HIV-1 Tat Enters T Cells Using Coated Pits before Translocating from Acidified Endosomes and Eliciting Biological Responses

Abstract

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...

Clathrin-mediated Endocytosis and Lysosomal Cleavage of Hepatitis B Virus Capsid-like Core Particles

“…However, at this time point the internalized particles did not co-localize with LysoTracker-red in lysosomes. Limited co-localization of the particles with the lysosomal marker was noted after 2 h whereas the particles extensively co-localized with LysoTracker red after 4 h. These results indicate that core particle routing to lysosomes is relatively slow, resembling the lysosomal trafficking kinetics of HIV-1 Tat in cells (25).…”

“…Thus, HBcAg cleavage followed by particle dissociation can facilitate release of the antigenic peptides and encapsulated ODN in lysosomes, allowing peptide loading on class II MHC molecules and TLR9 signaling, respectively. Furthermore, the acidic environment in lysosomes could possibly permit translocation of HBcAg and the delivered cargo to the cytoplasm, as was recently demonstrated for HIV-1 Tat and cationic peptides (25,32). Cytoplasmic translocation following lysosomal delivery could drive cross-presentation of peptides carried by the particle (9) and could allow delivery of encapsulated macromolecules into the cytosol and the nucleus.…”

Intracellular trafficking of nonviral vectors

“…In comparison, fulllength TAT enters T cells via clathrin-coated pits and endosomes after binding cell surface receptors utilized by HIV for infection. 123 In contrast, CHO cells, which lack T-cell-specific receptors, take up full-length TAT via proteoglycans. 113 Clearly in the examples just described, an endocytic ligand may utilize a particular cell entry route in its native context, but a vector directed by the same ligand may enter cells through a different internalization pathway.…”

The Glutamine-rich Region of the HIV-1 Tat Protein Is Involved in T-cell Apoptosis

“…Tat is efficiently taken up by a variety of cells (23,25,(27)(28)(29) and may enter T-cells by clathrin-mediated endocytosis (30).…”