Bacterial Shiga-like toxins are virulence factors that constitute a significant public health threat worldwide, and the plant toxin ricin is a potential bioterror weapon. To gain access to their cytosolic target, ribosomal RNA, these toxins follow the retrograde transport route from the plasma membrane to the endoplasmic reticulum, via endosomes and the Golgi apparatus. Here, we used high-throughput screening to identify small molecule inhibitors that protect cells from ricin and Shiga-like toxins. We identified two compounds that selectively block retrograde toxin trafficking at the early endosome-TGN interface, without affecting compartment morphology, endogenous retrograde cargos, or other trafficking steps, demonstrating an unexpected degree of selectivity and lack of toxicity. In mice, one compound clearly protects from lethal nasal exposure to ricin. Our work discovers the first small molecule that shows efficacy against ricin in animal experiments and identifies the retrograde route as a potential therapeutic target.

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.

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...