able to interfere with PL biosynthesis and exhibit antimalarial activity against the intraerythrocytic stage of Plasmodium falciparum in vitro (24). More particularly, 36 compounds which...

The human immunodeficiency virus, type 1, transactivating protein Tat is a small protein that is strictly required for viral transcription and multiplication within infected cells. The infected cells actively secrete Tat using an unconventional secretion pathway. Extracellular Tat can affect different cell types and induce severe cell dysfunctions ranging from cell activation to cell death. To elicit most cell responses, Tat needs to reach the cell cytosol. To this end, Tat is endocytosed, and low endosomal pH will then trigger Tat translocation to the cytosol. Although this translocation step is critical for Tat cytosolic delivery, how Tat could interact with the endosome membrane is unknown, and the key residues involved in this interaction require identification. We found that, upon acidification below pH 6.0 (i.e. within the endosomal pH range), Tat inserts into model membranes such as monolayers or lipid vesicles. This insertion process relies on Tat single Trp, Trp-11, which is not needed for transactivation and could be replaced by another aromatic residue for membrane insertion. Nevertheless, Trp-11 is strictly required for translocation. Tat conformational changes induced by low pH involve a sensor made of its first acidic residue (Glu/Asp-2) and the end of its basic domain (residues 55-57). Mutation of one of these elements results in membrane insertion above pH 6.5. Tat basic domain is also required for efficient Tat endocytosis and membrane insertion. Together with the strict conservation of Tat Trp among different virus isolates, our results point to an important role for Tat-membrane interaction in the multiplication of human immunodeficiency virus type 1.

The Tat protein is required for efficient HIV-1 (human immunodeficiency virus type 1) transcription. Moreover, Tat is secreted by infected cells, and circulating Tat can affect several cell types, thereby contributing to HIV-1 pathogenesis. We monitored Tat secretion by transfected CD4+ T-cells. A Tat chimaera carrying an N-glycosylation site did not become glycosylated when expressed in cells, while the chimaera was glycosylated when mechanically introduced into purified microsomes. These data indicate that secreted Tat does not transit through the endoplasmic reticulum. The use of pharmacological inhibitors indicated that the Tat secretion pathway is unusual compared with previously identified unconventional secretion routes and does not involve intracellular organelles. Moreover, cell incubation at 16 degrees C inhibited Tat secretion and caused its accumulation at the plasma membrane, suggesting that secretion takes place at this level.

The aim of our study was to investigate the impact of macroautophagy on exosome secretion. Exosomes are small membrane vesicles released in the extracellular space upon fusion of multivesicular endosomes with the plasma membrane. They were initially discovered as a way to remodel the reticulocyte plasma membrane before entering the blood circulation (Current Opinion in Hematology 2010, 17:177-183) and are now essentially studied as mediators of intercellular communication. Using iTRAQ proteomics, we compared the protein composition of purified exosomes secreted by cells impaired or not for macroautophagy by Atg5 depletion, during serum starvation conditions or complete medium culture. We show that the absence of serum modifies exosomal content, especially inducing secretion of two cytoplasmic protein complexes, namely proteasomal 19S regulatory particle (RP) and components of noncanonical translation preinitiation complex (PIC). This process is enhanced when autophagy is impaired by Atg5 depletion. Moreover, we show that the proteasome 20S core particle (CP) is released in the extracellular space. However, in striking contrast to what seen for its 19S RP regulator, release is independent of the exosomal vesicles, Atg5 expression and cell culture conditions. Exosome secretion can thus be considered as a cell process that participates in and reflects cell homeostasis, and care must be taken when studying potential extracellular function of exosomes due to the possible copurification of proteasome 20S CP. K E Y W O R D S endosomal microautophagy, exosomes, proteasome, translation preinitiation complex

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