HIV-1 Tat enters T-cells using coated pits before translocating from acidified endosomes and eliciting biological responses

Agnès Vendeville¹, Fabienne Rayne¹, Anne Bonhoure¹, Nadir Bettache¹, Philippe Montcourrier¹, and Bruno Beaumelle¹^*

¹ UMR 5539 CNRS, Département Biologie-Santé, Case 107, Université Montpellier II, 34095 Montpellier Cedex 5, France

^* Corresponding author. E-mail address: beaumel{at}univ-montp2.fr.

The HIV-1 Tat protein is secreted by infected cells. ExtracellularTat can affect bystander uninfected T-cells and induce numerousbiological 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 responsesdirectly upon binding to signaling receptors at the plasma membraneor following delivery to the cytosol. The pathway that enablesTat to reach the cytosol is also unclear. Here we visualizedTat within T-cell coated pits and endosomes. Moreover, inhibitorsof clathrin/AP-2 mediated uptake such as chlorpromazine, activatedRhoA, or dominant-negative mutants of Eps15, intersectin, dynaminor rab5 impaired Tat delivery to the cytosol by preventing itsendocytosis. Molecules neutralizing low endosomal pH or Hsp90inhibitors abolished Tat entry at a later stage by blockingits endosomal translocation, as directly shown using a cell-freetranslocation assay. Finally, endosomal pH neutralization preventedTat from inducing T-cell responses such as NF- ${kappa}$ B activation,apoptosis and interleukin secretion, indicating that cytosolicdelivery is required for Tat signaling. Hence, Tat enters T-cellsessentially like diphtheria toxin, using clathrin-mediated endocytosisbefore low-pH induced and Hsp90-assisted endosomal translocation.Cell responses are then induced from the cytosol.